Recent Advances in Nutrition- Reproduction Interactions in Dairy Animals

JOURNAL OF VETERINARY & ANIMAL SCIENCESTABLE OF CONTENTS

Abstracts of International Symposium on Dairy Animal Reproduction (ISDAR), March 30-31 2015,

Lahore Pakistan

Sr.No.

Title PagesNo.

I-01 New Approaches in Estrus and Ovulation Synchronization Programs in Cattle and Buffaloes Baruselli P. S.

1

I-02 Role of Ultrasonography in Management of Dairy Cattle Reproduction and Disease Diagnosis

Jaswant Singh and Gregg P. Adams

1

I-03 New Approaches of Estrus Synchronization to Improve Reproductive Performance in DairyBuffaloes

Nasim Ahmad

2

I-04 New Extension Approaches for Improving Small Dairy Holder in PakistanHassan Mahmood Warriach 2

I-05 Recent Developments in Understanding and Approaches to Deal with Reproductive Disordersin Dairy Animals

Parkash S Brar

3

I-06 Management of Reproductive Disorders in Buffaloes and CowsMuhammad Aleem and Muhammad Usman Mehmood 3

I-07 Updates on Vaginal Prolapse in Dairy AnimalsMuhammad Saleem Akhtar 4

I-08 Recent Advances in Bovine Semen CryopreservationMuhammad Anzar 4

I-09 Role of Early Onset of Puberty in Selection of Donor Bulls for Use in Artificial Insemination

Manzoor Ahmad

4

I-10 Recent Advances in Cryobiology of Bubaline Semen 5

Abstracts of International Symposium on Dairy Animal Reproduction (ISDAR), March 30-31, 2015, Lahore Pakistan

S. Murtza H. AndrabiI-11 Regulation of Puberty in Buffalo Bulls

M. Shahab, N Ahmad and M Arslan 6I-12 Nutrition – Reproduction Interactions in Buffaloes

Bhuminand Devkota 6I-13 Recent Advances in Nutrition- Reproduction Interactions in Dairy

AnimalsMuhammad Subhan Qureshi 7

I-14 Prospects of Nutrition and Reproduction in Dairy Cattle and BuffaloM. A. Rashid, M. I. Malik 7

I-15Transfer of Sperm and Embryo by Laparoscopic Method: A Technique to Improve Fertility in

Small Ruminants 8

Melih Aksoy, Ejaz Ahamd and Zahid Naseer

I-16Reproductive Biotechnologies in Dairy Animals: from Lab to Field8 Amjad Riaz

I-17Reproductive Disorders in Dairy Animals9 Nemat Ullah

I-18Recent Advances in Clinical Embryology9

Tayyab Rahil

ORAL PRESENTATIONO-01Improving Conception Rate following Application of Fixed-Time Artificial InseminationProtocol in Buffalo during Non-Breeding Season 10

S. S. Ghuman and D. S. Dhami

O-02Effect of Butylated Hydroxytoluene (BHT) Supplementation on Semen Quality atDifferentStages of Cryopreservation of Beetal Buck Sperm 10

A. Rehman, A. Sattar, A. Riaz, M. Ahmad, M. Ijaz, M. Shahzad, M. Ahmed andM. U. Mehmood

O-03Repeat Breeding Problems in Dairy Cows of Bangladesh11

M. J. D. Sarder, M. M. Rahman and M. H. Islam

O-04 Effect of Different Dietary Energy Levels on Dry Matter Intake, Milk Production, Milk


Composition and Reproductive Hormonal Level of Early Lactating Dairy Cattle 11

S. Sultan

O-05Investigations on Fertility Related Biomarkers in Water Buffalo Bull Semen to Reduce Male-Factor Losses during Peak Breeding Season 12

H. Ahmed, S. M. H. Andrabi,and S. Jahan

O-06Effects of Lycopene on Ram Sperm Parameters during Liquid Storage

M. N. Bucak, Ş. Güngör,N. Başpinar, K. Çoyan and Ş. Dursun 12

O-07 Effect of Timing of AI on Pregnancy in Estrus Synchronized Nili-Ravi Buffaloes: A

Preliminary Study 13

S. Abbas, A. Husnain, M. Hassan and N. Ahmad

O-08Prevalence and Therapeuticsof Iodine Deficiency as bortifacient in Pregnant Goats

W. Yaqub,, M. S. Khan, A. Aslam, M. Ijaz,M. Avais, J. A. Khan and M. Z. Shabbir14

O-09Efficiency of Fatty Acid-Free Defined System for In Vitro Maturation of Buffalo Oocytes 14

A. Azam, A. Husna, Q. Shahzad,T. Akhtar, E. Haq, N. Ullah and S. Akhter

O-10Effect of Skim Milk & Egg Yolk in Tris Based Extender on Liquid Storage of Buck Semen

15A. Mushtaq, M. R. Ullah, N. Hussain, M. Ahmad, M. Z. Tahirand N. Ahmad

POSTER PRESENTATIONP-01The Influence of Season on Surface Follicle Count and Oocyte Quality in Buffalo Ovaries

16 K. K. Ahirwar, V. K. Gandotra, N. Singh, D. Pathak, O. Singh, S. S.Ghuman and P. S. Brar

P-02L-Cysteine Supplementation Enhanced Post-Thaw Quality of Spermatozoa in Achai and 16

Holstein Fresian Bulls

Zakirullah, H. Khan, M. S. Qureshi, M. Younas, A. Gohar, R. U. Khan, S.S. Afridi, H. Rehman andU. Saddique, I. M. Khan and A. Khan

P-03Effects of Cysteamine on Ram Sperm Parameters during Liquid Storage


M. N. Bucak, Ş. Güngör,N. Başpinar, K. Çoyan andŞ. Dursun 17

P-04 Response of Cryopreserved Nili Ravi Buffalo Bull Semen to Gallic Acid Addition in Semen

Extender 17

M. Tariq, I. Rabbani, M. S. Yousaf, H. Zaneband S. Iqbal

P-05Reducing Glycerol with Trehalose Supplementation Increases Survival of Bull Spermatozoa

18A. A. Channa, Z. Ashraf, N. Ahmad, M. Ahmad, M. Ahmed and A. Z. Durani

P-06Effect of Feeding TMR on Reproductive Performance of Non Descript Dairy Cattle

18M. I. Khan, M. A. Mirza andB. Shahid

P-07Serum Profiles of Certain Oxidants, Antioxidants, Reproductive and Stress Hormones in

Buffaloes (Bubalus Bubalis) 19

R. Kausar, A. Hameed, M. Hussain, S. A. Khanum and N. A. Qureshi

P-08Effects of Different Equilibration Times on Cell Membrane and DNA Integrity of Sperm afterCryo-Reservation in Cattle Breeds 19

A. Khan, M. S. Qureshi, R. U. Khan, U. Saddique, I. M. Khan, S. M. H.Andrabi, A. Gohar, M. Younas, H. Khan, H. Rehman and Zakirullah

P-09Relationship of Age, Breed and Libido with Semen Traits of Cattle Bulls20 H. Rehman, M. S. Qureshi, R. U. Khan, U. Saddique, A. Khan, A.

Gohar, M. Younas, H. Khan,I. M. Khan and Zakirullah

P-10Cross Breeding Promotes Deterioration of Semen Quality in Cattle2

0 I. M. Khan, M. S. Qureshi, R.U. Khan, U. Saddique, A. Khan, S. M. H.Andrabi, A. Gohar, M. Younas, H. Khan, H. Rehman and Zakirullah

P-11Determination of Prevalence of Reproductive Diseases in Small Ruminant by RetrospectiveStudy at Northern Barind Tract in Bangladesh 21

M. H. Islam, M. J. D. Sarder, M. Rahman and M. A. H. Sarkar

P-12Fertility Improvement in Cross-Bred Dairy Cows through Supplementation of Vitamin E as

Antioxidant 21

I. Khan, M. S. Qureshi, S. Akhtar, I. Ali and Ghufranullah


P-13A Strategy to Improve Cryo-Survival and Fertility of Buffalo Bull Spermatozoa

22A. Razzaq, A. Sattar, A. Riaz, M. Avais, M. Ahmad, A. R. Malik, A. G.Tarin, M. U. Mehmood andM. Younis

P-14Studies on Uterine Microbial Flora of Sahiwal Cattle during Oestrus22

H. Rehman, M. Rabbani, A. A. Sheikh, N. Ahmad, S. Raza and J. MuhammadP-15Comparative Reproductive Performance of Beetal Goats in Accelerated and Annual Kidding

Systems 23

N. Ahmad, K. Javed, M. Abdullah, A. S. Hashmi, A. Ali and T. Hussain

P-16Use of Butylated Hydroxytoluene an Antioxidant in the Buffalo Semen Extender23

T. Munir, M. Aleem, A. Ijaz, I. K. Khan and M. Aftab

P-17Effects of Flxseed Suplementation on Semen Quality and Quantity in DifferentDairy Cattle

Breeds 24

H. Khan, M. S. Qureshi, I. Khan, Ihsanullah, S. Rehman, T. Mahsud and S.Afridi

P-18 The Way to Preserve the Genome of Valuable Animal: Epididymis Gamete Retrieval inSlaughtered Ram and its Post-Thaw Evaluation 24

U. Riaz, A. Husnain, M. I. Naveed, U. Farooq and A. Riaz

P-19Effect of Varying Concentrations of Bovine Serum Albumin and Egg Yolk on Post-Thaw

Quality of Beetal Buck Semen 25

M. Shahzad, A. Sattar, N. Ahmad, A. Rehman, M. Ahmad, M. S. Khan, M.Ahmed, A. A. Channa and S. Iqbal

P-20 Effect of Different Levels of Concentrate Mixture for Attaining Early Puberty in Sahiwal

Heifers 25

Z. Iqbal, M. Abdullah, K. Javed, J. A. Bhatti, N. Ahmad and F. Ahmad

P-21Influence of Crude Proteins Levels on Milk Production, Blood Urea Nitrogen and SerumProgesterone Profile in Mid Lactating Jersey Cows 26


T. Ahmadand S. Sultan

P-22Testicular Biometry, Scrotal Circumference, Serum Testosterone and Semen Characteristics

in Achai and Jersey Bulls 26

A. G. Khan, M. S. Qureshi, H. Khan, M. Younas, A. Gohar, S. Akhtar, R. U.Khan, H. Rehman andU. Saddique

P-23Dystocia Due to Parapagus Monster in a Murrah Buffalo27

R. K. Chandolia andG. Singh

P-24Effect of Cholesterol Loaded Cyclodextrin on Sperm Survival of Buffalo Bull during Stages of

Cryopreservation 27

M. Ahmed, A. Sattar,M. H. Fayyaz and M. Ahmad

P-25 Effect of Bypass Fat Supplementation on Productive and Reproductive Performance of High

Yielding Murrah Buffaloes 28

N. Saxena, V. Mudgal, C. Mohan, K. Kumar, M.L. Sharma, S.K. Sarkar, J. K.Singh, A. K. Pandeyand D. Lal

P-26Seasonal Pattern of Calving in Nili-Ravi Buffaloes 28

A. Waheed, M. Tariq, S. Ahmad, U. Hassan, B. Mustafa, M. M. Tariq and H.Nawaz

P-27Distribution of Buffaloes Affected with Peri-Partum Reproductive Disorders, Maintainedunder Different Production Systems 29

M. A. Ali, L. A. Lodhi, A. Waheed, M. Tariq, F. Hassan and S. Ahmad


P-28 Distribution of Buffaloes Affected with Peri-Partum Reproductive Disorders, with Reference

to Feeding Pattern 29

M. A. Ali, L. A. Lodhi, M. Tariq, A. Waheed, F. Hassan and S. Ahmad

P-29 Environmental Factors Affecting some Reproductive Traits in Holstein Friesian x SahiwalCrossbred Cattle in Pakistan 30

T. Mahmood, K. Javed, A. Ali, N. Ahmad, A. H. Saleem, M. Z. Farooq and S.Ahmad

P-30Uterine Didelphys in Buffalo-A Case Study30 U. Iqbal, M. Ahmad, M.Nawaz, A. Mehmood and F. Awan

P-31Constraints Limiting the Conception Rates in Cows Inseminated with Frozen Semen

30 Md. R. Gofurand A. M. Bhuyan

P-32Effect of GnRH Analogue Administration at the Time of A.I on Conception Rate in Nili Ravi 31

Buffaloes

M. Binyameen, S. Anwar, B. E. Azam, M. Ahmad, A. Rehman and A.U. Khan

P-33 Effect of Exogenous Growth Hormone on Semen Quality, Hematological Variables and SerumTestosterone Concentrations in Nili-Ravi Buffalo Bulls 31

A. Masoood, I. Ahmad,M. Ahmad, N. Ahmad, Z. I. Qureshi and Z. Ahmad

P-34Granulosa Cell Tumour of Ovary in Buffalo-A Case Study32

F. Awan, M. Mubeen, U. Iqbal and M. Ijaz

P-35 Comparative Evaluation of Rose Bengal Plate Test and ELISA for the Diagnosis of Brucellosisin Cattle at Private Dairy Farms of Punjab 32

M. S. Rafiq, M. Ijaz, A. Z. Durrani, K. Mehmood, A. J. Sabirand S. H.Farooqi

P-36A Case Report; Fetal Mummification Accompanied with Viable Kids at Full Term in Teddy x

Beetal Doe 33

A.Husnain, A. Rehman and M. A. Sattar


P-37Pinhole Technique – A Novel Way to Castrate Male Animals33 M. Fazili

P-38Interventions during and Post AI to Improve Conception Rate in Repeat Breeder Sahiwal 33

Cows

M. S. Akhtar, I. Hussain, M. H. Lashari, L. A. Lodhi, I. Ahmad, S.Inayat, S. A. Muhammadand S. Ullah

P-39Ultra-Rapid Freezing Protocol Improves the Post-Thaw Quality and In Vivo Fertility of Water

Buffalo Bull Spermatozoa 34

H. K. Rehman, H. Ahmed and S. M. H. Andrabi

P-40Evaluation of Sperm Quality Parameters of Fresh and Frozen-Thawed Water Buffalo Semenin Relation to Field Fertility during Peak Breeding Season 35

H. Ahmed, S. M. H. Andrabi,H. K. Rehman and S. Jahan

P-41Duck Egg Yolk Plasma in Extender Improves the Freezability of Buffalo (Bubalus bubalis) 35

Bull Spermatozoa

F. Tehseen, , S. M. H. Andrabi, H. Ahmed, A. Riaz and N. AhmadP-42 Effect of Various Concentrations of Catalase in Extender on Cryopreservation of Buffalo Bull

Spermatozoa 36

L. Ali, A. U. Khan, and S. M. H. Andrabi

P-43Effects ofAsphaltum on Libido, Serum Testosterone, Hematology and Biochemical

Metabolites in Lohi Ram 36

Y. Saeed, M. Ahmad andT. G. Shafia

P-44Juvenile In-Vitro Embryo Transfer from 4- To 8-Week-Old Kundi Buffalo Calves

M. A. Kakar,, S. Jamali, M. K. Baloch and M. T. M. Kiani 37

P-45Replacement for Commercial Dairy Farm: Intensive Breeding Program for Pre-pubertal

Buffalo Heifers 37

A. Husnain and N. Ahmad


P-46Effect of Clomiphene Citrate and Human Chorionic Gonadotropin (hCG) on OvulationInduction in Pre- Pubertal Sahiwal Heifers 38

S. A. A. Bukhari, S. Ali, I. Ahmad, Z. I. Qureshi, M. Zubair,andU. RehmanP-47Duration of CIDR Placement’s Effect on Estrus Induction in Dairy Holstein Cows

C. Öztürk, Ş. Güngör,M. N. Bucak and M. B. Ataman 38

P-48Impact of Intensity of Biostimulation on Oestrus Incidence, Conception Rate in PostpartumNili-Ravi Buffaloes during Peak Breeding Season 39

N. Zaidiand M. Anwar

P-49 Estrus Synchronization does not Improve Fertility in Chronically Repeat Breeding Sahiwal Cows 39

M. Hassan, A. Razzaq, A. Husnain, A. A. Channa and N. Ahmad

P-50Predominance of Bovine Trichomoniasis in Multan District, Adjacent Areas, Pakistan

40S. Murtaza, M. A. Raza, M. S. Akhtar, M. M. Ayaz, M. A. A. Farooq, M. Aliand F. Ali

P-51Effects of Ebselen on Ram Sperm Parameters during Liquid StorageKenan Çoyan, Mustafa Numan Bucak, Şükrü Güngör,Nuri Başpinar, Şükrü Dursun, and Pınar İli 40

Author Index

42Scientific Review Committee

43


J. Vet. Anim. Sci. (2015), Vol. 5(Suppl. 1)

The Local Organizing Committeeof the ISDAR 2015 has beensolely responsible for thereview of the abstractscontained herein. The abstractswere peer reviewed and edited byMushtaq Ahmad and Muhammad ZahidTahir

INVITED PRESENTATIONS

I-01 New Approaches in Estrus and OvulationSynchronization Programs in Cattle and BuffaloesP. S. Baruselli

Department of Animal Reproduction, FMVZ-USP, São Paulo-SP, Brazil [email protected]

Reproductive performance in a dairyherd has a significant impact onproduction and profitability. Althoughartificial insemination (AI) andembryo transfer (ET) are an importanttool to accelerate genetic gain andmilk yield, the widespread use thesereproductive biotechnologies arelimited by factors which reduce thepercentage of cows submitted for AIand ET such as a low efficiency ofestrous detection and postpartumanestrous. The use of ovulationsynchronization programs for AI and ETin dairy herds has been reportedacross multiple studies toconsistently increase the servicerate, decrease the interval fromcalving to first service and also toincrease proportion of pregnant cowssooner after the end of the voluntarywaiting period.The control of theemergence of follicular waves and of

ovulation at predetermined times,without the need for estrus detection,has facilitated the management andimproved the efficiency of AI and ETprograms in dairy herds.Follicularwave development and ovulation can besynchronized by treatments withprostaglandin and GnRH or acombination of estradiol andprogestogen/ progesterone.Synchronization protocols dramaticallyimprove reproductive success byoffering the possibility of planningthe application of assistedreproductive technologies andallowingproducers to breed more cowsin less time. Although there aresimilarities in estrous cycle lengthbetween cattle and buffaloes,reproductive differences between thesegenetic groups are well documented.For this reason, one cannot assumethat outcomes obtained in cattle (Bostaurusor Bos indicus) would be the same asin buffalo. Conclusively, the estrussynchronization treatments mayfacilitate the application of assistedreproductive biotechnologies, thusallowing a more efficient geneticimprovement and reproductive outcomein cattle and buffalo herds.Key words: Animal reproduction, Dairy,Cattle, Buffalo

I-02 Role of Ultrasonography in Management ofDairy Cattle Reproduction and Disease DiagnosisJ. Singh* and G. P. Adams

Veterinary Biomedical Sciences, Western College ofVeterinary Medicine, University of Saskatchewan,Saskatoon, Canada*[email protected]

Abstracts of International Symposium on Dairy Animal Reproduction (ISDAR), March 30-31, 2015, Lahore Pakistan 1


Use of ultrasonography in bovinepractice began in mid80’s and is now aroutine practice for large farms inbothdeveloped and developing countries.Transrectal ultrasonography is nolonger an adjunct to rectal palpation.It provides valuable informationregarding dynamic changes in ovaries(follicle number, follicle size,corpus luteum changes) and uterus(echotexture, fluid accumulation,coiling) that are predictive of thestage of estrous cycle.Ultrasonography is particularly usefulfor early pregnancy diagnosis,assessing fetal health, twinning, andfetal sex diagnosis. Ultrasonographyof testes and accessory sex glands(vesicular gland, prostate,bulbourethral glands) in male animalsprovides diagnostic and prognosticvalue for many disease conditions.Likewise, ultrasonography is usefulforassessing the response to treatmentfor ovarianand uterine diseases(luteal cysts, follicular cysts,pyometra, mummified featus, etc.). Itis noteworthy that the use ofultrasonography is not associated withhigher embryonic loss. Imaging theovaries in cattle has led to anunderstanding of ovarian dynamics inthis and other species, includinghumans. These advances have usheredthedevelopment of new protocols forovarian synchronization, fixed-timeartificial insemination, andsuperstimulation. Advances in Dopplerimaging (Spectral, Color, Power),ultrasound biomicroscopy, 3dimensionalimaging, tissue stress analysis,andechotexture analysis will ultimatelyenable us to determine the healthstatus of individual follicles andtheir contained egg and allow

determination of the precise stage ofthe estrous cycle based on a singleexamination.

Key words: Ultrasonography,Reproductive diseases,

Cattle reproductionI-03 New Approaches of Estrus Synchronization to Improve Reproductive Performance in Dairy BuffaloesN. Ahmad

Department of Theriogenology, Faculty of VeterinaryScience, University of Veterinary and Animals Sciences, Lahore [email protected]

Buffaloes contribute more than 65% oftotal milk production in Pakistan.They are often blamed for having slowreproduction. Series of systematicexperiments were conducted in ourgroup at UVAS, Lahore to see if itsreproduction can be enhanced throughestrus and ovulation protocols. Ourpremier study showed that buffaloescan be successfully synchronized withoptimum fertility usingeither PGF2alpha alone (detected oestrus) orusing (Ovsynch protocol) during lowbreeding season, to calveduring theperiod when milk availability is short(Warriach et al 2008). Then we movedon to synchronization with CIDR basedprotocols particularly during summerwhen most buffaloes are anestrous.Administration of EB and GnRH afterCIDR removal resulted in tightersynchrony (less variability)andimproved fertility in anoestrousbuffaloes (Naseer et al., 2010). Thisimplied great potential in fertilityimprovement in anoestrous buffaloes.These results got confirmed in a studybased on large number of buffaloes



which indicated that administration ofEB in conjunction with CIDR improvesestrus intensity and pregnancy rate inmultiparous buffaloes. Similarly,there was a trend (P>0.05) of moresynchronous timing of ovulation(62.9±1.8 vs 72.4±3.6hrs) and higherovulation rate (91% vs 64%) after EBadministration (personalcommunication). Because ovulationstudies in normal buffaloes arescarce, we determined it and foundthat timing of ovulation was 35.3±0.21hours from the onset of standing heat.More interestingly, highest pregnancyrate, 53% (15/28) was observed inbuffaloes inseminated at 24 h,followed by 37% (10/27) at 12 h. Theseare exciting new findings which implythat AM-AM rule (24 hr) is moresuitable for buffaloes (Riaz et al.2014). In order to enhance ovulationand pregnancy rates we found thataddition of eCG at the time of CIDRremoval in FTAI program increased theestrus response, ovulation rate andpregnancy per AI in Nili-Ravibuffaloes (Naveed et al., 2014). Onthe question of reducing embryo lossin buffaloes we enhanced progesteroneafter AI on day 7 throughadministration of hCG. Resultsrevealed that hCG successfully inducedovulation and improve overallconception rate (Husnain et al.,2013). In conclusion, variousinnovations in synchronizationprotocols indicate clearly thatpregnancy per AI can be significantlyenhanced, both breeding and lowbreeding season, in buffaloes. Thisoutcome is more pronounced when thebody condition of buffaloes is good.However, its implications remainchallenging for a small holder buffaloproducer. (Acknowledgements: Author

gratefully acknowledge financialassistance provided by the HEC Pak-USProject under S&T agreement).

Key words: Estrus synchronization,Hormones, Fixed timeinsemination

I-04 New Extension Approaches for Improving Small Dairy Holder in PakistanH. M. Warriach

Project Manager, ASLP Dairy ProjectUniversity of Veterinary & Animal Sciences, Lahore [email protected]

Pakistan is a developing country whoseeconomy primarily depends onagriculture. Livestock forms 55% shareof agriculture in national GDP. Dairyfarming forms the major segment oflivestock industry in Pakistan.According to an estimate there are 9million small holder dairy farmers inPakistan. Livestock department isprimarily focusing on providingveterinary care and treatment whilemeagre efforts are being done toincrease the production of animals.Most of small holder dairy farmers areunaware of basic principles oflivestock management. We launched adairy extension program in 56 villagesof Pakistan under the umbrella ofAgriculture Sector Linkages Program(ASLP). We conducted monthly farmermeetings in selected villages tospread awareness on livestock health,management, nutrition andreproduction. We noticed that adoptionof simple extension messages had asignificant impact on the milkproduction of animals. For example,simply untying of animals resulted in1.5 liter increase in milk productionper animal per day. If we can achieve



the same in whole lactating herd ofthe country, we can bring a revolutionin total milk production. Similarly,high calf mortality is a big issue inPakistan which can be easily dealtwith by adopting simple calf rearingpractices like provision of ad libcolostrum to calves. We experiencedthat individual visits of extensionworker to farms on regular basis canhelp to develop trust and motivatedairy farmers to adopt the extensionmessages. Moreover, we observed thatit is of utmost importance to adoptwhole family approach in dairyextension because women and childrenare equally involved in small holderdairy production system. In thisregard, monthly female farmer meetingsand involvement of local schools indairy extension proved to be quiteuseful. Lastly, informationcommunication technology (ICT) can beused to spread mass awareness.Television, radio, social networkingand text messaging on cell phones areeffective tools to spread extensionmessages at mass level.Key words: Communication, Extension,

Small Dairy holder,Reproductive managment

I-05 Recent Developments in Understanding and Approaches to Deal with Reproductive Disorders inDairy AnimalsP. S. Brar

Veterinary Gynaecology and Obstetrics, Guru Anad DevVeterinary and Animal Sciences University Ludhiana,Punjab, Indias [email protected]

Dairying, an integral part of humanlivability, is undergoing fast

structural changes, especially inAsia. Although the milk yield in dairycattle has considerably increased overthe last 50 years,there has also beena temporal increase in the incidenceof infertility. With the invent ofnewer knowledge of animal reproductiveprocess, and development andapplication of better diagnostictechniques, our abilities tounderstand, and to address the variousreproductive disorders have alsoimproved. The underlying causes formost of the reproductive failures liein inappropriate and faulty animalmanagement and nutritionalinadequacies. Precise role of thevarious components of the nutrition iscomplex and yet remains to be fullyunderstood.This information though isvery vital to the development ofappropriate feeding strategies foruncompromised harmonious health,production and reproductive processes.We have observed that dietarydeficiency of polyunsaturated fattyacids affected the duration of oestrusin dairy cattle by altering theproduction of prostaglandins fromendometrium. Further, the various feedsupplementations with higher energysources have not always beensuccessful in preventing or curinginfertility. Studies have revealedthat increasing the proportion ofproteins in animal diet, althoughimproved milk yield but decreasedtheconception rates by altering theuterine environment. Some of thesuccessful efforts in improvingfertility included supplementaryfeeding of ionophoreswhich increasedcirculatory glucose, decreasedentericmethane production and improvedfertility in dairy bovines. Theoestrus synchronization protocols



developed and tested in our laboratoryhave been successful to quite anextent in by-passing the problem ofestrus detection andimprovingfertility. Studies have also suggestedthat changes in the duration ofoestruscycles, often associated withinfertility in high yielding cows, mayhave occurreddue to luteal deficiencyowing to increased progesteroneketabolism. Newer techniques have alsobeen developed to facilitate stressfree and uneventful relief in certainobstetrical conditionssuch as uterinetorsion. Further studies are warrantedon better understanding of climatechange, and various biotic and abioticstressors vis-à-vis reproductiveefficiency in dairy cattle.

Key words:Reproductive disorders, Reproductive

efficiencey, Fertility, Bovines

I-06 Management of Reproductive Disorders in Buffaloes and CowsM. Aleem* and M. U. Mehmood

Department of Theriogenology, University of Veterinary and Animals Sciences, Lahore, Pakistan *[email protected]

Reproductive disorders occurfrequently in dairy animals andseverely affect both theirreproductive efficiency andprofitability. In Pakistan, dairyfarming mainly based on buffaloes andcattle (Sahiwal,Cholistaniand crossbred) population. Recently exoticanimals (Friesian, Jersey and Friesianx Jersey) are imported to meet theincreasing demand of milk production.Some ofthe most common reproductivedisorders confronting dairy animalsinclude anestrus, uterine torsion,

metritis, prolapse, cystic ovariandegeneration (COD), retained fetalmembrane (RFM), repeat breeding (RB)and abortion. Incidences of thesedisorders vary in different breeds andspecies. Like, anestrusis the majorcondition in dairy buffalo and inexotic cows repeat breeding andabortion are commondisorders.Occurrences of these disordersincrease in summer months.Mostlikely,inadequate feeding is the primereason in the former case and loweredheat tolerance and poor management inthe case of latter. Uterinetorsion,prolapsed and dystocia arecommon disorders faced in buffaloesparticularly of smallfarmers.Diagnosis generally based onphysical and clinical examination andsurgical procedures like caesareansection are relatively uncommon duelack of resources, expertise and postoperative care.Likewise, the majorproblems of high yielding pure exoticdairy breeds are cystic ovariandegeneration, anestrus, repeatbreedingand footrot. Neverthelessreproductive disorders aresignificantly lower in exotic crossbred cows. However, incidence ofreproductive disorders can and arebeing reduced by avoiding summercalving in exotic breeds with theadaptation of balanced nutrition, andusing estrus synchronization protocolswith the application of bestmanagement practices. Moreover,extension and educationalprogrammesfocusing herd management andpreventive veterinary practices areessential to improve the reproductivedisorders in dairy animals.

Key words: Cystic ovariandegeneration, Infertility,

Summer stress



I-07 Updates on Vaginal Prolapse inDairy AnimalsM. S. Akhtar

Faculty of Veterinary Sciences, Bahauddin ZakariyaUniversity, Multan, Pakistan [email protected]

Prolapse of vagina is the most commonmaternal gestational complication indairy animals that includes outwardprotrusion of the vaginal floor, thelateral walls and a portion of theroof of the vagina through the vulvarlips. The basic cause appears to bethe weakening or relaxation of theconstrictor vestibuli muscle and atonyof the vaginal musculature. Theincidence of pre-partum cervico-vaginal prolapse has been higher (3-13%) in water buffalo. The incidenceis higher in summer season and is morein stall fed animals. Postulatedetiologies for a higher incidenceinclude low levels of plasma copper,selenium and zinc, low levels of serumcalcium and phosphorous and increasedlevels of plasma estradiol during lategestation.Less common etiologicalfactors include vaginitis and urinarytract infection. Vaginal prolapse ismore common between 8-10 months ofpregnancy in buffalo.The protrusion ofan ugly looking soiled mass is anobvious clue for diagnosis made by theowner.Except in extreme or severecases, the prognosis is generally fairto good for the life of the animal,and the fetus, if treatment is promptand aftercare is good.The vulvar trussis of practical value in pregnantdairy animals and is a frequently usedmethod for prevention of recurrence ofcervico vaginal prolapse. The trussmade of leather, wide cloth and ropesare good.A wide variety of sutures

have been suggested for retaining theprolapsed organ in dairy animalsincluding the buhner suture, thebootlace suture, flessa sutures, andthe interrupted horizontal mattresssuture.Two techniques for surgicalrepair of vaginal and cervicalprolapse include vaginopexy (Minchev’smethod) and cervicopexy(Winklers operation).Care of animalswith prolapse include little ofexercise, feeding of less bulky diets,reduction in estrogenic feeds andfrequent watch of these animals.Recentlya modified Buhner’s technique,using sterile infusion set tubing assuture material, is suggested to beeffective in retention of the mass incattle.

Key words: Dairy animals, Vaginalprolapse, Incidence,

Etiology, TreatmentI-08 Recent Advances in Bovine Semen CryopreservationM. Anzar

Research Scientist (Cryobiology), Agriculture and AgriFood Canada, Saskatoon Research Center, Saskatoon, Saskatchewan S7N OX2, Canada [email protected]

Cryopreservation is a long-termstorage of living cells or tissues atextremely coldtemperature.Cryopreservation ofgermplasm (semen, oocytes and embryos)plays an important role in theconservation ofanimal geneticdiversity and exploitation ofgenetically superior animals.Frozensemen is extensively used inartificial insemination programsthroughout the world. The success ofsemen cryopreservation largely dependsupon species and breeds within



species. During semencryopreservation, sperm undergovarious processes like dilution,cooling, and freezing and thawing.During these processes, sperm face thebiophysical events, i.e. osmoticshock, solution effect andintracellular ice formation. Theseevents affect sperm structures whichultimately result incells’malfunctioning. Since the inception ofsemen freezing technology,cryobiologists have been trying tominimize sperm damage due to thebiophysical events. Recently, the pre-freeze treatment of mammalian spermwith cholesterol-loaded cyclodextrinimproved the post-freeze survival ofsperm in various species. Generationof reactive oxygen species (ROS)during semen processing leads to lipidperoxidation of sperm plasma membraneand death. The addition ofantioxidants in semen extender helpsto neutralize the adverse effect ofROS on sperm health. Due to the bio-security risks associated with animalproducts, the scientists are eagerlylooking for semen cryopreservationprocedure without animal protein (eggyolk or milk). There is always a scopeof improvement in semencryopreservation technology. Highquality of frozen-thaw semen ensuresgood fertility in the field.

Key words: Cryopreservation, Reactiveoxygen species,

Semen

I-09 Role of Early Onset of Pubertyin Selection of Donor Bulls for Usein Artificial InseminationM. Ahmad

Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore [email protected]

Pakistan has the best dairy buffalobreeds in the world (NiliRavi andKundi). In spite of the fact thatartificial insemination (AI) wasintroduced in the country in 1955, itsbenefits like in advanced dairy world,have so far failed to show similarenhancement in Pakistan for buffaloproductivity over the last 65 years.The main reason for this has been theuse of donor bulls without exactlyknown genetic values.The semenproduction unit, Qadirabad (Sahiwal),since 1972 is maintaining a largenumber of donor buffalo bulls forproduction of semen doses to meet theinsemination demands of the field. In1980, a research project startedinvestigating the state of fertilityof the stock. The data analyzed as ofnormal bulls showed an average bodyweight of 511 ± 11.82 kg achievingfirst semen ejaculation by 35.22 ±1.53 months of age. These averages forabnormally developing bulls maintainedduring the same period (1972-80) forbody weight and age at first semenejaculation were 548 ± 22.25 kg and41.2 ± 2.62 months, respectively.During the conduct of this work,definite relationships between age andbody weight, age and scrotalcircumference (cm), scrotalcircumference and semen productionability and sexual behavior indicesand onset of puberty were established(Ahmad et al. 1983 and Arsalan et al.1987). Following establishment of theselection criteria, two studies onnormally developing young bulls raisedunder optimal feeding and managementand observing/ recording the various



sexual behavior indices after 15 monthof age till onset of puberty wereundertaken. One such study was atsemen production unit Qadirabad(Sahiwal) involving 13 young candidatebulls during 1981 to 1983. Five youngbulls of this group donated semenbefore two years of age (from 21 to 23month of age). The other study wasundertaken at NARC, Islamabad during1984-86 on 16 growing bulls. Six bullsof this group gave semen ejaculatebefore two years of age (from 16 to 24months of age). These were remarkablefindings and were worthy of follow upfor selection of “Performance TestedBulls” capable of yielding semenaround two years of age.In early 2014,it was decided to make a “Calf RaisingCenter” operational atBuffalo Research Institute, Pattoki(District Kasur). As a first step, theexisting status of bull raising foruse in AI at different sites wereevaluated by recording their age, bodyweight, scrotal circumference and ageat first semen ejaculation. At BRI, agroup of 18 young bulls brought fromdifferent sites and different ownerswas raised as a future donor bulls.Their ages, body weights (kg) andscrotal circumferences (cm) variedfrom 25 to 47 months, 250 to 413 kgand 17.5 to 25.0 cm respectively.These recorded values for differentages were much less than theirqualifying standards. They were farmore than two years of age, yet noneof them was able to show the sign ofonset of puberty.At SPU, Qadirabad,young bulls being raised under goodmanagemental condition, 30 of themvarying in age from 25 to 36 monthshad body weights and scrotalcircumferences varying from 270-540 kgand 19 to 29 cm respectively. The

second group of 11 bulls rangedbetween36 to 47 months had body weights andscrotal circumferences varying from395 to 553 kg and 22.5 to 29 cmrespectively. None of these 41 growingbulls whose ages were much beyond twoyears had so far not yielded any semenejaculate. Among adult donor bullgiving regular weekly semenejaculates, showed that their ages atfirst semen ejaculation were 39 and 50months (best bull and the oldest).Among four available progeny testedbuffalo bulls, their ages at firstsemen ejaculation were 37, 37, 39 and39 months. All these findingssuggested that in years following 1987till to-date, selection and rearingprograms of breeding buffalo bullsfailed to follow the targets ofproducing “Performance Test Bulls”capable of showing onset ofpuberty by around two years of age.This situation was causing lessproduction of fertile semen doses anddelaying the age of production ofprogeny tested bulls.Since August2014, at CRC six candidate bull calvesare being raised in four age groupsunder a uniform environment ofnutrition, health cover andmanagemental practices. From birthonward their monthly body weights andthrice monthly scrotal circumferencesare being recorded. After 15 month ofage in group of eight they are takenout twice weekly in a special openpaddock area to observe the expressionof various sexual behavior indices(sexual interest (SI), mounting (M),penile protrusion (PP) and semenejaculation (SE). So far, four youngbulls have expressed onset of pubertyat 20, 21, 23 and 23 months of ages.Nine are showing partial penile



protrusion and two have shown mountingbehavior. These recording willcontinue till such time these bullsexpress the process of onset ofpuberty. The data thus obtained willbe tabulated that at what age theycompleted the period of onset ofpuberty. This exercise will continuein a cyclic manner as a routine of the“Calf Raising Center”. Those showingonset of puberty will leave CRC forSPU for further raising and progenytesting. New ones completing the ageof 15 months will join the exercisefor detection of arising signs ofsexual behavior indices.

Key words: Young Buffalo Bulls; Onsetof puberty; Calf RaisingCenter; Semen productionUnit; Sexual Behavior Indices(Sexual Interest) (SI),Mounting; Penile Protrusion(PP) and SemenEjaculation (SE)

I-10 Recent Advances in Cryobiologyof Bubaline SemenS. M. H. Andrabi

Animal Reproduction Laboratory, Animal SciencesInstitute National Agricultural Research Centre,Islamabad, Pakistan [email protected]

Cryopreservationof bovine semeninvolves severalcritical steps thatcanaffect sperm structure andfunctions. Plasmalemma is the primarysite of injury, which occurs duringthe freeze-thaw processes, resulting insubstantial loss of viablespermatozoa. Cryopreservation may alsoaffect the sperm DNA stabilityresulting in decreased fertilizationrates. Successfulcryopreservation ofbovine spermatozoadepends on several

factors, like biochemical makeup ofsemen, season of collection,composition of extenders (buffersandcryoprotectants), cooling/freezing andthawing rates. Therefore, thispresentation details the recentadvances in cryobiology of bovinesemen to minimize the “adverseaffects”.

Key words: Buffalosperm,Cryoprotectants, Thawing

I-11 Regulation of Puberty in Buffalo Bulls

M. Shahab1, N Ahmad2 and M Arslan3

1Department of Animals Sciences, Quaid-i-AzamUniversity, Islamabad; 2Department of Theriogenology, University of Veterinary and AnimalSciences, Lahore and 3Pakistan Academy of Sciences, Islamabad [email protected]

Puberty is hallmark of developmentwith great physiological importance.This milestone is especially importantin farm animals as it makes possiblereproduction and production. Pubertyis orchestrated by synchronizedneural, endocrine and somatic signals.While the underpinning of puberty hasbeen well studied in several dairyspecies surprisingly little is knownabout the pubertal dynamics in waterbuffalo which is a prime dairy animalin Asia. This talk focuses on somatic,morphologic, behavioral and endocrinedeterminants of puberty in male waterbuffalo based on our earlier work withthis model and intends to give ideasfor the future research in the area.Longitudinal (birth to puberty) andcross-sectional studies indicate thatin Nili-Ravi male buffalo meanpubertal age, body weight andtesticular volume are 22.8±1.1vmonths, 421±19 kg and 188±12 cm,



respectively. The attainment ofpuberty is related to the time offirst ejaculation with motile sperm.Correlations among age, body weightand testicular volume are >0.95(P<0.01). Increase in body weight andtesticular size is markedly greaterfrom 8-15 months of age than duringany other period of development. Asignificant (P<0.05) relationship isfound between age at puberty and thetime when a maximal increase intesticular growth rate is observedduring the prepubertal period. Seasonof birth does not influence (P>0.05)age, body weight or testicular volumeat puberty. Mean serum testosteroneconcentrations remain low from birthto 12 months of age (0.3±0.1 ng/ml). Amarked rise in testosterone isobserved at 14 months of age (2.7±0.9ng/ml) followed by a second peak at 18months of age (3.3±1.2 ng/ml).Increased androgenic activity leads toestablishment of spermatogenesis, asreflected by appearance of significantnumber of spermatids, occurring by 18months of age. A significant increasein sexual behavior score alsomanifests around this time. Serumestradiol levels increase during theearly part of prepubertal developmentattaining a mean concentration of 27±3pg/ml at 5 months of age and remainmore or less unchanged until the ageof 15 months. The levels declinethereafter and remain low throughoutthe rest of the pubertal development.Interestingly a pulsatile pattern ofestradiol secretion is observed inneonate and juvenile males whereas theprepubertal animals show an absence ofsuch a pattern. Collectively foregoingresults suggest that in the buffalotesticular quiescence extends from 0to 7 months of age, followed by a

period of rapid testicular growth andconsiderable androgenic activity alongwith decreased estradiol tone.Enhanced testosterone secretionestablishes spermatogenesis andinduces sexual behavior. Lot needs tobe studied regarding developmentalgene expression in the buffalohypothalamus that is the penultimatetrigger of initiation of puberty.

Key words: Puberty, Hormones, Age,Testicular growth

I-12 Nutrition-reproduction Interactions in BuffaloesB. Devkota

Department of Theriogenology, Agriculture and Forestry University, Chitwan, Nepal [email protected]

Buffalo rearing is very important forrural development in many countries.The buffalo production system,however, is a subsistence type in mostof the countries. These buffaloes aregenerally kept only under roughagefeed with an occasionalconcentratesupplement that put them undernutritional stress. Studies in ruralbuffaloes in Nepal using nutritionalindices of body condition score (BCS)and some blood metabolic parametersindicated that buffaloes were undersevere nutritional stress during thepre-monsoonfodder-scarce dryseason ofFebruary to June. The effectsonreproductive inability were related tosub-normal levels of some minerals andtotal protein in the blood. Also, theproportion of anestrus buffaloes withpoor BCS was greater (P<0.01) duringthe dry season as compared with thefodder-rich monsoon or post-monsoonseason. Nutritional status alsoaffected on the result of assisted



reproduction in anestrus buffaloes. Insilently ovulated buffaloes bearing CLin the ovary, treatment with PGF2 waseffective producing higher estrusdetection and pregnancy rates ascompared to the conventionaltreatment. However, BCS of thebuffaloes before treatment affected(P<0.05) pregnancy rate aftertreatment. Pregnancy rate was alsoadversely influenced by low serumconcentrations of calcium (P<0.01) andtotal protein. When the effect ofprogestin based CIDR co-synch protocolwas compared betweenthe group ofanestrus buffaloes with BCS of >2.5and <2.5 during the dry season,thefirst AI pregnancy rate was higher(P<0.05) in buffaloes with >2.5 BCSthan in<2.5 BCS buffaloes. Therefore,understanding of nutrition –reproduction interaction is importantwhile implementing modern techniquesto improve reproductive efficiency ofthe buffalo.

Key words: Dry season, Reproductiveperformance, Fodders

I-13 Recent Advances in Nutrition- Reproduction Interactions in Dairy AnimalsM. S. Qureshi

Faculty of Animal Husbandry and Veterinary Sciences University of Agriculture, Peshwar-25120, Pakistan [email protected]

Reproduction is the luxury function ofmammalian bodiesand has got the lastpriority in nutrients partitioning indairy animals, after lactation andhealth.Appropriate intake ofnutrients, associated with desirableconcentrations of blood metabolites,especially glucose, is needed for anormal reproductive cyclicity. The

interaction between reproduction andnutrition becomes critical during thepostpartum periods in high yieldingdairy animals when most of thenutrients are utilized forlactogenesis and little are left forsupporting reproductive activities.Theprimary objective of rearing dairyanimals has been the production ofmilk. Selective breeding has been inpractice and lactation has beenconsidered as the most importanteconomic trait. The two traits oflactation and reproductive efficiencyhave been found to be associated witheach other; hence the selection formilk yield also led to enhancedfertility. However, beyond certainlevels of milk production the drain ofnutrients from the circulating bloodresults in cessation of reproductiveprocess. The declining fertility withthe enhanced lactation yield duringthe last several decades has beenfound both under total mixed ration inthe USA as well as pastoral feeding inthe New Zealand. The postpartuminitiation of lactation coincides witha decreased feed intake and theanimals enter into a state of negativeenergy balance (NEBAL). The energydeficiency is met out by the processof lipolysis, leading to loss of bodycondition score (BCS). Pulsatilerelease of LH and response of thefollicle to LH surge are hindered.Such a condition leads to a delayedovulation and lowered conceptionrates. Plasma levels of insulin-likegrowth factor-I are directly relatedto energy status and is critical toovarian follicular development. Duringthe early NEBAL period, the ability offollicles to produce sufficientestradiol for ovulation seems todepend on the availability of insulin



and IGF-I in serum and the changingenergy balance profile. Leptin hasbeen discovered as a product of whiteadipose tissues, reflecting the energystatus of the body. Leptinconcentrations are lower during adeclining BCS and vice versa; beingfavorable during the risingconcentrations. Hence leptin has beencalled as the permissive factor forthe reproductive process. The animalsget out of NEBAL when the nutrientsintake exceeds utilization and the BCSstarts raising. It leads to resumptionof ovarian cyclicity and conception.Poor nutrition and weight loss incattle causes a decrease in bloodprogesterone concentrations. Onepossibility is that greater milkproduction in dairy cattle isnegatively affecting bloodprogesterone concentrations andcausinginfertilityindairycows. Lowerplasma concentrations of progesteronehave been reported in cows with highermilk yield. There are severalpotential mechanisms for decreasedprogesterone in highproducing cows.Progesterone concentrations in bloodare determined by rates of secretion,metabolism and clearance throughliver. In dairy buffaloes, crudeprotein intake (CPI) was positivelycorrelated with the postpartumestrusand ovulationintervals (POI).CPI excess torequirements was lower inanimals which expressed oestrus thanthose which remained anoestrus.Prepartummetabolizable energy (ME)intake was higher in animals observedin oestrous than those remaininganoestrous. The animals resuming tooestrus had a narrow and almostconstant CP/ME ratio, while theanoestrus animals had a widelyfluctuating ratio, ranging from 10.7

to 13.1 g/MJ. CP/ME was relatedpositively with POI. Increasing energyintake increased BCS (r=0.16) andduration of expulsion ofplacenta(r=0.19) and discharge oflochia (r=0.24) butdecreasedpostpartum ovulation interval(r=-0.27, p<0.01).Reproductive processis regulated by the hypothalamus-pituitary gonadal axis and itfunctions only if the hypothalamuspituitary adrenal (or stress) axis issilent. Dairy cattle face thechallenge of stress associated withhigher metabolic rates, lactation,higher ambient temperatures andmalnutrition. Oxidative stress resultsdue to imbalance between theproduction of reactive oxygen speciesin higher quantities than theirclearance through the antioxidantsystem. It causes damage to thecellular function, decreasing theproduction of hormones and geneproducts boosting the reproductiveprocess. Antioxidants nutritionalsupplementation helps the dairyanimals in maintaining reproductiveactivities.

Key words: Reproduction; nutrition,stress, fertility, ovariancyclicity, protein, energy,antioxidants

I-14 Prospects of Nutrition and Reproduction in Dairy Cattle and BuffaloM. A. Rashid*, M. I. Malik

Department of Animal Nutrition, University of Veterinary and Animal Sciences, Lahore, Pakista *[email protected]

Improved reproduction is one of themain contributors to the profitability



in the dairy production. Among theseveral other factors, nutritionalmanagement has been found to play avital role in achieving reproductivetargets. It is generally accepted thatnutritional management is the mainlimiting or controlling factor forreproduction in many types of domesticlivestock. However, this relationshipis complex and has given variableresponses. Over the past few decadesadvancements in animal selectionimproved the animal productivitysignificantly with negative impact onreproductive traits.To date, the areaof nutrition and reproduction in dairycattle has been recognized andinvestigated extensively and reviewed.Studies suggest that reproductiveefficiency and fertility can bedirectly or indirectly affected by thenutritional factors like energy,proteins, fats, minerals and vitamins.On the contrary to dairy cattle,buffalo reproduction is characterizedby low fertility, longer calvinginterval, seasonal breeding, poorestrous behavior, delayed age ofpuberty and age at first calving inheifers. Main reason for poorreproduction in buffalo has been thepoor nutrition. To date, limitedpublished work is available on theeffect of nutrition on reproduction inbuffalo. The purpose of current paperis to review and highlight thenutritional factors that can possiblyaffect the reproduction, with specificemphasis on dietary components such asprotein, energy, fat and vitamins orminerals in dairy cattle and buffalo.

Key words: Nutrition, Reproduction,Cattle, Buffalo

I-15 Transfer of Sperm and Embryo by

Laparoscopic Method: A Technique toImprove Fertility in Small RuminantsM. Aksoy*, E. Ahamd and Z. Naseer

Department of Reproduction and AI, Faculty of VeterinaryMedicine, Adnan Menderes University, Aydin, Turkey*[email protected]

Assisted reproductive technologieslike artificial insemination (AI) andembryo transfer (ET) are beingemployed as rapid tools to extendproductivity of livestock industry.The vaginal and trans-cervical methodsof AI and ET are routinely practiced;however, the fertility rate in termsof pregnancyis low following thesemethods in small ruminants.In case ofAI, the sperm have to travel fartherto the point offertilization;therefore, large numbersof sperm (100-300 million sperm) arerequired per insemination. Conversely,in sperm sorting business andgenetically engineered sperm toproduce transgenic animals, a verylimited numbers of sperm are availablefor insemination. Similarly, thedirect deposition of frozen thawedembryos is required for successfulpregnancy. In this scenariolaparoscopic method of sperm andembryo transfer is advantageous, sinceit is efficient (3-5 min are requiredper animal) and provide optimalpregnancy rate by using minimum numberof frozen thawed sperm. The overallpregnancy rates with laparoscopic AIand ET are reported upto 50-60 % and45 % respectively. This pregnancy rateoccasionally reached to 85% duringbreeding season. Furthermore, thelaparoscopic AI is reported as themethod of choice to get higher



pregnancy rate in nulliparous sheepand goat. In conclusion thelaparoscopic method of AI and ET hasprofoundly paved the way to improvefertility in small ruminants.

Key words: Laparoscopic AI, Embryotransfer, Small ruminants

I-16 Reproductive biotechnologies in dairy animals: from lab to fieldA. Riaz

Department of Theriogenology, University of Veterinary& Animal Sciences, Lahore [email protected]

In Pakistan, the corporate dairies,though more inclined to exoticanimals,but also claim for compromisesdue to climatic shifts. In contrast,the indigenous dairy animalsare bestaccustomed to our environment buteconomically not suitable forcorporate setup due to geneticdiversity. To equilibrate the geneticvariation, an efficient reproductiveperformance is required to reduce thegeneration interval. The reproductiveefficiency could be improved byadopting the recent advances inbiotechnologies. For fieldapplication, artificial insemination(AI) technique is most famous.Forbuffalo, the different inseminationtimes (24h after heat signs) emphasizeus further to investigate ovarianphysiology in local animals. Parallelto AI for male genome, embryo transfer(ET) is being performed to harvest thegenetic potential from female side. ETis not successful in our local animalsdue to poor super-ovulation responseand is because of poor ovarianreserves. The efforts are being madeto establish criteria for assessing

ovarian reserves. The fieldapplications of AI, ET and in-vitroproduced embryos is being appreciatedbecause of the availability of sexedprogeny based on sexed semen.An in-vitrofertilization (IVF) technique has madeit possible to harvest maximum numberof embryos by ultrasound guidedaspiration of oocytes (OPU) directlyfrom ovaries. IVF also providesopportunities to use relatively lownumbers of sperm to produce viableembryos. This allows for theutilization of high value semen andmay provide significant opportunitieswhen coupled with sexed semen. Anothermost attractive biotechnology isanimal cloning. It has potential toproduce carbon copies of elitegenetics but its applications arelimited to experimental purposesbecause of poor efficiency. Keeping inview the future prospects we need toestablish our basic skills in thisfield of fourth generationreproductive biotechnology. Inconclusion, thereproductivebiotechnologies are powerful tools forbreed improvement but need furtherinvestigations related to indigenousdairy animals.

Key words: Embryo transfer, Ovum pickup, Artificial insemination

I-17 Reproductive Disorders in Dairy AnimalsN.Ullah

Project Executive, Genetic Improvement Centre (GIC)PLDDB, LES Khizerabad, District Sargodha, Pakistan [email protected]

Reproductive disorders affecting thefertility of main dairy animals isdiscussed in this paper. Dataconcerning reproductive disorders in



cattle and buffalo have been gatheredfrom different livestock farms andveterinary clinics in Pakistan. Repeatbreeding, to combat anoestrus, uterineprolapse, retention of foetalmembranesand dystokia have beenrecorded as major disorders affectingfertility of dairy animals. Measureshave been suggestedto combat thesedisorders and keep the fertility ofdairy animals within acceptablelimits.

Key words: Fetal membranes, Prolapse,Dairy animals

I-18 Recent Advances in Clinical EmbryologyT. Rahil

Reproductive Biology and Obstetrics and Gynecology Baystate Medical Center/Tufts University, Springfield Massachusetts, USA [email protected]

Objective of this presentation is toreview recent advances in clinicalembryology. Since the first child,Louis Brown was born through in vitrofertilization (IVF) in 1979; selection ofdevelopmentally competent embryos hasbeen the focus of clinicians. Embryoselection based on morphologicalevaluation has some clinical relevancebut is highly subjective. In clinicalembryology selection ofdevelopmentally competent embryosusing preimplantation genetic testingemerged in early 2000s. Implantationfailures, recurrent pregnancy losses,and developmental defect like Down’ssyndrome (Trisomy21) are attributed to embryoaneuploidy. More than 50% of the firsttrimester aborted fetuses areaneuploidy (Jacob 1992). Chromosomalabnormalities may range up to 60% in

cleavage stage embryos from womenyounger than 35 years to 80% in women41 years and older (Munne´ et al;2007). Genetic testing of embryos forchromosomal abnormalities and singlegene defect helps to select a normalembryo. Time lapse imaging of in vitrogrowing embryos is emerging as a toolto select developmentally competentembryos. A risk model of aneuploidy bystudying the embryo morphokinetics hasbeen reported (Campbell et al 2013).Fertility preservation in male hasbeen in practice for many decades.However, female fertility preservationthrough oocyte cryopreservation formedical as well as social reasons isrelatively a recent phenomenon. Liverbirths as a result of embryo producedfrom cryopreserved oocytes have beenreported. A detailed account ofselection of developmentally competentembryos using embryo genetic andmorphokinetic techniques will bepresented. Female fertilitypreservation by vitrification ofoocytes will also be discussed.

Key words: IVF, Fertility, Genetictesting



ORAL PRESENTATIONS

O-01 Improving Conception Rate followingApplication of Fixed-Time ArtificialInsemination Protocol in Buffalo during NonBreeding SeasonS. S. Ghuman1* and D. S. Dhami2

1Department of Veterinary Gynaecology and Obstetrics,Guru Angad Dev Veterinary and Animal SciencesUniversity, Ludhiana-141004, India2Veterinary Officer,Civil Veterinary Hospital, Badbar, Barnala-148106, India *[email protected]

Small dairy farmers raising 1-3bovines account to 3/4th of livestockwealth and 68% of milk production inIndia. Moreover, only 19.2% buffaloownership in India is with largefarmers. Thus, to have a major impacton total milk production in India,strategies need to fous on improvingthe reproductive potential of buffaloreared by small farmers. Applyingfixed-time AI (FTAI) protocol inbuffalo may provide a potentialalternative for increasing their lifetime productive period. However, themajor bottlenecks in wide applicationof FTAI at small farmer’s doorstepare, a) poor conception rate duringnon-breeding season, and b) failure ofnon-conceived buffalo to return toestrus following FTAI. In addition, ifa buffalo fails to conceive, a farmerhas to bear a loss of $4.2/d in termsof loss of milk and other managementalexpenses. Thus, the small farmers canbe conveninced to adopt FTAI if aprotocol with good conception rate isavailable. The present study during

non-breeding season aimed atcomparison of fertility outcome of twoFTAI protocols in buffalo reared bysmall farmers. Group-I (Protocol cost:$11.63): Buffalo (n=24) were subjectedto Ovsynch protocol (d0 and d9, 20µgGnRH analogue each; d7 500µg PGF2α,both hormones by i.m. route; AI 16hafter d9 GnRH). Group-II (Protocolcost: $26.16): Buffalo (n=96) wereadministered (i.m.) 2mg estradiolbenzoate on d0 and 500µg PGF2α on d9.Sustained progesterone release device(1.38g progesterone) was placedintravaginally from d0 to d9. On d11,20µg GnRH analogue and 500IU eCG wereadministered (i.m.). AI was done 16hafter GnRH analogue admnistration. Inboth groups, pregnancy was confirmedby ultrasound aided diagnosis on d60post-AI and the buffalo failing toconceive and returning to estrus werere-inseminated at observed spontaneousestrus without any additional hormonaltreatment. Statistical analyses wereperformed using MINITAB release 13.2statistical software. Numerical datadifferences were consideredsignificant at P<0.05 using Chi-square(χ2) test. The conception ratefollowing FTAI in group-I and group-IIwas 20.83% (n=5/24) and 53.50%(n=51/96; p<0.05), respectively. Also,the conception rate following re-insemination in group-I and group-IIwas 25.00% (n=1/4) and 61.65%(n=16/26), respectively. Overall (1st AIand reinsemination) conception rate ingroup-I and group-II was 25.00%(n=6/24) and 69.80% (n=67/96; p<0.05),respectively. In summary, althoughestradiol/ progesterone-based FTAIprotocol is costlier compared toovsynch, the fomer is economical dueto much better conception rate inbuffalo during non-breeding season.



Key words: Buffalo, Conception,Estradiol, FTAI, Season

O-02 Effect of Butylated Hydroxytoluene (BHT)Supplementation on Semen Quality atDifferentStages of Cryopreservation of Beetal Buck SpermA. Rehman1*, A. Sattar1, A. Riaz1, M. Ahmad1, M.Ijaz2, M. Shahzad1, M. Ahmed1 and M. U. Mehmood1

1Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan, 2Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan *[email protected]

Butylated hydroxytoluene (BHT) is asynthetic analogue of vitamin E andplays an important role as anantioxidant. The objectives of thisstudy were two fold 1) to optimize theconcentration of BHT in semen extenderand 2) its effect during differentstages of cryopreservation of Beetalbuck sperm. A total of fourteenejaculates were collected from twoBeetal Bucks during seven successiveweeks from January to March.After each collection the ejaculatesfrom both bucks with motility ≥ 60 %,concentration > 2×109sperm/mL andabnormality rate <10% were pooled anddivided into five aliquots. Thealiquots were extended (50×106/0.5mlstraw) at 37°C with Tris-citric acidfructose (TCF) having15 % egg yolk and 5% glycerol,additionally supplemented withdifferent concentrations of BHT (0,0.5, 1.0, 1.5 or 2.0 mM). The extended

samples were cooled to 4°C for 90 min,equilibrated at 4°C for 4 hrs, frozenin liquid nitrogen (LN2) vapor (4 cmabove the LN2, for 7 min) and plungedinto LN2 for storage. Sperm evaluationassays, e.g., motility, viability,plasma membrane integrity (HOST),normal apical ridge (NAR) and DNAintegrity (acridine orange) wereevaluated at different stages ofprocessing (e.g. after dilution,before freezing and post thawing)using phase contrast and fluorescentmicroscopes. Data were analyzedthrough ANOVA under CRD andsignificant differences were comparedby using Duncan’s Multiple Range Test.Theaddition of BHT has no significanteffect after dilution and beforefreezing as all sperm evaluationassays were same (P>0.05) amonggroups. However, BHT, at an inclusionlevel of 1.0mM improved significantly(P<0.05) post thaw motility (52.14 ±1.01), live % of spermatozoa (61.29 ±2.31), PMI (58.71 ± 2.37) and NAR(60.71 ± 1.01) as compared to controland other treatments groups. Post thawDNA integrity of group 1mM and 0.5mMwere same but significantly (P<0.05)higher than control and othertreatments groups. It is concludedthat optimum concentration of BHT inTCF semen extender is 1.0 mM and itonly improves post thaw sperm qualityin Beetal Buck.

Key words: BHT, Beetal Buck,Cryopreservation

O-03 Repeat Breeding Problems in Dairy Cows of BangladeshM. J. D. Sarder1*, M. M. Rahman2and M. H. Islam1



1Department of Animal Husbandry and Veterinary Science,University of Rajshahi, Rajshahi-6205, Bangladesh,2Institute of Biological Sciences, University of Rajshahi,Rajshahi-6205, Bangladesh*[email protected]

Repeat breeding (RB) is a largeproblem in the dairy cattle leading togreat economic loss for the dairyproducer due to more inseminations,increased calving interval and highculling rates. The objective of thepresent study was to evaluate theprevalence of RB in cows and toidentify organisms in the uterinesamples along with their antibioticsensitivity tests.. The data collectedby using questionnaire and directinterview of the farmer. A total 1207dairy cows of Rajshahi, Bangladeshwere studied during the period fromAugust, 2011-January, 2013. Theoverall prevalence of RB was 20.4%.Genotypes of cow had significanteffect (P<0.05) on prevalence of RB.Age also had significant effect(P<0.05) on RB. The RB of cows werelowest in L×SL (3.0%), <4 years age(3.3%) and good body condition score(2.7%) groups. The RB of cows werefound highest in L×F (8.5%), 6 to <8years age (8.7%), 3rd parity (6.4%) andArtificial Insemination method(15.4%). A total 100 cows have beenselected for uterine sample fromrepeat breeder cases at Rajshahidistrict. A significant number ofbacteria were identified from diseasessample and they were Fusobacterium spp14 (45.16%, p=0.000), Actinomyces spp10 (32.25%, p=0.003), Escherichia coli12 (38.70%), Pseudomonas spp 11(35.48%) and Haemophilus spp 9 (29.0%,p=0.012). Amongst antibiotics,

Azithromycin and Ciprofloxacin (99.38%and 93.20%, respectively) were highlysensitive. In contrast, Penicillin(51.85%) and Oxytetracycline (25.2%)were highly resistance. The presentstudy suggested that genotype of L×SL,<4 year age, good body conditionsupport significantly reduces theprevalence of RB in dairy cows. Also alot of microorganisms identified whichhave very harmful effect on fertilitystatus of dairy cows in this studyarea. Moreover, Azithromycin andCiprofloxacin are very effective drugsagainst the organisms of Repeatbreeder cows.

Key words: Repeat breeder, Cows,Antibiotics sensitivity test,Causal agents

O-04 Effect of Different Dietary Energy Levels on Dry Matter Intake,Milk Production, Milk Composition and Reproductive Hormonal Level of Early Lactating Dairy CattleS. Sultan

Department of Animal Nutrition, Faculty of AnimalHusbandry and Veterinary Sciences, University of Agriculture Peshawar KPK, Pakistan [email protected]

The aim of the study was to evaluatethe effect of three different dietaryenergy levels on dry matter intake,Milk production, composition andreproductive hormonal level of earlylactating Holstein Friesian cows. Ninemultiparous early lactating (10±5 daysin milk) cows were selected on thebasis of nearly the same body weight(410±10) and milk production (12±01).These cows were randomly divided intothree groups with three animals ineach group under a completely



randomized design. Diets formulatedwere iso-nitrogenous with 16% CPcontents and varying dietary MElevels: Group A meeting 100% NRCrecommendations, Group B with 88% ofNRC recommendations and group Ccontaining 112% of the NRCrecommendation for early lactatingcows. The research trial was continuedfor a period of 75 days includedadaptation period and data wereanalysed by using the GLM procedure(Proc GLM; SAS®, Version 8.02; SASInstitute Inc., Cary, NC) as describedby steel and torrie (1981). Resultsfor dry matter intake shows high value(P<0.05) in or group C animals(13.78±0.17 kg/day) in high energy112% treatment, followed by group A(13.14±0.12 kg/day) and less DMI wasfor B (12.68±0.11kg/day) animals. Ahigher (P <0.05) milk yield(14.80±0.10 lit/day) was reported inthe tretment C compared to the groupdA and B (13.01±0.10 kg/day). For milkcomposition 100 ml fresh sample ofeach cow was collected daily andanalyzed by automatic milk analyzer(lacto scan) In milk composition high(P <0.05) milk fats (3.91±0.02)percentage was found at low energydiet B, followed by A (3.75±01) and C(3.58±0.03), whereas no statisticaldifference recorded between treatmentsfor Milk protein, lactose and solidnot fat. For blood serum hormonaldetermination 5 ml blood from all cowswere collected after 15 days each andthen analyzed. Serum progesteronehormones were significantly (P<0.05)increased with increasing dietaryenergy High progesterone level ofdairy cows were observed in group C(0.73 ng/ml) followed by group A and Bhaving 0.21 and 0.16ng/ml and overallmean of serum luteinizing profile was

significantly higher (0.75ng/ml) ingroup C compared to group A and B is0.17 and 0.16 ng/ml respectively withfeeding of high energy ME (112%) diet.It is therefore concluded that feedingrations hihg in ME contents at 112 %of NRC recommendations improved DMIMilk yield and improved reproductiveefficiency in term of hormonal level.

Key words: Dietary energy, DMI, Milkproduction, Milk composition,Reproductive Hormones

O-05 Investigations on Fertility RelatedBiomarkers in Water Buffalo Bull Semen toReduce Male-Factor Losses during Peak Breeding SeasonH. Ahmed1, 2, S. M. H. Andrabi1*, and S. Jahan2

1Animal Reproduction Laboratory, Animal SciencesInstitute, National Agricultural Research Centre,ParkRoad, Islamabad, Pakistan, 2Reproductive PhysiologyLaboratory, Department of Animal Sciences, Quaid-i-Azam University,Islamabad, Pakistan *[email protected]

A high fertility bull is aprerequisite for running anyreproductive or breed improvementprogramme. With the wide spreadapplication of artificial inseminationin water buffalo, the demand for bestmales have increased considerably.Therefore, the present study wasdesigned to assess the fertilityrelated biomarkers in water buffalobull semen to reduce male-factorlosses. Semen was collected from fiveadult buffalo bulls of acceptable



fertility with artificial vaginaduring peak breeding season. Afterinitial evaluation, semen samples ofeach bull were split and dilutedeither in PBS-0.1% BSA for freshanalysis or in Tris-citric acidextender for cryopreservation.Freshand frozen-thawed semen samples wereanalyzed by (i) Computer Aided SpermAnalyzer (CASA) for sperm motilityparameters, velocity distribution andkinematics, (ii) Acridine Orange Assayfor sperm DNA integrity, (iii) NeutralComet Assay for sperm DNAfragmentation and (iv) FluorescentProbes, propidium iodide and"5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolylcarbocyanine iodide" (JC-1) for spermviability and mitochondrialtransmembrane potential, respectively.The experiment was repeated six times.For in vivo fertility, data of at least100 inseminations per bull werecollected under controlled fieldconditions. Straight line velocity(VSL, 123.67±3.20 µm/s) of buffalospermatozoa in fresh semen samples hada significant correlation with in vivofertility (62.84 %; r=0.76, P<0.05),whereas tail length (TL, 68.55±3.11µm) and olive moment (OM, 12.82±1.08µm) of sperm DNA in fresh semensamples had a significant negativecorrelation with fertility (r=-0.49,P<0.05 and r=-0.72, P<0.01,respectively). Sperm kinematics i.e.,average path velocity (VAP, 71.97±1.38µm/s), VSL (61.37±1.37 µm/s),straightness (STR, 84.37±0.80 %) andlinearity (LIN, 56.45±1.27 %) offrozen-thawed semen samples weresignificantly correlated with in vivofertility (r=0.52, P<0.05; r=0.67,P<0.01; r=0.67, P<0.01 and r=0.58,P<0.02, respectively). Similarly, % of

viable spermatozoa with highmitochondrial transmembrane potential(V/HP, 29.34±1.24 %) in frozen-thawedsemen samples were significantlycorrelated with in vivo fertility(r=0.89, P<0.01). Neutral comet assayparameters i.e., TL and OM in frozen-thawed semen samples had a significantnegative correlation with fertility(TL, 78.44±0.98 µm, r=-0.63, P<0.01and OM 17.27±0.56 µm, r=-0.50;P<0.04). The best predictive equation(R2 adjusted=65.0%, P<0.01) offertility obtained for fresh buffalosemen after step forward multipleregression analysis included twoparameters i.e., TL and OM. The bestpredictive equation (R2 adjusted=83.9%,P<0.001) of fertility for frozen-thawed buffalo semen included threeparameters i.e., rapid subpopulation,VAP and VSL. The best singlepredicator of fertility for fresh andfrozenthawed buffalo semen was OM (R2

adjusted=43.7%, P<0.04) and CASAprogressive motility (R2 adjusted =43.1%, P<0.04), respectively. Thepresent study identified the potentialsperm quality parameters that couldserve as biomarkers of fertility inwater buffalo semen during peakbreeding season. Moreover, theprognostic values of buffalo spermquality parameters as predictors of invivo fertility were better forfrozenthawed semen compared to freshsemen.

Key words: Buffalo spermatozoa,Viability, Mitochondrialtransmembrane potential, Invivo fertility,CASA



O-06 Effects of Lycopene on Ram Sperm Parameters during Liquid StorageM. N. Bucak1, Ş. Güngör1*,N. Başpinar2, K. Çoyan3 and Ş. Dursun4

1Department of Reproduction and ArtificialInsemination, Faculty of Veterinary Medicine, SelcukUniversity, Konya, Turkey, 2 Department of Biochemistry,Faculty of Veterinary Medicine, Selcuk University, Konya, Turkey, 3 Department of Hıstology andEmbryology, Faculty of Medicine, Pamukkale University,Denizli, Turkey, 4 Rebuplic of Turkey Mınıstry of Food,Agrıculture and Lıvestock Bahri Dağdas InternationalAgrıcultural Research Instıtute, Konya, Turkey*[email protected]

Lycopene addition of extender would beuseful for liquid storage of ramsemen. The aim of this study was toinvestigate the effects of threelevels lycopene on ram spermparameters during liquid storage at5°C up to 72 h.Five Merino rams (2-3 years of age)were housed at Selcuk UniversityFaculty of Veterinary MedicineResearch and Application Farm, Konya,and maintained under uniform feedingand housing conditions. A total numberof 30 ejaculates (10 ejaculates foreach ram) were collected from the ramswith the aid of an artificial vaginatwice a week, according to AI standardprocedures. The ejaculates containingspermatozoa with >80% forwardprogressive motility andconcentrations higher than 2x109

spermatozoa/ml were used in the study.Nine ejaculates for each ram wereincluded in the study. Ejaculates werepooled at 37°C and pooled ejaculate

was divided into four equal aliquotsand diluted (37°C) with the Tris baseextender, containing lycopene at threedoses (0.5, 1.0 and 2.0 mM) and noadditives (control), at a finalconcentration of approximately 2 x 108

sperm. Subjective sperm motility wasdetermined at phasecontrastmicroscope, viability (SYBR14/PI),andmitochondrial activity (JC1-PI)were determined at fluorescentmicroscopy after 0, 24, 48 and 72 h ofstorage at 5°C.The study wasreplicated nine times. Results wereexpressed as the mean ± SEM. Data forsperm motility, viability andmitochondrial activity were analyzedby analysis of variance, followed byTukey’s post-hoc test to determinesignificant differences betweengroups. Differences with values ofP<0.05 were considered to bestatistically significant. Statisticalanalyses were performed by using SPSS11.5 package program. The extendersupplemented with 0.5 mM lycopene(51.97±0.53) resulted in highermitochondrial activity, in comparisonto the control (47.17±1.85) group at72 h of storage. Lycopene at 0.5 mM(62.50±1.11) led to higher spermmotility when compared to 2 mMlycopene (49.16±4.72) group at 72 h ofliquid storage (P<0.05). Spermviability was better in 2mM lycopene(78±6.79) than the control, 0.5mM and1mM lycopene groups (74.02±3.43,76.96±1.04, 72.75±0.66)respectively,with no significant differences(P>0.05) at 24 h storage. Our datasuggest that the lycopene can be addedto Tris base extender for improvingthe ram sperm motility, viability andmitochondrial activity during theliquid storage.



Key words: Ram sperm, Lycopene, Liquidstorage,

Fluorescent staining.O-07 Effect of Timing of AI on Pregnancy inEstrus Synchronized Nili-Ravi Buffaloes: A Preliminary StudyS. Abbas, A. Husnain, M. Hassan andN. Ahmad*

Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, 54000 Pakistan *[email protected]

The use of Controlled Internal DrugReleasing (CIDR) device has beenreported to synchronize and induceestrus in cows and buffaloes. Fixedtime artificial insemination (FTAI) isa value addition technique in CIDR-based synchronization programme andrequires an ovulation control. Limitedinformation is available on timing ofAI following CIDR-GnRH synchronizationprotocol to achieve optimal fertility.In this background the objective ofpresent study was to determine theeffect of timing of AI on pregnancyrate in Nili-Ravi Buffalo synchronizedwith CIDR-GnRH protocol. The study wasconducted during breeding season at acommercial dairy farm. Thirteen adultNili-Ravi buffaloes having 400-450 Kgbody weight, BCS (2.64±0.06), parityranges from 2 to 4 and more than 90days postpartum were used in thisstudy. Each buffalo was fitted withCIDR (1.38 g progesterone Pfizer Co,USA) on random stage of estrus cycle..On day 6 after CIDR insertion,luteolytic agent PGF2α (2 mL; i.m) wasinjected to all animals. CIDRdevice was removed on day 7 and GnRH(2 mL; i.m) was administered on day 8.Estrus detection was done throughteasing and confirmed by visual

observation. Buffaloes were thenrandomly allocated into three groupsto get AI dose either at 12 h (n=4),24 h (n=5), or 36 h (n=4) after theadministration of GnRH. Follicularsize of the largest follicle wasmeasured from PG injection to 48 hafter administration of GnRH (day 10)through ultrasonography at 12 hinterval. Pregnancy diagnosis wasconducted using ultrasonography on 35th

days after insemination. Data fortiming of ovulation and size of theovulatory follicle was analyzed byusing One way ANOVA and for estrusresponse ovulation and pregnancy perAI was analyzed by using Z-test forpopulation proportion. Resultsrevealed that estrus response was 100%(13/13). Overall mean size of dominantfollicle just before ovulation, timingof ovulation after GnRH injection andovulation rate were 13.2±0.38,32.77±1.46 h mm and 84%respectively. . Pregnancy per AI waslower (25%, 1/4) when inseminatedearlier (12 h), maximal (50%, 2/4)when bred at 24 h and zero (0%, 0/3)when inseminated later (36 h). It isconcluded that buffaloes may beinseminated at 24 h GnRHadministration following CIDR-GnRHsynchronization protocol for achievinghigher pregnancy.

Key words: Synchronization, CIDR,Buffalo, GnRHO-08 Prevalence and Therapeutics ofIodineDeficiency as Abortifacientin Pregnant GoatsW. Yaqub1,*, M. S. Khan1,A. Aslam2, M. Ijaz1,M. Avais1, J. A. Khan1and M. Z. Shabbir3

1Department of Clinical MedicineandSurgery,



Lahore.2Department of Pathology,3Quality Operations Lab., University of Veterinary and Animal Sciences, Lahore.*[email protected]

Iodine deficiency is one of the majorissues inpregnant goats in Lahore,Pakistan. Low levels ofiodine resultin decreased plasmaTriiodothyronine(T3) and Thyroxin(T4)hormones leading to highrisk ofabortions, still births, andcongenital anomalies in pregnantgoats. The present study was carriedout to 1) determine the prevalence ofiodine deficiency in pregnant goats,and 2) evaluate the effectiveness ofThyroxin and minerals mixture intreating iodine deficient pregnantgoats. Plasma samples from 300pregnantgoatswere collected from Lahoremetropolitan and subsequently testedthrough ELISA for T3 and T4hormones.Overall 84(28%) of the pregnant goatsrevealed significantly low (P<0.05)levels of plasma T3 and T4 hormones. Outof these 84 pregnant goats,18(21.4%)aborted.For therapeutic trials, 24pregnant goats, having low levels of T3

and T4 hormones, were randomly dividedinto 3 groups of 8 viz. A, B and C.Goats in group A were treated orallywith Thyroxin at 0.2mg/20kg/day whileanimals in group B were given mineralmixture at 50gm/animal/day orally. Themembers in groupCserved as positivecontrol (untreated). The plasma levelsof T3 and T4 were determined at day 0(pretreatment) and day 15 (posttreatment) using ELISA.Data onprevalence was analyzed using PearsonChi Square Test whereas T3 and T4values in different treatment groupswere analyzed by one way ANOVA.On day0 (4.5months pregnancy), plasma T3

levels of goats in groups A, B and C

were 51.71±12.18, 59.93±14.44and53.1±13.58ng/dL, respectively.Plasma T4 levels at day 0 were1.79±0.46, 1.94±0.77and 1.70±0.69ug/dL in groups A, B and C,respectively. On day 15, plasma T3

levels of goats in groups A and B(98.64±5.70and 139.79±26.64ng/dL,respectively)were significantlyincreased (P<0.05) compared to groupC. Similarly, plasma T4levels of goatsin groups A and B (4.54± 0.43and6.13±1.19ug/dL, respectively) weresignificantly increased(P<0.05)compared to group C. When compared T3

and T4 levels of goats in groups A andB, a significant increase (P<0.05) wasobserved in group B. At kidding, allthe goats in groups A and B gave birthto normal kids without goiter orcongenital anomaly/deformity. On theother hand, 3 of the goats in group Caborted during trial period and 5 ofthe goats gave birth to kids sufferingfrom severe goiter. In conclusion,iodine deficiency is prevalent inpregnant goats in Lahore and isassociated with abortions and goiter.For treatment, mineral mixture is moreeffective than thyroxin in treatingiodine deficiency in pregnant goats.

Key words: Goiter, Thyroxin, MineralMixture, ELISA

O-09 Efficiency of Fatty Acid-Free DefinedSystem for In Vitro Maturation of Buffalo OocytesA. Azam1*, A. Husna1, Q. Shahzad2,T. Akhtar3, E. Haq2, N. Ullah4and S. Akhter1

1Department of Zoology, PMAS-Arid Agriculture University,



Rawalpindi-46300 Pakistan, 2Buffalo Research InstitutePattoki, Lahore-54000 Pakistan, 3Livestock TrainingInstitute, Bahadurnagar, Okara-56300 Pakistan, 4Punjab livestock and dairy development board LES khizarabad, Sargodha-40100 Pakistan *[email protected]

The evaluation of medium for in vitromaturation of buffalo oocytes hasprogressed towards more definedconditions instead of use of complexmedium with serum, for the studiesevaluating effect of specificcomponent in the medium. Therequirement of a fatty-acid freedefined medium is intensified if theeffect of specific fatty-acidcomponent of medium is to beinvestigated. The objective was toevaluate the effect of fatty-acidfreebovine serum albumin (FAF-BSA)alone or supplemented with epidermalgrowth factor (EGF) in the in vitromaturation medium on in vitro maturationof oocytes and further development ofthe embryos. The cumulus oocytecomplexes (COCs) were collected fromslaughterhouse ovaries by aspiratingfollicles, and kept in maturationmedium for 24 h. Three types ofmaturation media were evaluated: TCM-199 supplemented with 0.02 IU/mL FSH,1 µg/mL estradiol-17β (E2), 50 µg/mLgentamicin and 1) 10% fetal bovineserum (FBS), 2) 6 mg/ml FAF- BSA or 3)6 mg/ml FAF- BSA + 10.0 ng/mLepidermal growth factor (EGF). Aftermaturation, Tyrode’s albumin lactatepyruvate (TALP) and Syntheticoviductal fluid (SOF) media were usedfor fertilization and further culturerespectively. Data was recorded inpercentages and analyzed by ANOVA at5% level of significance. LeastSignificance Difference (LSD) test was

used to compare treatment means.Thepercentage of COCs with expandedcumulus was higher (P < 0.05) in FBSand FAF-BSA+EGF supplementedmaturation medium compared with FAF-BSA alone. No difference (P>0.05) wasobserved in meiotic resumption[Germinalvesicle (GV), Germinal-vesicle breakdown (GVBD), Metaphase-I(MI)] and nuclear maturation(Metaphase-II (MII)] in all threetypes of maturation media. Thepercentage of embryos capable ofcrossing 8-16 cell block and reachingup to morula/blastocyst was higher (P< 0.05) in FBS and FAF-BSA+EGFsupplemented media compared with FAF-BSA alone. This study shows that lowerdevelopment rates of FAF-BSAsupplemented defined maturation mediumwere restored and enhanced by addingEGF and comparable with serum. Thismedium can be efficiently used instudies evaluating effect of specificfatty acid component in the medium.

Key words: In vitro maturation, Serum freemedium,

Defined medium, Epidermalgrowth factor

O-10 Effect of Skim Milk and Egg Yolk in TrisBased Extender on Liquid Storage ofBuck SemenA. Mushtaq, M. R. Ullah, N. Hussain, M.Ahmad, M.Z. Tahirand N. Ahmad*

Department of Theriogenology, University of Veterinary & Animal Sciences, Lahore 54000, Pakistan *[email protected].

Artificial insemination is a majortool for wide spread use of highgenetic potential of the male and use



of liquid semen is less laborious andeconomical way of semen extensioncompared to frozen semen. Theobjective of present study was tocompare the effect of Tris Citric acidEgg Yolk (TCEY: Tris 1.73gm,CitricAcid1.93gm, Egg Yolk 20ml) and SkimMilk Tris Citric Acid (SMTC: Skim Milk0.9gm, Tris 1.73gm, Citric Acid1.93gm) semen extenders on buck semenparameters (motility, live sperm &membrane Integrity) at 4 ˚C from 0 to96 hours of storage. Semen wascollected from two Beetal bucks oncein a week for four consecutive weeks(4 replicates) through artificialvagina. The ejaculates qualifying theminimum criterion (75% motility, 80%live sperm & 80% membrane integrity)were pooled and divided into twoaliquots i.e. one diluted with TCEYand second with SMTC. After extensionsemen samples were cooled to 4º C andthen incubated at 4º C for 96 hours.Motility, live ratio (Eosin andNigrosine staining), HOS (HyperOsmotic Swelling) & NAR (Normal ApicalRidge) were assessed at 12 hoursinterval from 0 to 96 hr ofincubation. Semen samples were pre-warmed at 37˚C before evaluation. Datawas analyzed by repeated measure ANOVAin SAS Enterprise Guide 4.2. Spermmotility and HOS were higher (p˂0.05)from 12 to 96 hours in SMTC comparedto TCEY (17.5% ±3.22 vs. 3.75%±2.39 ;33.75% ±0.75 vs. 25.5% ±1.94respectively at 96 hours).Moreover,live sperm and NAR were higher(p˂0.05) from 0 to 24 hours in SMTC(72% ± 2.38 vs. 60.75% ±2.95; 70.25±1.93 vs. 55% ±1.87 respectively at 24hours) than TCEY after 24 hours till96 hours their difference was same. Itis concluded that skim milk basedextender can maintain higher sperm

quality as compare to semen stored inegg yolk based extender during liquidstorage at 4ºC for 72 hours forfruitful AI & maximum for 96 hours.

Key words: Buck semen, Tris Citrate,Skim Milk extender, liquidstorage.



POSTER PRESENTATION

P-01 The Influence of Season on Surface Follicle Count and Oocyte Quality in Buffalo OvariesK. K. Ahirwar1, V. K. Gandotra1*, N. Singh2, D.Pathak3, O. Singh3, S. S. Ghuman1 andP. S. Brar1

1Department of Veterinary Gynaecology and Obstetrics,2Department of Animal Genetics andBreeding,3Department of Veterinary Anatomy, GuruAngad Dev Veterinary and Animal Sciences University,Ludhiana-141004, India*[email protected]

The influence of season wasinvestigated on surface ovarianfollicle and oocyte quality inbuffalo. From an abattoir, buffaloovaries were collected (n=142) duringsummer (July-August, n=66) and winter(DecemberJanuary, n=76) season.Ovaries were examined for the surfacefollicles and oocyte quality. Thesurface follicles of each ovary werecounted and measured using vernier’scallipers. These were classified assmall (<5 mm), medium (5-8 mm) andlarge (>8 mm) follicles. The mediumand large surface follicles wereaspirated using 18G needle attachedwith a 10 ml disposable syringecontaining 0.5 ml cumulus-oocytecomplex (COC) holding media (dPBS + 3-5% calf serum) at 37˚C. UsingSPSS-17, data was subjected to t-test.During winter season, average folliclecount per ovary was higher than thatin summer (7.61±0.37 vs 5.92±0.25;p<0.05). Between seasons, thepercentage of small, medium and large

size follicles was different (summer:77.7, 15.3 and 6.9% vs winter: 73.4,16.1 and 10.5%, respectively; p<0.05).During winter season as compared tosummer, average oocyte recovery rate(2.07±0.12 vs 1.72±0.12; p<0.05) andthe percentage of grade-A oocytes washigh (43.7 vs 22.8%; p<0.05). Duringsummer season, the recovery of grade-Boocytes was higher than that in winter(35.9 vs 20.9%; p<0.05). Themicroscopic examination of ovaries (10each in summer and winter) revealedtertiary follicle with intactgranulosa cell layers (9.05±0.32;layer thickness, 181.52±9.27µm) inwinter and loosened granulosa celllayers (7.45±0.23; layer thickness,209.69±6.90µm) in summer.Vacuolization was observed between thegranulosa cell layers and antralfluid, as well as in between thegranulosa cells in summer season andthe vacuolization was absent in winterseason. Also, many apoptotic bodies inovarian granulosa cell layer were onlyobserved in summer season. In summary,season has a significant influence onthe number of follicles on ovariansurface, oocyte recovery rate andoocyte quality in buffalo.

Key words: Buffalo, Follicle, Oocyte Quality, Ovary,

SeasonP-02 Cysteine Supplementation Enhanced Post-Thaw Quality of Spermatozoa in Achai and Holstein Fresian BullsZakirullah1, H. Khan1*, M. S. Qureshi1, M. Younas1, A. Gohar2, R. U. Khan1, S. S. Afridi1, H.Rehman1andU. Saddique1, I. M. Khan, A. Khan 1



1Faculty of Animal Husbandry and Veterinary Sciences,The University of Agriculture, Peshawar, Pakistan,2Semen Processing Unit,Government Cattle Breeding andDairy Farm Harichand, Charsadda, Pakistan *[email protected]

Production of Reactive oxygen species(ROS) during the process ofcryopreservation is associated withlipid peroxidation of the spermmembrane that adversely affects spermmotility and fertility. The objectiveof the current study was to establishthe optimal concentration of L-Cysteine for cryopreservation of Achaiand Holstein Fresian bull semen underthe subtropical condition of Peshawarregion. Experiments were executed onsemen of six matured bulls, threeAchai- the indigenous breed and threeHolstein Fresian- exotic breed atGovernment Cattle Breeding and DairyFarm Harichand Charsadda. Semen fromthe experimental bulls of either breedwere collected with the help ofartificial vagina maintained at 42°Cand processed separately breed wise.Two consecutive ejaculates werecollected from each bull at weeklyinterval for 3 weeks in April- May2014. Immediately after collection,the ejaculates were subjected to grossand microscopic examination. Semensamples having more than 70% motilitywere selected for further processing.The semen was split into four aliquotsin which tris-citric acid extender(TCA) supplemented with variousconcentrations of L-Cysteine was usedto reach 0 mMolml−1 (group I, control),0.5 mMolml−1(group II), 1.0mMolml−1

(group III) and 1.5mMolml−1 (group IV).Diluted semen was cooled to 4°C for 2h, equilibrated for 4 h at 4°C, filled

in straws(0.5ml) at 4°C, kept inliquid nitrogen vapours for 10 min andthen stored in the liquid nitrogen(-196 oC) for assessment. Post-thawedmotility of frozen semen straws wasassessed using standard procedure.Thawing of frozen semen straws wascarried out at 37°C for 30 seconds ina hot-water bath. Sperm viability andacrosomal integrity were determined bydual staining procedure i.e Trypan-blue and giemsa stains. The supravitalstain trypan-blue distinguished liveand dead spermatozoa whereas giemsastain was used to evaluate theintegrity of the acrosomal membrane.Similarly plasma membrane integritywas assessed using the hypoosmoticswelling (HOS) test. The data wasstatistically analyzed with two-wayanalysis of variance using SPSSversion 16, Chicago, IL, USA. Thegroup differences were compared by theDuncan's Multiple Range Test.During current study, the progressivemotility and viability of spermatozoawas significantly higher in 1.5mM or1.0mM L-Cysteine supplemented groupsin comparison with 0.5mm or 0.00 mM.Also current finding indicated thatthe number of HOST+ve spermatozoa at1.5 or 1.0 mM was greater incomparison with lower concentration of0.5 or 0.00mM L.Cysteine. Furthermore,significant difference for acrosomalintegrity was recorded in semensamples treated with 1.5 mM L-Cysteinefollowed by 1mM, 0.5mM and control.The current study demonstrated that1.0 or 1.5mMolml−1 was the mostfavorable concentration of L-Cysteineto be added to the TCA extender forimproving the quality of frozen–thawedAchai and Holstein Fresian bull semen.



Key words: L.Cystein, Achai, HolestainFrerian, Bull, semen, postthaw

P-03 Effects of Cysteamine on Ram Sperm Parameters during Liquid StorageM. N. Bucak1*, Ş. Güngör1, N. Başpinar2,K.Çoyan3andŞ. Dursun4

1Department of Reproduction and ArtificialInsemination,Faculty of Veterinary Medicine, Selcuk University,Konya, Turkey, 2Department of Biochemistry, Facultyof Veterinary Medicine, Selcuk University,Konya, Turkey, 3 Department of Hıstology and Embryology, Faculty ofMedicine, Pamukkale University, Denizli, Turkey, 4

Ministry of Food, Agriculture and Livestock, BahriDağdas International Agricultural Research Institute,Konya, Turkey*[email protected]

The aim of this study was toinvestigate the effects three levelsof cysteamine, on ram sperm parametersduring liquid storage up to 72 h at 5°C. Five Merino rams (2-3 years ofage) were housed at Selcuk UniversityFaculty of Veterinary MedicineResearch and Application Farm, Konya,and maintained under uniform feedingand housing conditions. A total numberof 30 ejaculates (10 ejaculates foreach ram) were collected from the ramswith the aid of an artificial vaginatwice a week, according to AI standardprocedures. The ejaculates containingspermatozoa with >80% forwardprogressive motility andconcentrations higher than 2x109

spermatozoa/ml were used in the study.Eight ejaculates for each ram wereincluded in the study. Ejaculates were

pooled at 37°C and pooled ejaculatewas divided into four equal aliquotsand diluted (37°C) with the Tris baseextender, containing cysteamine atthree doses (0.5, 1.0 and 2.0 mM) andno additives (control), at a finalconcentration of approximately 2 x 108

sperm. Subjective sperm motility wasdetermined at phase-contrastmicroscope, viability (SYBR14/PI),andmitochondrial activity (JC1-PI)were determined at fluorescentmicroscopy after 0, 24, 48 and 72 h ofstorage at 5°C.The study wasreplicated eight times. Results wereexpressed as the mean±SEM. Data forsperm motility, viability andmitochondrial activity were analyzedby analysis of variance, followed byTukey’s post-hoc test to determinesignificant differences betweengroups. Differences with values ofP<0.05 were considered to bestatistically significant. Statisticalanalyses were performed by using SPSS11.5 package program. The extendersupplemented with 1mM cysteamine(78.33±2.10) resulted in highermotility, in comparison to the controlgroup (70.83±1.53) for 48 h of storage(P<0.05). Sperm viability was betterin 0.5 mM cysteamine (76.69±4.04)thancontrol (69.08±3.88). Formitochondrial activity, 1 mMcysteamine gave the higher rate(73.51±4.39), compared to control(69.47±3.68), with no significantdifferences (P>0.05) up to 72 h ofstorage. This study highlights theprotective effect of cysteamine (0.5and 1 mM) on sperm quality during theliquid storage of ram semen.

Key words: Ram sperm, Cysteamine,Liquid storage.

Fluorescent staining



P-04 Response of Cryopreserved NiliRaviBuffalo Bull Semen to Gallic Acid Addition in Semen ExtenderM. Tariq1, I. Rabbani2*, M. S. Yousaf2, H. Zaneb3andS. Iqbal4

1Gomal College of Veterinary Scieces, Gomal University Dera Ismail Khan, 2Department of Physiology, University of Veterinary and Animal Sciences Lahore, 3Department of Anatomy and Histology, University of Veterinary and Animal Sciences Lahore, 4Semen Production Unit, Qadir Abad, Sahiwal.*[email protected]

In buffaloes, the ratio of fertilitywith cryopreserved semen as comparedto cattle is lower as buffaloes aremore prone to lipid peroxidation.Gallic acid (GA), a polyphenol, isknown to carry excellent antioxidantproperties. The objectives of thisstudy were to improve buffalo bullsemen quality with the use of GA as anantioxidant during the process ofcryopreservation and to determine theoptimal concentration of GA as anantioxidant for buffalo bull semen.For this study, four (4) healthyfertile Nili-Ravi buffalo bullsbetween 5-10 years of age, withejaculates having 5-8 ml volume and>80% progressive motility wereselected. GA with concentrations, 0,15, 30, 45, 60 and 100µM was added toa 25 ml extended semen respectivelyand incubated prior tocryopreservation at 37°C for 5 min toallow GA to be absorbed inspermatozoa. The antioxidative effectsof GA were evaluated indirectly interms of spermatozoa motility,viability (Eosin-negrosin stainmethod), spermatozoa plasma membrane

and acrosomal integrity (Hypo-osmoticswelling test and Apical Ridge methodrespectively) and DNA status throughAcridine orange staining assay. Thedata were analyzed using one wayANOVA, followed by Duncan multiplerange test, p < 0.05 was consideredsignificant. Results of this studyshowed that addition of GA to semenextender did not improve the buffalobull spermatozoa quality parameterssignificantly as compared to thecontrol group. However, it wasobserved that addition of GA at aconcentration of 15 µM, improved themotility, viability and plasmamembrane integrity (p < 0.05) ofspermatozoa. Whereas, increasingconcentration of GA from 30µM to100µM, showed toxic effects. Inconclusion, GA did not produce anysignificant protection against cryo-injury in buffalo bull semen.

Key words: Cryopreservation, GallicAcid, Reactive oxygen species

P-05 Reducing Glycerol with TrehaloseSupplementation Increases Survival of Bull SpermatozoaA. A. Channa1*, Z. Ashraf1, N. Ahmad1, M. Ahmad1,M. Ahmed1 and A. Z. Durani2

1 Department of Theriogenology, 2Department of ClinicalMedicine and Surgery, University of Veterinary and AnimalSciences, Lahore-Pakistan*[email protected]

Glycerol is commonly usedcryoprotectant for the preservation ofspermatozoa. Apart from cryoprotectivequality of glycerol, toxic effects on



spermatozoa have been also reported.The addition of non-penetratingcryoprotactants like trehalose isreported to enhance the post-thawsemen quality in bovine, caprine,canine and Equine. Therefore, thepresent study was designed to minimizesperm damage by adding differentconcentrations of trehalose (0, 50,100, 150, 200mM) in Tris-based eggyolk extender containing 3.5% either7% glycerol. For this, semen wascollected from three Sahiwal bulls (4-8 years of age) and was extended withdifferent concentrations of glyceroland trehalose and cooled at 4°C for 2h. The semen was filled in 0.5 mlstraw ant kept on liquid nitrogenvapors for 7 minutes and finallystored in liquid nitrogen. The postthaw sperm motility, viability (eosin-nigrosin), membrane integrity (HOSsolution), acrosome integrity(formaldehyde citrate) and DNAintegrity (acridine orange) werecarried out and statistically analyzedby two-way ANOVA. Reducing glycerolconcentration from 7 to 3.5% (0mMtrehalose; Control) declined overallpost-thaw semen quality. Thesupplementation of 150mM trehalose(3.5% glycerol) and 100mM trehalose(7% glycerol) both groups were equalbut gave significantly (P < 0.05)better post-thaw (plasma membrane,acrosome and DNA integrity) spermsurvival as compared to Contro group.The post thaw semen parameters in100mM trehalose (7% glycerol) weresignificantly (P<0.05) better as:motility (57.00 ± 1.22% vs. 42.00 ±1.22%), viability (67.60 ± 0.93% vs.51.00 ± 1.30%), plasma membraneintegrity (59.00 ± 0.89% vs. 41.40 ±1.17%), acrosome integrity (55.00 ±0.71% vs. 40.00 ± 0.71%) and DNA

integrity (97.35 ± 0.25% vs. 96.51 ±0.37%) in comparison with Control.Significantly (P<0.05) high DNAintegrity was observed in all thetreatment groups as compare toControl. Intact acrosome 45.20 ±0.86%, was observed higher in (3.5 %Glycerol; 0mM trehalose) as compare to40.00 ± 0.71% in (7 % glycerol; 0mMtrehalose), is the indication thatglycerol may decrease the acrosomeintegrity. It is concluded thatreducing 7% glycerol concentration to3.5% supplemented with 150mM trehaloseyielde better post-thaw parameters infrozen-thawed semen of cattle.

Key words: Trehalose,Cryopreservation, Spermatozoa,

Sahiwal bull

P-06 Effect of Feeding TMR on Reproductive Performance of Non Descript Dairy CattleM. I. Khan1*, M. A. Mirza1 andB. Shahid2

1Department of Animal Husbandry, Livestock DevelopmentResearch Centre (LDRC) Muzaffarabad, 13100, Pakistan, 2

Department of Zoology, University of Azad Jammu andKashmir, Muzaffarabad, 13100, Pakistan *[email protected]

The low reproductive performance ofnondescript dairy cattle in Azad Jammuand Kashmir is because of poor geneticmakeup and poor nutrition. Therefore,the present study was conducted todetermine the effect of TMR feeding onfertility of dairy cows maintained atLivestock Development Research CentreMuzaffarabd. The seventeen multiparouscows (2-5 parity), with body conditionscore of (3.5 - 4) having the same



reproductive status were divided intotwo groups. The control group (9 cows)was fed with the conventional rationconsisting of wheat straw, cotton seedcake, wheat bran, and molasses. A nonlactating cow weighing 250 kg bodyweight was fed with an allowance ofration to provide the quantity of thenutrients at a rate of Dry Matter 5.9Kg, TDN 3.5 Kg, CP 0.62 Kg, Ca 0.02kgand P 0.01kg. The treatment group (8cows) was fed only the TMR at the rate2 % of the body weight of animal. TMRwas containing crude protein 11.5 %,Fat 5%, TDN 58%, Crude Fiber 15.7% andAsh 10 %. The experiment was conductedfrom January 2014- July 2014 duringthe low breeding season. Fertilityrate in term of number of services perconception was studied in both thegroups. In control group 55.5 % cowsshowed the cyclicity where as 44.4%did not showed estrus or heat andremained silent during theexperimental period. Similarly numbersof services per conception were 3.5 incontrol groups. In treatment group62.5 % cows showed the estrus where as37.5 % remained silent. In treatmentgroup the total numbers of servicesper conception were 1.5. The data wasanalyzed by chi square test thatreveled that TMR feeding significantly(P < 0.05) increased the incidence ofcycilicity in treatment group. Thenumbers of services per conceptionswere significantly (P< 0.05) lower intreatment group compared to controlgroup. Feeding of TMR improve thereproductive performance ofnondescript dairy cattle.

Key words: TMR, Dairy Cows, Fertility,Low breeding

Season

P-07 Serum Profiles of Certain Oxidants,Antioxidants, Reproductive and StressHormones in Buffaloes (Bubalus Bubalis)R. Kausar1*, A. Hameed1, M. Hussain1, S.A. Khanum1and N. A. Qureshi2

1Animal Science Division, Nuclear Institute forAgriculture and Biology, Faisalabad 38000, Pakistan2 Department of Wildlife & Fisheries, GC University,Faisalabad 38000, Pakistan *[email protected]

A preliminary study was conducted todetermine certain oxidants,antioxidants and hormonal profiles incyclic, acyclic and conceivedbuffaloes during low breeding season.Eight buffaloes at SB farm,Faisalabad, were synchronized usingOvsynch protocol and the buffaloesobserving estrus were inseminated. Theday of AI was specified as d 0. Theblood sampling was started on d 0 upto 20 days, with 3-days gap betweeneach sampling. Pregnancy diagnosis wasmade through rectal palpation after 2months of AI. The levels of serumcatalase (CAT), superoxide dismutase(SOD), total antioxidant capacity(TAC), total oxidant status (TOS),progesterone and cortisol wereestimated. The estrus synchronizationrate was 62.5%. The progesteroneprofile showed that two buffaloes wereconceived; three observed cyclic andthe 3 remained acyclic.The CATactivity was lower (P<0.05) in cyclicand conceived buffaloes on d 0 thanacyclic animals, whereas it was higherat mid-luteal phase in conceivedanimals. The SOD activity wasconsiderably higher on d 0-8



(22.08±5.98 to 30.38±2.85 U ml-1), thendecreasing gradually till d 20 in allbuffaloes. In acyclic buffaloes, theTOS was significantly higher (P<0.05)on d 0-8 and d 16 than acyclicanimals. The TAC was higher (P<0.05)in conceived buffaloes at early lutealphase and in cyclic animals at mid-luteal phase. A parallel trend wasobserved in the levels of TOS and TACin conceived buffaloes, bothincreasing and decreasing at the sametime, with significantly higher levelsat early-luteal phase on d 4. Thecortisol was higher (P<0.05) inacyclic than cyclic and conceivedbuffaloes on d-0. It was concludedthat cyclic, acyclic and conceivedbuffaloes exhibited different trendsin the levels of oxidants,antioxidants and reproductive andstress hormones, indicating therelationship of oxidative stress withreproductive process. There is a needto further investigate thisrelationship to improve reproductionin buffaloes.

Key words: Buffalo, Hormones,Oxidants, Antioxidants,Reproduction

P-08 Effects of Different Equilibration Times on Cell Membrane and DNA Integrity of Spermafter Cryo-Reservation in Cattle BreedsA. Khan1, M. S. Qureshi1, R. U. Khan1*, U. Saddique1,I. M. Khan1, S. M. H. Andrabi2, A. Gohar3, M.Younas1, H. Khan1, H. Rehman1and Zakirullah1

1Faculty of Animal Husbandry and Veterinary Sciences,

University of Agriculture, Peshawar, Pakistan,2AnimalReproduction Laboratory, Animal Sciences Institute,National Agricultural Research Centre, Islamabad,Pakistan. 3Semen Processing Unit, Harichand, Charsada,Pakistan*[email protected]

Equilibration time in semen processingplays a vital role in semenqualitative and quantitativeparameters. Nine mature bulls wereselected in two (n=2) different breeds(Holstein- Friesian, Jersey) to findout the effect of differentequilibration times on semen qualityparameters. Artificial vagina withtemperature 42oC was use to collectsemen. Four semen samples were takenfrom each bull per week. Semen sampleswere extended in egg yolk extender(1:4), cooled, equilibrated fordesired times and stored in liquidnitrogen. Semen was thawed at 37oCafter 24 hours of storage andevaluated for sperm motility(subjective), seminal pH, dead spermpercentage, hypo osmotic swelling test(HOST), acrosomal integrity and DNAdamage (Acridine orange test). Datawas subjected to two way analysis ofvariance (Breed and equilibrationtime). Different equilibration timesincluded were 2 hours (T1), 4 hours(T2) and 6 hours (T3) for each semensample. Highest (P<0.0012) spermprogressive motility (33.45±0.31%) andsperm plasma membrane integrity(70.95± 0.33%) was observed for T2 inJersey breed. Sperm DNA integritydecreased (P<0.0011) linearly from T1(95.91± 0.14%) in Jersey to T3(84.87±0.22%) in Holstein-Friesian.Seminal pH was significantly(P<0.0013) high in T1 (6.82± 0.01) in



Jersey, followed by T2 (6.47±0.01) inHolstein-Friesian and T3 (5.90± 0.01)in Jersey and Holstein-Friesian. Thedead sperm percentage wassignificantly (P<0.0011) low in groupswith semen sample treated with T2(52.37±0.56%) in Jersey compared toother group. Significantly (P<0.0011)high sperm individual motilitypercentage (74.16±0.23%) and acorsomalintegrity (68.41±0.32%) was observedfor T2 in Jersey breed. It isconcluded that 4 hours equilibrationtime preserved cell membrane and DNAintegrity of sperm and could be usedfor better postthawed semen quality.Moreover, Jersey breed showed the bestsemen quality at differentequilibration times.

Key words: Cattle breed, Semen quality, Sperm

integrity, DNA damage

P-09 Relationship of Age, Breed andLibido with Semen Traits of Cattle BullsH. Rehman1, M. S. Qureshi1, R. U. Khan1*, U.Saddique1, A. Khan1, A. Gohar2, M. Younas1, H. Khan1 and I. M. Khan, Zakirullah1

1Faculty of Animal Husbandry and Veterinary Sciences,University of Agriculture, Peshawar, Pakistan,2Semen Processing Unit, Harichand, Charsada, Pakistan *[email protected]

The present study was conducted tofind libido in different breeds indifferent age and their correlationwith semen quality. Three maturecattle bulls from each of Friesian,Jersey, Achai, Cross (Friesian xSahiwal) and Sahiwal in different age

(1-3, 3-5 and above 5 years) wereselected. For age grouping, data ofall the breed was pooled. Libido wasmeasured as 1. Time (seconds) theentering arena until mounting (P1)2.Time (seconds) from mounting theteaser until developing of erection(P2) 3. Reaction time (seconds) (P3).Semen was collected twice a week andanalyzed for semen volume, spermconcentration, sperm motility, deadsperm percentage (Eosin-negrosin dye)and hyposmotic swelling test (membraneintegrity). Data was subjected tothree factorial analyses (breed, ageand libido) and Pearson’scorrelation. The results revealed thatlibido score in term of P1 (6.6±0.46Sec), P2 (1.88±0.07 Sec) and P3(9.57±0.49Sec) was significantly high(P<0.05) in Jersey breed while crossbred bulls showed the lowest libidoscore. Jersey bulls showedsignificantly high (P<0.05) semencharacteristics including spermconcentration (1560.0±59.00 x106/ml),membrane integrity (77.42±0.97%) andlivability (83.32±8.32%). Libidocalculated at different age groupsshowed that older animals having ageabove five years exhibited the bestlibido (P1, P2 and P3) compared to theyounger animals. Similarly, semencharacteristics including semen volume(6.50±0.73 ml) and membrane integrity(74.00±1.40%) were significantly high(P<0.05) in older bulls (above 5years) compared to the young animalswith no significant effect (P>0.05) onsperm concentration, motility and deadsperm percentage. Membrane integrityshowed significantly high positivecorrelation with sperm concentration(r=0.52) and significantly lownegative correlation with dead spermpercentage (r= -0.28). We concluded



from the results that Jersey breedshowed higher libido score and semenquality. From the results we concludedthat libido and semen quality washigher in Jersey bulls compared to theother breeds. Moreover, libido andsemen quality increased withincreasing age (up to 8 years).

Key words: Cattle bulls, semenquality, libido, age, breed.

P-10 Cross Breeding Promotes Deterioration of Semen Quality in CattleI. M. Khan1, M. S. Qureshi1, R.U. Khan1*, U.Saddique1, A. Khan1, S. M. H. Andrabi2, A. Gohar3,M. Younas1, H. Khan1, H. Rehman1 and Zakirullah1

1Faculty of Animal Husbandry and Veterinary Sciences,University of Agriculture, Peshawar, Pakistan,2AnimalReproduction Laboratory, Animal SciencesInstitute,National Agricultural Research Centre,Islamabad, Pakistan, 3Semen Processing Unit,Harichand, Charsada, Pakistan *[email protected]

Nine normal mature bulls havingaverage age was 3 ± 0.14 years wereselected in three (n=3) differentgroups of Sahiwal,Holstein- Friesian andCross-bred (Sahiwal x Holstein-Friesian) tocompare the semen quality of pure andcrossbred cattle bull. Semen sampleswere collected twice a week (totaleight semen samples) by means ofartificial vagina at 42°C andevaluated. Semen samples wereindividually diluted with extender(egg yolk extender) and tested forindividual motility, progressive

motility (under microscope), plasmamembrane integrity (HOST), acrosomaland DNA integrity (Acridine orangetest), seminal pH and dead sperm ratio(Eosin Negrosin dye) in fresh andthawed semen. Semen samples wereprocessed and cryopreserved for 24hours. Semen samples were thawed at37°C and evaluated (manually) forabove semen quality parameters infrozen thawed semen. Data wassubjected to one way analysis ofvariance. High sperm individualmotility (85.12±0.51%), progressivemotility (20.25±0.47%), plasmamembrane integrity (82.25±0.43%),acrosomal integrity (79.91±0.51%), andDNA integrity (98.54±0.14%) wereobserved in fresh semen in Sahiwalbulls, which showed significant(P<0.01) difference among the othercattle bulls. High dead sperm ratio(51.87±0.50%) deterioration of plasmamembrane integrity through HOST test(38.29±0.45%) and DNA (92.45±0.28%)was observed in frozen semen in Cross-bred bulls, which showed significant(P<0.01) difference among the othercattle bulls. It is concluded thatSahiwal bulls showed best semenqualitative characteristics in freshand frozen semen conditions followedby Holstein Friesian, while Cross-bredbulls’ semen samples showed the lowestsemen quality in fresh and frozenconditions.

Key words: Cattle, Pure breed, Cross-bred, Semen

quality

P-11 Determination of Prevalence ofReproductive Diseases in Small Ruminant by



Retrospective Study at Northern Barind Tract in BangladeshM. H. Islam1*, M. J. D. Sarder1, M. Rahman1 and M.A. H. Sarkar2

1Department of Animal Husbandry and Veterinary Science,University of Rajshahi, Rajshahi-6205, Bangladesh,2Controller Department, University of Rajshah, Rajshahii6205, Bangladesh*[email protected]

The retrospective study was done toidentify the reproductive diseases inrelation to age, sex and season ofsmall ruminant. The survey conductedin the veterinary clinic andvaccination camp of the study area.Records of 265 clinical cases of smallruminant by closed questionnairesmethod under this study, from July2012 to June 2013 were analyzed toassess the importance of existingreproductive diseases. The computerprogram SPSS were used to analyzed andchi square & F test were used forlevel of significance. The sex wassignificantly relation with variousreproductive diseases and disorders.The female small ruminant was higher(84%) prevalence than male (15.5%).The maximum diseases frequency was 87(32.8%) in anoestrus condition andlowest 5 (1.9%) in Prostatitis ofsmall ruminants. The others diseaseshighest to lowest rate were 42(15.8%), 27(10.2%), 21 (7.9%), 18(6.8%), 15 (5.7%),14 (5.3%), 12 (4.5%)and 7 (2.6%) affected with abortion,retained placenta, destrocia,cervicitis, Urinary tract infection &mastitis, urolethiasis, repeatbreeding and orchitis, respectively insmall ruminants. The highest andlowest disease occurrence rate were

anoestrus (20.4%) & repeat breeding(1.1%), anoesturs (7.9%) & prostatitis(0%) and anoestrus (4.5%) & cervicitis(0%), orcaitis (0%), urolethiasis (0%)and prostatitis (0%) was observed inkid (< 6monts), young (6 to 12 months)and old (>12months) ages of smallruminant. Similarly the seasons hadsignificant effect (P<0.05) and thehighest & lowest incidence rates ofreproductive disorders were anoestrus(9.1%) & destrocia (0%), anoestrus(9.1%) & orcaitis (0%), and anoestrus(14.7%) & mastitis (0%), dystocia(0%), and prostatitis (0%), insummer, rainy and winter season. Themost frequent reproductive diseasesand disorder were of small ruminantswere found in female, kids and rainyseason at northern Barind tract inBangladesh.Key words: Reproductive Diseases,

Small Ruminant, Age, Sex andSeasons

P-12 Fertility Improvement in Cross-Bred DairyCows through Supplementation of Vitamin E as AntioxidantI. Khan1*, M. S. Qureshi1, S. Akhtar1, I. Ali2 and Ghufranullah3

1Faculty of Animal Husbandry and Veterinary Sciences,The University of Agriculture, Peshawar-25120,Pakistan,2Institute of Biotechnology and Genetic Engineering,The University of Agriculture, Peshawar-25120, Pakistan.3Directorate General (Research), Livestock and Dairy Development, Khyber Pakhtunkhwa, Peshawar. *[email protected]

Heavy milk production along withenvironmental thermal stress causesstress to the dairy cows that resultinto poor fertility. This study was



conducted to investigate the role ofvitamin E in combating stress,reflected by general (cortisol),thermal (heat shock protein, HSP-70)and oxidative (malondialdehyde, MDA;Glutathione per oxidase, GPx; Superoxide dismutase, SOD) stress. Theexperiment was conducted on thirty sixearly lactating dairy cows, comprisingindigenous (9 Sahiwal, 9 Achai), 9cross-bred (Sahiwal x HolsteinFrisian) and 9 Holstein Frisianlocated at livestock research anddevelopment station Surezai, districtPeshawar, Pakistan. Vitamin E wassupplemented to dairy cows in feed ata dose rate of 1000 IU/ cow/ day for40 days. Cows were synchronized andsampling was conducted on day-zero(control) before Vitamin Esupplementation, day-20 and day-40 ofvitamin E supplementation during thediestrus phase of the estrus cycle.Results indicated that concentrationsof SOD and GPx increased (P < 0.001)with vitamin E supplementation fromday-0 to day-40. SOD and GPxactivities varied (P < 0.05, P <0.001, respectively) between breeds,more prominent in local and crossbredcows. The stress markers (plasma MDA,HSP-70 and serum cortisol) decreased(P < 0.001) with vitamin Esupplementation. Crossbred and localcows showed higher response (P< 0.001)to vitamin E supplementation comparedto HF in stress parameters.Progesterone concentration increased(P < 0.001) with vitamin Esupplementation from day-0 to day-40.Progesterone concentrations increasedmore notably in Crossbred and HF (P<0.05). However, highest value wasobserved for progesterone in Achajcows. Vitamin E supplementationreduced the number of dominant

follicles in all cows (P< 0.01) fromday-0 to day-40. The effect of vitaminE on follicular growth was moreintensive in crossbred than HF andlocal cows (P< 0.05). The stressmarkers cortisol was positivelycorrelated with HSP-70 (r= 0.285, P=0.003) and MDA (r= 0.225, P= 0.013).HSP-70 and MDA were also positivelycorrelated (r= 0.371, P= 0.000) witheach other. Cortisol were negativelycorrelated with SOD (r= -0.388, P=0.000) and GPx (r= 0.330, P=0.000).MDA and HSP-70 were also negativelycorrelated with SOD (r= -0.399,P=0.000 and r= -0.355, P=0.000) andGPx (r= -0.447, P= 0.000 and r= -0.498, P= 0.000) respectively.Progesterone was positively correlatedwith SOD (r= 0.404, P=0.000) and GPx(r= 0.443, P=0.000). The results ofthe present study reveals that vitaminE supplementation can increasereproductive performance in HF andcrossbred cows suggesting regularfeeding of antioxidant to thesebreeds.

Key words: Dairy, HSP-70, Stress,Fertility, Antioxidant

P-13 A Strategy to Improve Cryo-Survival and Fertility of Buffalo Bull SpermatozoaA. Razzaq1, A. Sattar2*, A. Riaz2, M. Avais3, M. Ahmad2, A. R. Malik1, A. G. Tarin2, M. U. Mehmood2 and M. Younis4

1Department of Animal Husbandry, Muzaffarabad, AzadGovt. of the State of Jammu & Kashmir, 2Department ofTheriogenology, University of Veterinary and AnimalSciences, Lahore, Pakistan, 3Department of CMS, University of Veterinary and Animal Sciences, Lahore, Pakistan, 4Semen Production Unit,



Qadirabad, Sahiwal, Pakistan *[email protected]

During cryo-processing of spermatozoa,cholesterol is an important componentin the regulation of membranefluidity. The objective of the presentstudy was to determine the improvementin cryosurvival of spermatozoa byaddition of cholesterol in Tris-citricacid (TCA) semen extender and alsopregnancy rate in Buffaloes. Buffalobulls were of Nili Ravi breed having5-7 years of age. Experiment-1:Cholesterol was added to TCA semenextender in the form of cholesterol-loaded cyclodextrin (CLC). Extendedsemen was incubated with CLC at roomtemperature for 15 minutes before theaddition of egg yolk and glycerol.Five replicates of each bull (n=3)were separately evaluated and furtherprocessed with at least 60 % spermmotility. Semen samples were dilutedat 37° C in extender containing either1 mg (Low), 2 mg (Medium), 3 mg (High)of CLC or without (Con) per mlcontaining 120 million spermatozoa.After extension at 37 °C, semen wascooled to 4°C in 90 min, equilibratedat 4°C for 2 hours, frozen in liquidnitrogen (LN2) vapor (4 cm above theLN2 for 7 min) and shifted to LN2 forstorage. Post thaw motility (PTM),live spermatozoa (LS), plasma membraneintegrity (PMI) and normal apicalridge (NAR) were assessed usingstandard procedures. Analysis ofvariance revealed that mean PTM and LSvalues were significantly (P <0.05)higher in Medium (44.33 ± 1.68 and53.60 ±2.03 %) as compared to Con (31.33 ±1.98 and 41.20 ±2.50 %) respectively. Mean PMI and NARvalues were significantly (P<0.05)

higher in High and Medium groups ascompared to Con. Experiment-2: Semendoses from Medium (n=50) and Con(n=50) were used for fertility trialin 100 Nili Ravi buffaloes in thefield area of Muzaffarabad at > 90days postpartum. Althoughnonsignificant (P>0.05), the pregnancyrate was higher (64 %) in buffaloesinseminated with semen of medium groupas compared to their Control (62%)counterparts It is concluded thataddition of cholesterol in Mediumconcentration (2 mg CLC) to Buffalobull semen can improve cryosurvival ofbuffalo bull spermatozoa but notfertility as compared to Con group.

Key words: CLC, Buffalo Bull,Spermatozoa

P-14 Studies on Uterine Microbial Flora of Sahiwal Cattle during OestrusH. Rehman1, M. Rabbani1, A. A. Sheikh1*,N. Ahmad2,S. Raza1 and J. Muhammad1

1University Diagnostic Lab, University of Veterinary & Animal Sciences, Lahore 54000, Pakistan, 2

Department of Theriogenology, University of Veterinary & AnimalSciences, Lahore 54000, Pakistan *[email protected]

Conception rate of cattle variousfactors including bacterial pathogensaffect the reproductive ability aftercalving resulting in subfertility.Post partum invasion of uterus bybacterial pathogens disturb uterinefunctionality leading to low fertilityof the cows. Current study wasenvisaged to find out the microbialload in the uterine lumen of Sahiwalcattle at the time of estrus, just



before artificial insemination. Atotal fifty (50)uterine samples ofnormal cyclic adult Sahiwal cows withno previous history of uterinedisorder, kept at Livestock Productionand Research Institute (LPRI),Bahadurnagar Farm, district Okara,were collected at the time of estrusprior to insemination using sterilizedsampling device consisted of a rod,normally used for artificialinsemination, having both the endsopen. At one open end, there was aconnection to a syringe which hasnormal saline in it and at the otherend there was half gelatin capsule toprevent the rod from contaminationwhile entering into the uterus. Theuterine samples were cultured onnutrient and blood agar under aerobicand an-aerobic conditions andidentification of bacteria was done onthe basis of morphology, stainingcharacteristics and biochemicalreactions as per guidelines ofBergey's Manual of DeterminativeBacteriology. Statistical analysis wasdone using SPSS 18.0 and frequency ofbacterial isolates were calculatedwhich showed isolation of total 11bacterial species from the uterinesamples. Out of these bacterialisolates, 28 (56%) isolates were E. coli,22 (44%) were Micrococcus spp., 19 (38%)were Bacillus spp., 16 (32%) wereStaphylococcus aureus, 5 (10%) wereStaphylococcus epidermidis, 10 (20%) wereStreptococcus spp., 2 (4%) were Pseudomonasspp., 11 (22%) were Citrobacter spp., 2 (4%)were Salmonella spp., 5 (10%) were Proteusspp. and3 (6%) were Corynebacterium spp.Statistical analysis showed thatsignificant association (p< 0.05) ofnon pregnant Sahiwal cattle withisolation of Streptococcus. Otherbacteria like

E. coli, Micrococcus, Bacillus, Staph aureus, S.epidemicus, Pseudomonas, Citrobacter,Salmonella, Proteus and Corynebacterium werenon significantly associated (p> 0.05)with pregnancy status of these animalswhen inseminated artificiallyindicating that E. coli,Micrococcus spp.,Bacillus spp., Staphylococcus aureus, S.epidemicus, Pseudomonas, Citrobacter,Salmonella, Proteus and Corynebacteriumarethe normal microbial flora in theuterus of Sahiwal cattle whileStreptococcus spp. are not normally presentas uterine flora which could result indecrease pregnancy rate.

Key words: Uterine microflora, Conception rate,

Uterine bacteriology, Sahiwalcattle.

P-15 Comparative Reproductive Performance ofBeetal Goats in Accelerated and Annual Kidding SystemsN. Ahmad1*, K. Javed1, M. Abdullah1, A. S. Hashmi2,A. AliandT. Hussain3

1Department of Livestock Production, University ofVeterinary and Animal Sciences Lahore 54000,Pakistan,2Institute of Biochemistry and Biotechnology,University of Veterinary and Animal Sciences Lahore54000, Pakistan, 3Department of Molecular Biology,Virtual University of Pakistan, Lahore. [email protected]

Beetal is a goat breed of Pakistan.This is popular for its milk andparticularly meat production of tenderquality. Fecundity is one of anothervalued economic trait in this breed.The present study was planned toevaluate the reproductive performanceof accelerated kidding verses annual



kidding system for a period of twoyears. A total of 50 Beetal goats wererandomly divided in two groups having25 animals each. Accelerated kiddingsystem means to have more frequentservices and kids per doe during aperiod of two years as compared toannual kidding system. Thesupplementary feeding was adopted forsuccessful conception rate andjudicious use of their naturalbreeding abilities. It was observedthat accelerated kidding system ismore profitable. The data wereanalyzed by complete randomized design(Steel et al., 1997) using proc GLMprocedure of SAS.1995 (SAS institute,cary, NC). The onset of estrus,kidding percentage and conception ratewas 8, 1.33 and 10%, respectively.However, the number of kids born,single and triplet births wereaugmented as 6, 1.94 and 20%,respectively in accelerated system. Itwas also found that twin births werereduced by 11.6% in acceleratedsystem. Maximum 1.64 ±0.81 servicesper conception (SPC) were observed.The litter size (LS) was observed as1.66 ±0.71which was significant(P<0.05) in accelerated kidding systemas compared to maximum 1.19 ±0.40(SPC) with 1.50 ±0.55 (LS) in annualkidding system. The shorter gestationlength was also recorded inaccelerated system. It is concludedthat accelerated kidding system inthis breed is economically doable.Further studies and research withlarger data set is needed to explorereproductive performance in Beetalgoats under accelerated kidding systemand to validate the findings ofcurrent study.

Key words: Reproductive performance,Services per conception,

Accelerated kidding system,Beetal goat, Pakistan

P-16 Use of Butylated Hydroxytoluene anAntioxidant in the Buffalo Semen ExtenderT. Munir*, M. Aleem, A. Ijaz, I. K. Khan and M. Aftab

Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan *[email protected]

Nili-Ravi breed of buffalo is one ofthe best breeds of the world. Duringfreezing, the semen quality is damageddue to the production of ReactiveOxygen Species (ROS) from the plasmamembrane of spermatozoa causing damageto the membrane integrity due to lipidperoxidation. The main objective ofthe present study was to determine thelevel of total antioxidants in thesemen and to optimize the quantity ofBHT in the semen extender of the NiliRavi Buffalo bulls. In this study,semen from Nili-Ravi buffalo bulls(n=10) was collected twice per week inthe month of October. The semen ofeach bull was diluted with Tris eggyolk citrate extender separately.Antioxidant BHT was added to theextended semen at the rate of 1.0mM,1.5 mM and 2 mM (n=20 per bull pertreatment) while Control wascontaining no BHT. The semen wasvisually analyzed with the help ofphase contrast microscope formotility, vitality (Eosin/Nigrosinstaining), plasma membrane (HOS assay)and acrosomal integrity (NAR), atthree stages i.e. after dilution,before freezing and after freezing.The semen was frozen manually at SPUQadir Abad. ABTS assay was used for



the determination of total antioxidantcapacity (TAC), in theSpectophotometric Laboratary, GCUniversity Lahore. The semen wastransported in thermos having ice fromSPU Qadir Abad to Lahore to limit ROSproduction during travelling. Datacollected were presented as mean ±SEM. Treatment groups were comparedusing one way ANOVA and results werecompared by using Duncan MultipleRange Test using SPSS (version 16.0);SPSS Chicago IL. Results of this studyshowed significant (P<0.05)improvement in sperm motility,vitality, plasma membrane andacrosomal integrity at 1.5 mM BHTconcentration as compared to Controlsample. Maximum TAC value (2.08 mM)was observed in the extended semencontaining BHT and minimum TAC values(0.58 mM ) in the post thaw semensample with BHT. It is concluded thataddition of antioxidants in thebuffalo semen improves the semenquality before and after freezing.Fortification of buffalo semenextender with BHT (@ of 1-1.5 mM)improved post thaw semen quality.However, further study is required toanalyze the fertility of buffaloesafter AI with BHT fortified semen.

Key words: Butylated Hydroxytoluene,Buffalo Semen

Extender, Nili-Ravi

P-17 Effects of Flaxseed Suplementation onSemen Quality and Quantity in Different Dairy Cattle BreedsH. Khan1, M. S. Qureshi1, I. Khan1*, Ihsanullah1, S. Rehman2, T. Mahsud2 and S. Afridi1

1Faculty of Animal Husbandry and Veterinary Sciences, University of Agriculture, Peshawar-25120,Pakistan.2Directorate of Livestock, FATA *[email protected]

Flaxseed is a good source of omega-3fatty acids. The current study wasdesigned to investigate the effects ofdietary flaxseed as a source of omega-3 fatty acids on quality of liquid andfrozen thawed semen in different dairycattle bulls. Nine bulls of 3-5 yearsage, 3 each from Holstein Frisian(HF), Jersey and Cross-bred (HF xSahiwal) were selected, and furtherdivided into three treatment sub-groups, 1) Control group FL-0 was fed6 kg concentrate, 6 kg wheat straw and40 kg green fodder as basal diet, 2)FL-I bulls fed with 50 g of importedrumen protected flaxseed plus basaldiet, 3) treatment FLL bulls fed 50 glocal non rumen protected flaxseedalong with basal diet. Trial wascarried out for a total of 11 weeks.Adaptation period was given to animalsbefore the commencement of experiment.Semen collection was started from thethird week of supplementation.Factorial design was used forstatistical analysis to find out theeffect of breed and flaxseedsupplementation. Seminal volume didnot vary among the treatment andcontrol groups in all breeds (P>0.05). Sperm concentration, massmotility, individual motility,viability and HOST of the fresh semenwere higher (P<0.05) in the bulls fedwith (FL-I) as compare to FL-0 and FL-L. Motility, viability and HOST ofpost-thawed semen were also higher(P<0.05) in the bulls fed with (FL-I)compared to FL-0 and FL-L. Total Spermoutput was significantly higher



(P<0.05) in HF bull fed with rumenprotected flaxseed (FL-I) comparedwith FL-L and FL-0 of the same breed.HF bulls of FL-I and FL-L group showedmore favorable response to flaxseedsupplementation than crossbred andJersey of the same group. Total spermoutput was correlated positively(P<0.05) with mass motility (r=0.48,P= 0.001), membrane integrity (r=0.58,P= 0.000), percent motility (r=0.63,P= 0.051) and seminal volume (r= 0.64,P= 0.000), whereas, seminal volume andconcentration of spermatozoacorrelated negatively (r= -0.75, P=0.000). The results of the presentstudy imply that feeding rumenprotected flaxseed improve the qualityof bull spermatozoa.

Key words:cattle Bull, Flaxseed, Semenquality, Dietarysupplementation, Fertility

P-18 The Way to Preserve the Genomeof Valuable Animal: Epididymis Gamete Retrieval in Slaughtered Ramand its Post-Thaw EvaluationU. Riaz1*, A. Husnain2, M. I. Naveed3, U. Farooq1 and A. Riaz2

1University College of Veterinary and Animal Sciences,Islamia University of Bahawalpur 63100, Punjab, Pakistan 2Department of Theriogenology, Universityof Veterinary and Animal Sciences Lahore 54000, Punjab, Pakistan. 3Livestock and Dairy DevelopmentDepartment, Punjab, Pakistan.*[email protected]

Post-mortem spermatozoa recovery is animportant technique for obtaininggermplasm reserves from geneticallyvaluable animals or endangeredspecies. It can be used to dilute offa huge loss in any breed conservation

program. The objective of the presentstudy was to evaluate pre-freeze andpost thaw sperm attributes collectedfrom ram epididymis (n=05). Testeswere collected from slaughter houseduring breeding season and transportedat refrigeration temperature in normalsaline. The evaluation was carried outwithin two hours of slaughtering.Sperms were obtained by slicing thetissue of cauda epididymis with ascalpel; fluid was collected andmeasured. The samples were carefullydissected free of blood clots andextraneous tissue. Semen samples werediluted with the Tris Citrate egg yolkextender. After extension, cooling andequilibration of semen for 2 hours at4 oC, filling was done in 0.5 ml Frenchstraws to attain a final concentrationof 100 × 106 spermatozoa per straw.Freezing of samples was carried out bykeeping the straws at the level of 4cm above liquid Nitrogen (LN) vaporsfor 7 min and finally frozen in LNthrough programmable biofreezer. Thesperm attributes included percentagesof sperm motility, viability, normalmorphology, plasma membrane integrity(PMI) and normal apical ridge (NAR).Results revealed that pre-freezepercentages of motility, viability,normal morphology, PMI and NAR were65, 70, 20, 70 and 66%, being 30, 38,25, 36 and 34 for post thawspermatozoa, respectively. The postthaw sperm attributes were found to bewithin an acceptable limit. It isconcluded that ram gametes attainedfrom their epididymis can be preservedwith acceptable quality. Furtherinvestigations can be directed towardsgamete handling and addition of somesupplements in extenders in order toimprove its preservation.



Key words: Conservation, EpididymalSperm, Ram

P-19 Effect of Varying Concentrations of BovineSerum Albumin and Egg Yolk on Post-Thaw Quality of Beetal Buck SemenM. Shahzad1, A. Sattar1*, N. Ahmad1, A. Rehman1, M.Ahmad1, M. S. Khan2, M. Ahmed1, A. A. Channa1 and S. Iqbal3

1Department of Theriogenology, 2Department of ClinicalMedicine and Surgery, University of Veterinary and AnimalSciences, Lahore 54000, Pakistan, 3Semen Production Unit,Qadirabad, Sahiwal, Pakistan *[email protected]

Bovine Serum Albumin (BSA) protectsspermatozoa from harmful effects oflipid peroxidation upon its additionin semen extenders. The aim of presentstudy was to optimize theconcentration of BSA with varyingconcentration of egg yolk in semenextender through the evaluation ofpost thaw quality of Beetal bucksemen. Two Beetal bucks were used forsemen collection for 7 weeks. Semenfrom both the bucks were collectedtwice a week whereas once a week semencollection was used for this study and2nd collection was used for anotherstudy going on in the Department.Ejaculates of both bucks having >70%motility, > 0.5 ml volume, >2.5×109 perml concentration and <10%abnormalities were pooled and dividedinto 9 aliquots. Tris Citrate Egg Yolk(TCEY) extender containing differentconcentrations of egg yolk (20, 15 and10 %) was prepared. Extender withlatter two concentrations also

received four concentrations (0, 1, 2and 3%) of BSA separately. Nine typesof extenders were mixed with abovesaid 9 aliquots of buck semen. Afterextension at 37°C, semen was cooled to4°C in 90 min, equilibrated at 4°C for4 hours, frozen in liquid nitrogen(LN2) vapors (4 cm above the LN2 for 10min) and shifted to LN2 for storage.Post thaw semen evaluation assays, i.e.,post-thaw motility (PTM%), livepercentage (LP%), morphologicalabnormalities (MA%), plasma membraneintegrity (PMI%), normal apical ridge(NAR%) and DNA integrity (DI%) wereconducted after 24 hours of storage.Two-way ANOVA was performed andsignificant differences between meanswere compared using Tukey’s test. Itwas observed that mean± SE values of PTM, LP, PMI, NAR andDI were significantly (P<0.05) higherin groups having 15 and 10 % egg yolkwith 1 and 2 % BSA, respectively ascompared to all other groups. Inconclusion, this study indicated thatthe addition of 15% egg yolk with 1%BSA and 10% egg yolk with 2% BSA inBeetal buck semen may enhance its postthaw sperm quality.

Key words: Beetal Buck, Semen, BSA,Egg yolk

P-20 Effect of Different Levels of ConcentrateMixture for Attaining Early Pubertyin Sahiwal HeifersZ. Iqbal1*, M. Abdullah1, K. Javed1, J. A. Bhatti1, N.Ahmad1 and F. Ahmad2

1Department of Livestock Production, University ofVeterinary and Animal Sciences, Lahore, Pakistan2Pir Mehr Ali shah Barani University, Rawalpindi *[email protected]



The objective of the study was toevaluate effect of varying levels ofconcentrate on the growth and age atpuberty in Sahiwal heifers. Theresearch was conducted at livestockexperimental station, Jahangirabad,Khanewal, Punjab, Pakistan. Sixtythree Sahiwal female young stocks405±23 days age were divided intothree groups (21 FYS, with 7 FYS perreplicate were offered green fodder(GF), GF+ Concentrate ration (CR)@0.5% of body weight (BW) and GF+CR@1%of BW. The feeding trial continuedtill age at which heifers on alltreatments were expected to reach atage of puberty and got pregnant. Theresults showed a significant (P<0.01))difference in mean dry matter intake,average daily gain, body measurementsand feed efficiency. The digestibilityof nutrients was greater (P<0.05) ingreen fodder with 1 % of concentrateration. Sahiwal heifers fed greenfodder with 1 % of concentrate rationattained puberty earlier than thosefed 0.5 % concentrate ration alongwith green fodder. Body height, bodylength and heart girth were found thehighest (P<0.05) in heifers fed ongreen fodder with 1 % concentrateration.Cost to gain ratio in heiferswas Rs. 97.18, 90.60 and 93.03 onGF,[email protected]% and GF+CR@1%,respectively. The best performance interms of increased dry matter intake,body condition score, feed efficiency,and weight gain and body measurementswas observed in the heifers fed ongreen fodder along with starter @ 1 %.The cost to gain ratio was observedcomparatively low in the heifers onthe treatments supplemented with 1%concentrate. It is further suggestedthat the heifers be supplemented with

concentrate @ 1 % of body weight toattain early maturity got pregnant.Key words: Concentrate Ration,

Sahiwal Heifers,Growth Performance, DryMatter Intake

P-21 Influence of Crude Proteins Levels on MilkProduction, Blood Urea Nitrogen andSerumProgesterone Profile in Mid Lactating Jersey Cows

T. Ahmad and S. Sultan*

Department of Animal Nutrition, Faculty of AnimalHusbandry and Veterinary Sciences, University of Agriculture Peshawar, Khyber Pakhtunkhwa *[email protected]

An experiment was conducted to theinfluence of different crude protein(CP) levels on milk production andmilk composition (fat, protein andlactose), blood urea nitrogen (BUN)and progesterone (P4) profile in mid-lactating Jersey cows. A total of ninecows at mid lactating stage (110±20days) were randomly selected for thetrail and divided into three groups.Each group was fed experimental diethaving 13, 16 and 19% crude protein ondry matter (DM) basis respectively.All the animals were fed individuallyon maize fodder and wheat straw basedration ad-libitum along with 5kg oftheir respective experimental diets.The experiment was continued for 75days including 15 days adaptationperiod and 60 days for datacollection. Dry matter intake was notsignificantly (p>0.05) effected in allthe groups but numerically high valueswere recorded for the group havinghigh level of CP. Body weight gain was



recorded significantly higher in group13%CP (14.0±2.08) and group 16%CP(6.0±2.08) as compared to group 19% CP(-4.67±3.18) throughout theexperimental period. Milk yield(kg/day) was significantly (p<0.05)higher in group 19%CP (11.8±0.23)compared to 13%CP (10.46±0.32) and16%CP (11.23±0.34). Similar trend wasrecorded for milk fat percentagehaving mean values of 3.99, 3.85 and3.75% fat in 19%CP, 16%CP and 13% CPgroup respectively. Mean percentage ofmilk protein and lactose were notsignificantly (p>0.05) effected in allthe treated groups. Blood ureanitrogen was significantly (p<0.05)higher in group 19% CP followed by16%CP and 13% CP groups having meanvalues 18.87, 15.93 and12.07 mg/dlrespectively. No significantdifference was found for progesteronelevel in the serum having mean valuesof 3.02, 2.91 and 2.76 ng/ml for 13,16 and 19% CP group respectively. Itis concluded that performance of cowsat mid-lactation can be maintained atlower dietary crude protein up to 13%in concentrates with maize fodderbased ration reflected by optimumlevels of milk yield, blood ureanitrogen and serum progesterone.

Key words: Crude Proteins, MilkProduction, Blood

Urea Nitrogen, SerumProgesterone Profile

P-22 Testicular Biometry, ScrotalCircumference, Serum Testosterone and Semen Characteristics in Achai and Jersey BullsA. G. Khan1, M. S. Qureshi1, H. Khan1*, M. Younas1, A. Gohar2, S. Akhtar1, R. U. Khan1, H. Rehman1 andU. Saddique1

1Faculty of Animal Husbandry and Veterinary Sciences,University of Agriculture, Peshawar, Pakistan,2SemenProcessing Unit,Government Cattle Breeding and DairyFarm Harichand, Charsadda, Pakistan *[email protected]

This study aimed to determine therelationship of scrotal circumference,age and body weight to testicularbiometry and to establish criteria forBreeding Soundness Evaluation (BSE) ofAchai (Indigenous breed) and Jersey(Exotic breed). Standard procedureusing measuring tape was used tomeasure the scrotal circumference andtesticular biometry of 8 Achai and 9Jersey bulls of various ages. Thegreatest scrotal area of circumferencewas recorded for measurement. Thelength of testes was measured fromdorsal to ventral side, width fromright to left side of the testes andthickness of testes from anterior toposterior side. The volume of thetestes were measured by4/3 π a b c, inwhich a, b, c related to, thickness/2,width/2 and length/2 of the testes,respectively. The weight of thetestes was calculated by multiplyingvolume with 1.038.The ejaculates werecollected twice a week from each bullfor 6 weeks starting from age groupsof four different breeds that was 16-36 (n=3), 37-48 (n=3), ≤49 (n=3)month. Semen volume, concentration,motility and progressive motility wererecorded through phase contrastmicroscope. Blood samples werecollected at three times; atbeginning, after 15 day and after 30of the experimental period to measuretestosterone level using ELISA. Datawas statistically analyzed through



analysis of variance and Pearsoncorrelation usingSPSS (version 16.0)statistical packages. Duncan MultipleRange Test was used to signify the agegroups of bulls breed-wise separately.During current study, significant(P<0.05) increase has been observed inlength, width and thickness of testes,scrotal circumference and body weightof both breed at adult age group (≤49month).Furthermore, testosterone level wasfound significantly higher (P<0.05) inboth indigenous and exotic breed. Theresult of the current studydemonstrated that ScrotalCircumference, Testicular Weight,Sperm Volume, Motility, ProgressiveMotility and Sperm output werepositive correlated with body weight.Therefore theses indices could be usedas practical indicators to selectbreeding bull during breedingsoundness examination under existingmanagement system.

Key words: Achai, Jersey, TestesBiometry, Scrotalcircumference, Testosterone,

P-23 Dystocia due to Parapagus Monster in a Murrah BuffaloR. K. Chandolia1* and G. Singh2

1Dept of Vet Gynaecology & Obstetrics, Lala Lajpat RaiUniversity of Vet & Animal Sciences, Hisar 125004, India2 Teaching Veterinary Clinical Complex, Lala Lajpat RaiUniversity of Veterinary and Animal Sciences, Hisar125004, India*[email protected]

A Murrah buffalo in its 2nd lactationwas presented to teaching veterinaryclinical complex, LUVAS, Hisar, India

with a history of a prolonged secondstage of labor with forceful abdominalcontractions and two forelimbs andhead were protruding from the vulva.On vaginal examination it was foundthat the second rib cage was present,perpendicular to the birth canal inthe uterus. Further detailedexamination confirmed the presence oftwo tails in the birth canal. Becauseone portion of fetus along with bothforelimbs and head were hangingoutside the vulvar lips. Therefore onexamination to right side of the fetustwo tails were present in the uterusand other portion of fetus was presenttransversely. Hence it was confirmedthat the fetus was a monster that wascausing dystocia. Since forcedextraction was not possible, thereforea fetotomy was performed under localanesthesia with 2% Lignocainehydrochloride injecting 710 mlepidurally between 1st and 2nd inter-coccygeal space. The head of thygesonfetotome was placed dorsally at thelumber region whereas loop of wire waspassed around the around the abdomenbehind the last rib and a full-termdead fetal monster was extracted. Thebuffalo was treated with an injectablecombination of 3.0 gm Ceftriaxone +1.5 gm Salbactum (Polcef forte), plusan injection of Melonex (Meloxicam 0.5mg/ kg b.wt), 30 ml intramuscularly,intra uterine bolus, Hyterus(combination of Nitrofurazone,Metranidazole, Povidone iodine, Ureaas anantiseptic and proteolytic agent)and supportive fluid therapy for 5days. The buffalo showed recoverywithout any complication. Thedevelopment of conjoined twins wasnearly complete. Two normal heads werepresent on two necks with normal eyesand ears. The twins were fused in the



thoracic region. There were fourforelimbs and an abnormal orientationof ribs. Two separate vertebralcolumns along the length of the animalended with two tails. There was asingle pelvis, and only two hind limbswere present. The calf had a singleanus and vulva. Conjoint fetuses wereattached with each other behind thexephoid region e.g. ventral abdomen asseen in photograph. Conclusion: Suchtype of cases can not be treated atfield level and forced extractionshould not be tried without knowingthe etiology of the dystocia. Thedecision should be taken immediatelywithout exhausting the animal.Key words: Murrah buffalo, dystocia,

parapagusmonster, India

P-24 Effect of Cholesterol Loaded Cyclodextrin on Sperm Survival of Buffalo Bull during Stages of CryopreservationM. Ahmed, A. Sattar,M. H. Fayyaz and M. Ahmad*

Department of Theriogenology, University of Veterinary and Animal Sciences, Outfall Road, Lahore, Pakistan *[email protected]

Sperm cryopreservation results in lossof sperm structural and functionalparameters.In vitro incorporation ofcholesterol into mammalian spermmembrane increases sperm membranefluidity at low temperatures. This isthe first study sought to evaluate theeffect of cholesterol loadedcyclodextrin (CLC) on buffalo spermquality parameters at different stagesof cryopreservation (i.e. afterdilution, before freezing, and afterthawing). Semen ejaculates from threebuffalo bulls were pooled (Replicates

= 6), treated either with CLC (2 and 4mg per 120 × 106 spermatozoa) orwithout CLC (control), incubated for15 minutes and diluted in tris-citricacid diluent. After dispensing in 0.5ml straws, cooled to 4oC and frozenusing standard procedure. Spermmotility, viability, plasma membraneintegrity (PMI; hypo-osmotic swellingtest), and normal apical ridge (NAR;formol citrate solution) wereevaluated (%) after collection/fresh(F), after dilution (AD), beforefreezing (BF) and postthawing (PT).Data were presented as mean ± S.E.Mand analyzed using analysis ofvariance taking replicate as randomfactor. Motility did not differ(P>0.05) between control (C0), CLC 2(C2) and CLC 4 (C4) groups from F, butreduced (P<0.05) due to cooling in C0,C2 and C4 (58.33±1.67, 65.00± 0.60,65± 0.46, respectively; P>0.05 betweengroups). PT motility of C0 and C2(43.44 ± 1.67 vs. 56.67 ± 1.67,respectively; P<0.05) was furtherreduced (P<0.05), while C4 showssimilar (P>0.05) motility (58.33 ±1.67) when compared to previousstages. In all groups (C0, C2, C4),sperm viability, PMI and NAR were same(P>0.05) at AD. At thawing, spermviability (57.33±0.67, 67.67±0.33,71.67±.67; c0, c2 and c4 respectively), PMI (55.00±1.00, 67.00±1.00 and70.67±0.67; C0, C2, C4 respectively)and NAR (56.00±3.51, 63.33±0.33 and67.33±0.67; C0, C2 and C4respectively) were reduced (P<0.05).Both CLC treatments (C2 and C4) showedno difference (P>0.05) in PMI from BFto PT, while PMI was reduced (P<0.05)in control. The concentration of CLCaffected post-thaw sperm quality in adose-dependent manner. The maindifference in sperm quality was



observed in PT stage. It is concludedthat CLC treatment for buffalo spermcryopreservation significantlyimproves cell cryosurvival andminimizes sperm damage at PT stage.Key words: Cholesterol LoadedCyclodextrin, Buffalo

Bull, Cryopreservation

P-25 Effect of Bypass Fat Supplementation onProductive and Reproductive Performance of High Yielding MurrahBuffaloesN. Saxena1*, V. Mudgal1, C. Mohan1, K. Kumar1,M.L. Sharma1, S.K. Sarkar2, J. K. Singh3, A. K. Pandey4 and D. Lal5

1Division of Animal Nutrition & Feed Technology, ICAR-CIRB, Hisar, 125001, Haryana, India, 2 Division of Design and Experiments, IASRI, New Delhi, 3Subcampus, ICAR-CIRB, Nabha, Punjab, India, 4Division ofAnimal Genetics & Breeding, ICAR-CIRB, Hisar,125001,Haryana, India, 5 Indian Council of AgricultureResearch, New Delhi, 110012, India * [email protected]

The study investigated the effect ofdietary supplementation of bypass faton productive and reproductiveperformance and blood biochemicalprofile of high yielding Murrahbuffaloes (Bubalus bubalis). Twenty-fourpregnant buffaloes with previousrecord of high productivity weredivided in two homogenous groups ofcontrol and treatment. The animals ofcontrol were fed basal diet consistingof a concentrate mixture, availablegreen fodder and wheat straw as perrequirements, while the animals of

treatment group were fed same rationsupplemented with bypass fat startingfrom one month prior of the expecteddate of calving to whole lactationperiod of 305 days. There were nodifferences in the dry matter andnutrient intake as well as milkcomposition except milk fat. Similarlyno difference in blood mineral contentand haematological parameters wasobserved except percentage ofmonocytes. However, biochemicalparameters such as enzymes alkalinephosphatase, creatinine phosphokinase,aspartate transaminase and amylasewere significantly different whileother parameters remained same. Therewas no response either positive ornegative on the reproductiveperformance of the buffaloesstatistically. Though supplementationof bypass fat statistically (P<0.05)improved the birth weight of calves,as well as production of fat-correctedmilk (6%) in high yielding buffaloes.It was concluded that bypass fatsupplementation increased the fat-corrected milk production and birthweight of calves when supplemented onemonth prior to calving till mid-lactation without any adverse effecton reproductive performance of highyielding buffaloes.

Key words: Buffalo, Bypass fat,Reproduction and

Production performanceP-26 Seasonal Pattern of Calving inNili-Ravi BuffaloesA. Waheed1, M. Tariq2*, S. Ahmad2, U. Hassan2, B. Mustafa2, M. M. Tariq3and H. Nawaz4

1Faculty of Veterinary Sciences, Bahauddin ZakariyaUniversity, Multan, 2University of



Agriculture,Faisalabad, Sub-Campus, Toba Tek Singh,3CASVAB, University of Balochistan, Quetta, 4Institute ofAnimal Science University of Agriculture, Faisalabad *[email protected]

Season of calving in buffalo showstrends in calving during differentseasons. This study was conducted toexplore seasonality of calving(calving frequency) in Nili-Ravibuffaloes. Data consisting of 17731calving records of buffaloesmaintained in three herds wereanalyzed. Herd 1, 2 and 3 contributed7667(43.2%), 3965 (22.4%) and6099(34.4%) records respectively inthe total data set. Data were dividedinto four seasons viz. Spring (Feb-Apr), summer (May-Aug), autumn (Sep-Oct) and winter (Nov-Jan) consideringthe month of calving. The four seasonshad 32.1% (n=5690), 20.5% (n=3637),34.6% (n=6137) and 12.8% (n=2267)calving records, respectively. Therewere significant (P<0.05) differencesamong frequencies of calving indifferent seasons. Most of the calveswere produced in autumn (34.6%)followed by spring, summer and winterseason. This distribution is helpfulin making management decisions forproviding feeding resources and otherprovisions for the buffaloes. Thisinformation is also helpful in makingmore appropriate breeding decisions.The results made it clear thatbuffaloes bred in winter (Nov-Jan) hadhigh reproductive efficiency. Itshowed impact of season onreproductive efficiency of Nili-Ravibuffaloes.

Key words: Season, Reproductiveefficiency, Nili-Ravibuffalo, Management

P-27 Distribution of Buffaloes Affected with PeriPartum Reproductive Disorders, Maintained under Different Production SystemsM. A. Ali1, L. A. Lodhi2, A. Waheed1**, M. Tariq3*, F.Hassan4 and S. Ahmad5

1Faculty of Veterinary Sciences, Bahauddin ZakariyaUniversity, Multan, 2Faculty of Veterinary Sciences,University of Agriculture, Faisalabad, 3University ofAgriculture, Faisalabad, Sub-Campus, Toba Tek Singh 4Department of Animal Breeding & Genetics, University of Agriculture, Faisalabad, 5FAST, Bahauddin Zakariya University, Multan.*[email protected]

Present study was conducted todetermine the distribution ofreproductive disorders in riverinebuffaloes. Data on calving disordersof buffaloes were collected from threeecological zones. The data were put todescriptive statistical analysis. Insouthern- irrigated zone, the highestnumber of buffaloes suffering fromcalving related reproductive disorderswere being maintained under ruralsubsistence small-holding followed bymarket-oriented small-holders andperi-urban commercial farms. Therewere significant differences among allproduction systems (P<0.05). Innorthern- irrigated zone the highestnumber of buffaloes who suffered fromcalving disorders was found in rural-subsistence smallholdings followed byperi-urban commercial farms, market-oriented small-holding and commercialfarms. However, number of buffaloeswhich suffered from such disorders wassignificantly higher (P<0.05) underruralsubsistence small-holding ascompared to marketoriented smallholdings and commercial farms butnonsignificantly higher than peri-



urban commercial farms. In arid-zone,significantly higher (P<0.05)percentage of buffaloes with calvingdisorders were found inruralsubsistence small-holdings ascompared to those in market-orientedsmall-holdings, peri-urban commercialfarms; the latter two did not differ(P>0.05). When pooled data wereanalyzed amongst with reference toproduction system, higher (P<0.05)percentage of buffaloes that sufferedfrom calving disorders was found inarid-zone followed by southern-irrigated zone and northern-irrigatedzone in rural-subsistencesmallholding; the difference amongthree agro-ecological zones wassignificant (P<0.05). In market-oriented smallholdings, highestproportion of affected buffaloes wasfound in southern-irrigated zonefollowed by northernirrigated zone andarid-zone; the difference among threezones was significant (P<0.05). Inperi-urban commercial farms, highestpercentage of affected buffaloes waspresent in northern-irrigated zonefollowed by southernirrigated zone andarid-zone; difference among threezones was again significant (P<0.05).As regards the commercial farms, nosignificant difference was found inthe three agro-ecological zones understudy.

Key words: Reproductive disorders,buffalo, ecological zones,household surveys

P-28 Distribution of Buffaloes Affected with PeriPartum Reproductive Disorders, with Reference to Feeding PatternM. A. Ali1, L. A. Lodhi2, M. Tariq3*, A.Waheed1, F. Hassan4 and S. Ahmad5

1Faculty of Veterinary Sciences, Bahauddin ZakariyaUniversity, Multan, 2Faculty of Veterinary Sciences,University of Agriculture, Faisalabad, 3University ofAgriculture, Faisalabad, Sub-Campus, Toba Tek Singh 4Department of Animal Breeding & Genetics, University of Agriculture, Faisalabad, 5FAST, Bahauddin ZakariyaUniversity, Multan*[email protected]

The present study was conducted toexplore calving related reproductivedisorders in Nili-Ravi buffaloes withreference to feeding regimes inPunjab, Pakistan. For data collection,the Punjab province was divided intothree ecological zones (southern,northern and arid). In the southernirrigated zone, the highest (P<0.05)number of buffaloes suffered fromcalving related reproductive disorderswas found in stall-fed buffaloescompared to open or grazing feedingpattern. In northern irrigated zonethe highest number of buffaloessuffered from calving disorders whichwere kept in open or grazing feedingpattern (P<0.05) as compared to stall-fed buffaloes. In arid zone, thehighest (P<0.05) number of buffaloessuffered from calving relatedreproductive disorders were those whowere stall-fed compared to open orgrazing feeding pattern. When the datain the three agro ecological zones ofPunjab were compared for stall fedbuffaloes, significantly lower(P<0.05) number of buffaloes withreproductive disorders was found inthe northern irrigated zone ascompared to the southern and the aridzone; the difference between thelatter two was however non-significant(P>0.05). The number of buffaloes whowere kept open or on grazing weresignificantly higher (P<0.05) in the



northern irrigated zone as compared tothe southern irrigated zone and thearid zone; the difference between thelatter two was however non-significant(P>0.05). Pooled data showed thatsignificantly higher number ofbuffaloes suffered from calvingdisorders was in stall fed feedingpattern as compared to those in openor on grazing (P<0.05).

Key words: Nili-Ravi Buffalo, Ecological zones, Calving

disorders, Feeding patternP-29 Environmental Factors Affecting someReproductive Traits in Holstein Friesian x Sahiwal Crossbred Cattlein PakistanT. Mahmood1, K. Javed2, A. Ali2*, N. Ahmad2, A. H. Saleem2, M. Z. Farooq2 andS. Ahmad3

1Buffaloes Research Institute, Pattoki, 2Department ofLivestock Production, University of Veterinary and AnimalScience, Lahore, 3Department of Animal Nutrition,University of Veterinary and Animal Science, Lahore *[email protected]

The objective of this study was toinvestigate the environmental effectson some reproductive traits inHolstein Friesian x Sahiwal crossbredcattle. Data on 2434 records of 533Holstein Friesian x Sahiwal crossbredcattle maintained at two differentLivestock Experiment Stations (LES,Rakh Dera Chahl and LES, RakhGhulaman) spanned over a period from1995 to 2011 were analyzed by MixedModel Least Squares Maximum Likelihood(LSMLMW) computer software. Theoverall least squares means for age atfirst calving, service period and

calving interval were 1404.39±68.85days, 215.96±20.73 days and 516.72 ±23.13 days, respectively. The year ofbirth, sire breed (Sahiwal, Friesian,Cross bred) and dam breed (Sahiwal,Cross bred) exerted significant effecton age at first calving (AFC). Theyear of calving, season of calving andlactation number significantlyaffected the service period incrossbred cattle. The calving intervalwas significantly affected by year ofcalving and lactation number .Theseason of calving, herd and level ofinheritance have nonsignificant effecton calving interval.

Key words: Environmental factors,Reproductive traits, Crossbred cows, Pakistan

P-30 Uterine Didelphys in Buffalo-ACase StudyU. Iqbal1*, M. Ahmad2, M.Nawaz3, A. Mehmood2 andF. Awan4

1Livestock and Dairy Development Department,Government of Punjab, 2Department of Theriogenology,University of Veterinary and Animal Sciences, Lahore –54000, 3Department of Theriogenology, PMAS AridAgriculture University, Rawalpindi – 46000, 4Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore *[email protected]

The basic aim of the study was todocument a case study of uterinedidelphys, a rare clinical conditionin Nili-Ravi buffalo. Approximately 54months old buffalo with the history ofrepeat breeding was brought to theSihala slaughter house, Islamabad.During ante-mortem examination, trans-



rectal palpation revealed theincreased cervical diameter withpoorly described external os. Uponpassing the AI rod, it was felt onlyin one side of narrow uterine body.After slaughtering, gross examinationrevealed the presence of two cervicalopenings. Upon dissection, it wasrevealed that the opening in separateuterine horn is due to presence ofband of tissue, although uterine hornconsistency was normal. Both ovarieswere cyclic. As far as biometricmeasurements are concerned cervix was7cm long with 7.5cm diameter. Whileovaries were 2.5 cm and 3 cm long with1cm diameter each. There was no commonuterine body to both horns. The lengthof right horn and left horn were 15 cmand 16.5cm respectively. This is aninteresting case which broadened thespectrum of causes of repeat breeding.

Key words: Uterine didelphys, Buffalo,Uterine horn,

Nili-Ravi, Case study

P-31 Constraints Limiting the Conception Rates in Cows Inseminated with Frozen SemenMd. R. Gofur*and A. M. Bhuyan

Department of Animal Husbandry and Veterinary Science,University of Rajshahi, Rajshahi-6205, Bangladesh *[email protected]

Herd-level management factors and cow-level factors have a lot of importancefor success of dairy farms. Economy ofdairy farming largely depends on ahigh pregnancy rate after artificialinsemination (AI) and high conceptionrate of dairy cows has a greatimportance to meet today’s expectationof dairy owners to obtain one calf per

cow per year. The study was undertakento determine the effects of breed,age, parity, feeding practice, bodycondition score (BCS), intervalbetween oestrus and AI, intervalbetween calving and AI, and milk yieldon first service conception rates inAI cows (n=308). The demographicfactors were recorded by interviewingthe farmers. All cows were examinedfor pregnancy diagnosis by per rectalpalpation of genital tract at 60-80days post-AI. The overall conceptionrate in cows was 52.6%. The conceptionrate was the highest (56.1%) in cowsof ˃3-5 years of age and the lowest(47.1%) in cows of 2-3 years of age.The highest (57.4%) conception ratewas in cows of parity 1-2 whereas thelowest (45.0%) was in cows of parity0. The conception rate in cows fedwith combination of green grass, strawand concentrate was significantly(P<0.05) higher (63.5%) than that withonly straw fed counterpart (38.5%).The conception rate in cows with BCS3-4 was significantly (P<0.05) higher(58.0%) than that of BCS 1.5-2counterpart (35.0%). The conceptionrate in cows received insemination at6-12 hours interval between oestrusand AI was significantly (P<0.05)higher (58.8%) than that of 13-24hours counterpart (40.4%). Themoderate yielding cows (>2-5 litresmilk) showed the highest (62.1%)conception rate. Feeding with greengrass, straw and concentrate forgaining good BCS and AI within 6-12hours of oestrus improve the firstservice conception rate in cows.

Key words: Conception, AI, BCS,Parity, Milk yield

P-32 Effect of GnRH Analogue Administration at the Time of A.I



on Conception Rate in Nili Ravi BuffaloesM. Binyameen1*, S. Anwar1, B. E. Azam1, M. Ahmad2,A. Rehman2 andA.U. Khan3 s

1Livestock and Dairy Development Department, Buffalo Research Institute Pattoki, 55300, Pakistan, 2Department of Theriogenology UVAS, Lahore, 5400,Pakistan,3Livestock and dairy development Department, DistrictBannu, 28100, Pakistan*[email protected]

Gonadotropin releasing hormone (GnRH)plays a major role in the growth offollicles and subsequent release ofpre-ovulatory LH surge. This study wascarried out to see the effect of GnRHanalogue administration at the time ofA.I on the conception rate in heifersexpressing first estrus and buffaloesat different postpartum estrus.Multiparous buffaloes expressing thefirst post-partum estrus were placedin group 1 (n=20). Similarlymultiparous buffaloes expressingestrus second (n=20), and third time(n=20) after previous A.I were placedin group 2 and group 3, respectively.Furthermore, 20 heifers expressingfirst estrus were placed in group 4.Each group was further divided intotwo sub-groups i.e. treatment andcontrol, with 10 animals in eachgroup. The buffaloes in each treatmentgroup received single injection ofGnRH analogue (Lecirelin, 50 mcg) atthe time of A.I, whereas, controlgroup did not receive any GnRHanalogue at the time of A.I. Pregnancytests were carried out on Day 45 afterA.I through transrectalultrasonography (Honda, Japan 7400,7.5 M Hz). All the data was analyzed

through SPSS version 13 and Chi squarewas applied to compare the results oftreatment and control groups.Differences in conception ratesbetween control and treatment sub-groups of groups 1 and 3 were non-significant (60% each). However, ingroup 2, conception rate was higher(P<0.05) in treatment sub-group (70%)as compared to control (60%).Similarly, in group 4 conception ratewas significantly higher (P<0.05) intreatment sub-group (60%) as comparedto control (40%). It can be concludedthat single injection of GnRH analogue(Lecirelin, 50 mcg) at the time of A.Iis beneficial in heifers expressingthe estrus first time and adultbuffaloes which are repeating thecycle 2nd time after previous A.I.Key words: Buffaloes, Estrus, Heifers,

GnRH, Conception.

P-33 Effect of Exogenous Growth Hormone onSemen Quality, Hematological Variables andSerum Testosterone Concentrations in Nili-Ravi Buffalo BullsA. Masoood1, I. Ahmad1,M. Ahmad1, N. Ahmad1*, Z. I. Qureshi1 and Z. Ahmad1

1Department of Theriogenology, University ofAgriculture, Faisalabad, 38040, Pakistan *[email protected]

In Pakistan, recombinant bovinesomatotropin (rbST) is used toincrease growth rate and milkproduction in beef and dairy animals,respectively. The aim of this studywas to examine possible effects ofrbST on semen quality, hematologicalvariables and serum testosteroneconcentrations in adult Nili-Ravi



buffalo bulls. Six adult Nili-Ravibuffalo bulls with clinically normalreproductive tract were divided intotwo equal groups. Bulls of thetreatment group were injected S/C withrbST (Boostin-S, LG Life Sciences,Korea) @ 500 mg fortnightly for 10weeks, while those of control groupreceived equal volume of normalsaline. Semen and blood collection wascarried out during 10-week treatmentperiod. Semen samples were collectedtwice per week and analyzed forphysical quality parameters. A totalof 120 semen samples, with 60 samplesfrom three bulls of each group (20samples per bull) were available.Blood samples were collectedfortnightly and analyzed for red bloodcell (RBC) count, hemoglobin (HB)concentration, packed cell volume(PCV), mean corpuscular volume (MCV),white blood cell (WBC) count,neutrophils, lymphocytes and serumtestosterone concentrations. Data wereanalyzed statistically, using ttest.Results indicated that rbST treatmentof Nili-Ravi buffalo bullssignificantly (P<0.05) increasedejaculatory volume (8.8±0.2 vs 7.7±0.2ml), sperm motility (72.6±0.4 vs67.4±0.7%), mass activity (3.35±0.07vs 2.52±0.08), sperm concentration(982.2±67.8 vs 731.9±50.5 million/ml),live sperm (80.1±0.3 vs 75.8±0.5%) andserum testosterone (4.02±0.21 vs2.37±0.07 ng/ml) compared to controlbulls. Among hematological variables,lymphocytes were increased (P<0.05),MCV, WBC count and neutrophilsdecreased (P<0.05), while Hb, RBCcount and PCV remained unchanged.Howver, values of these variables werewithin normal ranges for bulls. Bodyweight of these bulls could not berecorded. In conclusion, treatment of

Nili-Ravi buffalo bulls with rbSTimproved semen quality and increasedserum testosterone without adverseeffects on the general health ofbulls.Key words: Semen quality, Hematology,

Serumtestosterone, rbST, Nili-Ravibulls

P-34 Granulosa Cell Tumour of Ovaryin Buffalo-A Case StudyF. Awan 1*, M. Mubeen2, U. Iqbal3 and M.Ijaz2

1Department of Epidemiology and Public Health,University Of Veterinary and Animal Sciences, Lahore-54000, 2Departmentof Clinical Medicine and Surgery,University Of Veterinary and Animal Sciences, Lahore 54000, 3Livestock and Dairy Development Department, Government of Punjab. *[email protected]

The present study reports a rareclinical condition of sexcordoriginated granulosa cell tumor inNili Ravi buffalo. Approximately 7year old female buffalo with theanamnesis of repeat breeding andinfertility was brought to Lahore MeatProcessing Complex, Shahpur Kanjaranslaughter house, Lahore. Ante-mortemexamination through transrectalpalpation revealed the rounded, large,fluffy, firm mass having multiplecompartments with variable consistencyin the right ovary while the leftovary and the rest of the reproductivetract were normal on the basis of sizeand shape. This condition wassuspected to be a tumor or cyst.During necropsy, gross examinationrevealed that 400 g weighed rightovary (15x7x9 cm) had irregular



lobulations and bluish-red tinge withmany irregular, follicular-appearingcysts. After incision, the ovariansurface appeared to be red and hadfriable necrotic tissue with severalblood-clot having vascular spaces. Theadjacent outer cysts contained dark orred-brown serous fluid. The left ovary(1.2x0.2x0.4 cm) contained neitherfollicle nor corpus luteum.Histopathology of the tissue sectionsshowed the presence of amplenecrotized tissue . The cells wereslightly elongated with prominenthyperchromatic nuclei but withvariable size and shape. Multiplesections of the tissue had many cellsundergoing mitosis. . Cell cytoplasmvaried from scant to abundant. Basedon the histopathology and clinicalexamination, this condition wasconfirmed as granulosa cell tumour.The present study highlighteda rareclinical cause of anestrous andinfertility in Nili Ravi buffalo.

Key words: Granulosa cell, Sex-cordstromal tumour,

Nili-Ravi buffaloP-35 Comparative Evaluation of RoseBengalPlate Test and ELISA for the Diagnosis ofBrucellosis in Cattle at Private Dairy Farms of PunjabM. S. Rafiq1, M. Ijaz1*, A. Z. Durrani1,K. Mehmood2,A. J. Sabir1 and S. H. Farooqi1

1Department of Clinical Medicine and Surgery; University of Veterinary and Animal Sciences Lahore54000, Punjab, Pakistan, 2University College of Veterinary and AnimalSciences, Islamia University of Bahawalpur 63100, Punjab,Pakistan

*[email protected]

Brucellosis is mainly an importantdisease of cattle which ischaracterized by abortion andretention of placenta while in maleanimals it can lead to orchitis andinfection of accessory sex glands. Itis of major economic importance in thedeveloping countries that have not anational brucellosis eradicationprogram. Keeping in view theimportance of brucellosis andfollowing an outbreak in the area. Thepresent study was designed to find theseroprevalence and comparativeevaluation of Rose Bengal Plate Test(RBPT) and indirect Enzyme LinkedImmunosorbant Assay (ELISA) for thediagnosis of brucellosis in cattle.For this purpose 204 serum samples(male= 4; female= 200) were collectedfrom different private farms havingthis problem in their animals. TheRBPT test was performed by Bengal testkit (Synbiotics, USA) according tocompany’s instructionwhile an indirect ELISA kit (Svanova,Sweden) was used to identify Brucellaantibodies in the serum samples byfollowing the manufacturer’sinstruction. The data onseroprevalence was analyzedstatistically by Chi-square test usingStatistical Package for SocialSciences (SPSS). Overallseroprevalence of brucellosis incattle was 15.2% (p= 0.050).Antibodies detected by RBPT in maleand female animals were 0% (n=0) and2.5% (n=05), while antibodiesdetection by ELISA was 50% (n=02) and14.5% (n=29) respectively (p= 0.000).Study showed higher seroprevalence infemale animals detected by both RBPTand ELISA, whereas ELISA detected more



number of carrier animals as comparedto RBPT showing that ELISA isrelatively more efficient and can beused for mass screening of B. abortuscarrier animals. During the studynecessary control measures wereadopted because of the zoonoticimportance of this problem.

Key words: RBPT, ELISA,Seroprevalence, Brucellosis,

CattleP-36 A Case Report; Fetal MummificationAccompanied with Viable Kids at Full Term in Teddy x Beetal DoeA. Husnain*, A. Rehman and M. A. Sattar

Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, 54000, Pakistan *[email protected]

Fetal mummification has been reportedin several species, including thecattle, sheep, goat, horse, pig, dogand cat. Fetal mummificationaccompanied with viable kids at fullterm is not frequently happened evenin caprine, ovine and swine. A threeyear doe was presented atTheriogenology Clinic Ravi Campus-UVAS. She had delivered two live kids(one male and other female) lastnight. Owner brought her becausesomething was hanging from vulva andhe was confusing it with retainedfetal membrane/prolapse. The Doe waspurchased from flock three months ago.She has had two previous successfulparturitions. Feeding history andtemperature (102 F0) of the doe wasnormal. On physical examination itrevealed that hanging material isdried and dead fetus (Fig. 1 and 2)and some of fetal parts were presentin the vagina. The differential

diagnosis was made with abortion,stillbirth, maceration and hydropescondition. It is diagnosed asmummified fetus. Fetal mummificationoccurs when the fetus dies due togenetic defects, torsion orcompression of the umbilical cord,placental defects or infections duringthe second or third trimester ofgestation after the formation of theplacenta and substantial ossificationhas occurred but is retained in uterodue to a high blood level ofprogesterone.

Key words: Fetal Mummification, Viablekids, Teddy x

Beetal, Doe

P-37 Pinhole Technique – A Novel Way toCastrate Male AnimalsM. Fazili*

Teaching Veterinary Clinical Service Complex, Faculty of Veterinary Sciences and AH, Shere Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar, Kashmir, India. Pin code; 91-190006. *fazili_mr @yahoo.co.in

Castration is one of the mostfrequently performed surgicalprocedures in veterinary practice. Alltechniques of castration in animalshave several principles in common:adequate restraint, safety, goodanaesthesia, clean surgery, control ofhaemorrhage, and adequatepostoperative drainage. Traditionallythe ruminants are castrated by closed(Bloodless) techniques that includeuse of emasculatome (Burdizzo),elastic banding, elastrator, EZEbander or calicrate bander.Occasionally the open techniques likeshort scrotum method, Newberry knife



procedure and unilateral orchiectomy(for unilateral disorders) are alsopracticed.Pinhole castration analternative simple, cheap, quick,requiring no special instruments andfield applicable technique hasrecently been described. In alaterally recumbent animal, afterinfiltrating local anesthesia, in situspermatic cord ligation is achieved byrestraining the cord laterally withinthe scrotal sac and passing suturethrough a hypodermic needle insertedcaudal to cranial at the neck of thescrotum and adjacent the medial marginof the restrained spermatic cord. Theneedle is removed leaving the suturein place and the spermatic cordrepositioned medially. Then the needleis reinserted through the originalholes and the suture passed backthrough the needle, which iswithdrawn. The suture ends are tiedligating the spermatic cord, leavingthe knot subcutaneously. Instead ofthe hypodermic needle, traumaticsuture needle of a larger size may beused for passing the silk suture. Thetechnique has given encouragingresults in male animals of severalspecies including ruminants. Modes ofevaluation, various benefits and shortcomings of the procedure when comparedto the conventional techniques will bediscussed during the presentation.

Key words: Castration, Pinhole,Spermatic cord

P-38Interventions during and Post AI toImprove Conception Rate in Repeat Breeder Sahiwal CowsM. S. Akhtar1, I. Hussain1, M. H. Lashari2, L. A. Lodhi3, I. Ahmad3, S. Inayat4, S. A. Muhammad5 and

S. Ullah6

1Faculty of Veterinary Sciences, Bahauddin ZakariyaUniversity, Multan, Pakistan, 2Department of LifeSciences, Islamia University of Bahawalpur, Pakistan3Faculty of Veterinary Sciences, University ofAgriculture, Faisalabad, Pakistan, 4Department of Dairy Technology, University of Veterinary and AnimalSciences, Lahore, 5College of Veterinary and AnimalSciences, Jhang, Sub Campus UVAS, Pakistan6Veterinary Officer, Livestock and Dairy DevelopmentDepartment, Taunsa Sharif, Punjab *[email protected]

The objective of present study was toknow the effect of gonadotropinreleasing hormone (GnRH) andrecombinant bovine somatotropin (bST)on pregnancy rates in repeat breedercattle. It was hypothesized thatadministration of GnRH and bST atinsemination would enhance conceptusdevelopment and improve the fertilityin repeat breeder cows. Allexperimental cows were clinicallyhealthy and had at least threeunsuccessful services before enteringthe study. Selected repeat breedercows with lactation number 2-5, bodycondition score 3 (0-5), suckled bycalves were utilized.Estrus detectionwas performed twice daily (7:00 am and6:00 pm) by visual examination toobserve behavioural signs during theexperiment. At natural estrus, a totalof 196 lactating Sahiwal cows with ahistory of repeat breeding weredivided into Treatment (n = 105) andControl (n = 91) group. Treatmentgroup cows were given a singleintramuscular injection of GnRH(Dalmarelin i.e. Lecirelin, FATROPharmaceutical Veterinary Industry,Italy) at the dose rate of 100 µg just



before artificial insemination (day0). The animals in the treated groupwere also injected subcutaneously with500 mg of bovine somatotropin(Boostin-250 manufactured by LG LifeScience Ltd.) at the time of AI andagain 14 days later. Control groupreceived a placebo injection (2 mlSaline Solution) at AI and again 14days later. Frozen-thawed semen from asingle bull was used and all cows inestrus were inseminated by the sametechnician. To determine serumprogesterone concentrations, blood waswithdrawn from 20 cows in each grouptwice weekly from day 0 to 42. Theserum progesterone concentrations weremeasured by ELISA. The data forconception rate was analysed by leastsquare analysis of variance where asprogesterone concentrations werecompared by split plot design. Allexperimental cows were examined forpregnancy by palpation per rectum ofthe uterus and its contents fordetection of amniotic vesicle at 40day after artificial insemination, andpregnant cows were reexamined 3 weeklater at 60 ± 1 day after artificialinsemination. Pregnancy diagnosis wasperformed by single technician.Conception rates were significantlyhigher (P < 0.05) in cows treated withGnRH and bST (43%) in comparison tocontrol cows (27%) at 60 ± 1 day afterAI. The progesterone concentrationsdiffered nonsignificantly (P > 0.05)between pregnant cows of treated anduntreated group. The findings ofpresent study demonstrated that GnRHat the time of artificial inseminationwhereas bST at AI and 14 days laterimproved conception rates in repeatbreeder cattle.

Key words: Repeat breeder cow; GnRH;Somatotropin;

Pregnancy rates; Progesterone

P-39 Ultra-Rapid Freezing Protocol Improves the Post-Thaw Quality and In Vivo Fertility of Water Buffalo Bull SpermatozoaH. K. Rehman, H. Ahmed and S. M. H. Andrabi*

Animal Reproduction Laboratory, Animal SciencesInstitute, National Agricultural Research Centre,ParkRoad, Islamabad, Pakistan*[email protected]

Cryopreservation affects thestructures and functions ofspermatozoa and the major damageoccurs during the freezing process.Optimum freezing protocol can reducethe vulnerability of spermatozoa tocryoinjuries. Therefore, the aim ofthe present study was to compare theeffect of three freezing protocols onpost-thaw progressive motility(computer assisted sperm analyzer;CASA), velocity distribution (CASA),kinematics (CASA), viability(fluorescent probe-propidium iodide),mitochondrial transmembrane potential(fluorescent probe-5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹtetraethylbenzimidazolylcarbocyanine iodide, JC-1), DNA(acridine orange test) and acrosome(wet-mount) integrity, and in vivofertility (pregnancy rate) of waterbuffalo bull (Bubalus bubalis)spermatozoa. Semen was collected inartificial vagina (~ 42ºC) from threeadult Nili-Ravi buffalo bulls.Qualifying semen ejaculates fromindividual bulls were diluted (37ºC,Tris-citric acid extender), cooled(37ºC to 4ºC in 2 hr), equilibrated



(4ºC for 4 hr) and packed (4ºC) in0.54 ml straws. Freezing of straws wascarried out either by (i) Staticvapour protocol; nonprogrammable (5 cmabove liquid nitrogen for 10 min, ~-2ºC to -70ºC and plunging in liquidnitrogen), or (ii) Rapid vapourprotocol; programmable (4ºC to -15ºC @3ºC per min, -15ºC to -80ºC @ 10ºC permin, holding at -80ºC for 1 min andplunging in liquid nitrogen), or (iii)Ultra-rapid vapour protocol;programmable (holding at 4ºC for 2min, 4ºC to -20ºC @ 10ºC, -20ºC to -100ºC @ 30ºC per min, holding at 100ºCfor 1 min and plunging in liquidnitrogen). The whole experiment wasrepeated three times. Three hundredinseminations (100 per freezing-protocol and 50 per bull) were made inbuffaloes (2nd to 3rd lactation) onnatural heat under controlled fieldconditions. Two-way analysis ofvariance revealed that post-thawprogressive motility (%, 23.05±1.89 vs.16.48±1.70), rapid velocity(%, 30.66±1.96 vs. 22.45±2.14),curvilinear velocity (µm/s,109.26±2.79 vs. 96.10±4.43), DNA (%,96.33±1.21vs.91.66±1.87)and acrosome

(%,64.11±1.22 vs. 58.88±1.30) integrity,and percentages of viable sperm withincreased mitochondrial transmembranepotential (28.11±0.59 vs. 23.83±0.43)were higher (P<0.05) with programmableultra-rapid freezing than the othertwo protocols. The data on fertilitywere compared by Chi-squarestatistics. The in vivofertility rate ofbuffalo bull spermatozoa cryopreservedwith programmable ultra-rapid protocolwas higher (68.0 vs. 55.1%; P<0.05)compared to other methods. Inconclusion, ultra-rapid freezingprotocol improves the post-thaw

quality and in vivo fertility of waterbuffalo bull spermatozoa.

Key words: Freezing rates, CASA, Viability, Fertility,

Water buffalo spermP-40 Evaluation of Sperm Quality Parameters of Fresh and Frozen-Thawed Water Buffalo Semen in Relation to Field Fertility during Peak Breeding SeasonH. Ahmed12, S. M. H. Andrabi1*,H. K. Rehman1 andS. Jahan2

1Animal Reproduction Laboratory, Animal SciencesInstitute, National Agricultural Research Centre,ParkRoad, Islamabad, Pakistan, 2Reproductive PhysiologyLaboratory, Department of Animal Sciences, Quaid-i-Azam University,Islamabad, Pakistan *[email protected]

Standard sperm quality parametersviz., visual motility and morphologyhave shown different associations withfield fertility. Therefore, theobjective of the present study was todetermine if specific in vitro tests forsperm and acrosomal membranes could beused in predicting in vivo fertility offresh and frozen-thawed buffalo semenduring peak breeding season. Semen wascollected from five adult buffalobulls with artificial vagina (~ 42°C)during the months of October andNovember. Freshly ejaculated semenfrom each bull was split for dilutioneither in PBS-0.1% BSA for analysis orTris-citric acid extender forfreezing. Aliquots diluted in Tris-citric acid extender werecryopreserved by following thestandard protocol for buffalo



spermatozoa. Fresh and frozenthawedsamples were analyzed for visualmotility (%), morphology (%)structurally and biochemically intactplasmalemma (%, supra-vitalhypoosmotic swelling test) and livesperm with intact acrosome (%, dualstainingTrypan blue and Giemesastain). At least 100 inseminations perbull were made by using frozen-thawedsemen under optimum field conditions.In vivo fertility of individual bull wasdetermined through conception rate(%). Pearson’s correlations suggestedthat visual motility(%) and percentage of viable spermwith intact acrosome in fresh semenwere significantly associated withfield fertility (r=0.71, P<0.01 andr=0.62, P<0.01, respectively). Infrozen-thawed semen, visual motility(%), supravital plasma membraneintegrity (%) and percentage of spermwith intact acrosome weresignificantly correlated with fieldfertility (r=0.77, P<0.01; r=0.85,P<0.01 and r=0.91, P<0.01,respectively), whereas percentage ofnonviable sperm with damaged acrosomeand percentage of sperm tailabnormalities were negativelycorrelated with field fertility (r= -0.87, P<0.01 and r= -0.55, P<0.05,respectively). The prognostic value topredict the field fertility by theequation of visual motility,supravital plasma membrane integrityand sperm with intact acrosome infresh and frozen-thawed buffalo semenwas highest (R2 adjusted=55.8%, P=0.02and R2 adjusted=82.6%, P=0.000,respectively). The present studyconfirms a strong association betweenin vitro tests to evaluate sperm andacrosomal membranes with fieldfertility in water buffalo.

Furthermore, a multiple combinedanalysis of these parameters could bethe best strategy to obtain a betterprognostic value of the fieldfertility of water buffalo semenduring peak breeding season.

Key words: Buffalo Spermatozoa,Viability; Plasma membrane integrity,

Acrosome integrity, In vivo fertility

P-41 Duck Egg Yolk Plasma in Extender Improves the Freezability of Buffalo (Bubalus bubalis) Bull SpermatozoaF. Tehseen1, 2, S. M. H. Andrabi1*, H. Ahmed1, A. Riaz2 and N. Ahmad2

1Animal Reproduction Laboratory, Animal SciencesInstitute, National Agricultural Research Centre,Islamabad, Pakistan, 2Department of Theriogenology, University of Veterinary and AnimalSciences, Lahore, Pakistan.*[email protected]

The present study was designed tocompare the effect of differentconcentrations of duck egg yolk plasma(DEYP;10, 15 and 20 %) with control (wholechicken egg yolk, WCEY 20 % and wholeduck egg yolk, WDEY 20 %) added toTris-citric acid (TCA) based extenderon visual motility (VMOT %), acrosomeintegrity (NAR %), supravital plasmamembrane integrity (SV-PMI %), CASA(Computer assisted sperm analyzer)motility parameters (progressive, PROG%; rapid, RAP %; medium , MED %; slow,SLOW %) and kinematics (average pathvelocity, VAP μm/sec; straight linevelocity, VSL μm/sec; curvilinearvelocity, VCL μm/sec; amplitude of



lateral head displacement, ALH μm;beat crossfrequency, BCF Hz) of frozen-thawedbuffalo bull spermatozoa at time 1 (5min incubation at 37ºC) and 2 (45 minincubation at 37ºC). For extraction ofplasma, WDEY was diluted with 0.17 MNaCl solution (1:1) and stirred for upto 1 h at 4°C. After that the mixturewas ultra-centrifuged (10,000 g for 45min) twice at 4°C. The finalsupernatant obtained was termed asDEYP with an acceptable purity. Semenejaculates from three buffalo bullswere collected at weekly intervalswith artificial vagina (42ºC) for aperiod of 3 weeks (3 replicates).Cryopreservation was carried out asper standard protocol for buffalospermatozoa. Analysis of variancerevealed that at time 1, sperm VMOTwas significantly higher in extendercontaining 15 % DEYP than the controls(58.33±2.79 vs. 43.33±2.44). At time 2,sperm VMOT was significantly higher inextenders containing 15 and 20 % DEYPcompared with the controls (33.75±5.91vs.17.08±3.65). Acrosome integrity andSV-PMI of buffalo spermatozoa weresignificantly higher at time 1 and 2in extender containing 15% DEYP thanWCEY 20 %(control) (time 1 NAR, 62.67±2.17 vs.45.00±2.86; time 2 NAR, 43.50±3.36 vs.26.83±4.91; time 1 SV-PMI, 52.50±1.34vs. 38.50±3.18; time 2 SV-PMI,33.67±2.85 vs. 15.00±1.48). At time 1,mean percentages of PROG sperm wassignificantly higher in extenderscontaining 15 and 20 % DEYP than thecontrols (11.41±2.04 vs. 3.95±0.63). Attime 2, sperm PROG motility wassignificantly higher in extenderscontaining 15 % DEYP compared with thecontrols (4.50±1.83 vs.0.67±0.37). Mean

percentages of RAP sperm weresignificantly higher at time 1 inextender containing 15% DEYP thancontrols (25.17±4.34 vs. 7.29±1.19). Attime 2, mean percentages of RAP spermwere significantly higher in extenderscontaining 15 and 20 % DEYP thancontrols (6.73±1.89 vs. 1.08±0.56). Atpost thaw time 1 and 2, VAP and VCL ofbuffalo sperm were significantlyhigher in extender containing DEYP ascompared with controls (time 1 VAP,64.76±2.73 vs. 50.71±1.86; time 2 VAP,49.07±3.16 vs. 35.70±3.02; time 1 VCL,114.36±6.36 vs. 84.54±4.22; time 2 VCL,87.03±4.15 vs. 63.53±7.41). Beat crossfrequency (Hz) of buffalo sperm atboth time 1 and 2 was significantlyhigher in extender containing DEYP ascompared with control (WCEY 20 %)(time1,28.33±0.94 vs. 25.18±1.54; time 2,33.46±1.14 vs. 22.38±2.55). Inconclusion, WCEY or WDEY can bereplaced with 15 % DEYP in TCA basedextender for cryopreservation ofbuffalo bull spermatozoa.

Key words:Acrosome integrity, Egg yolkPlasma, Bull spermatozoa.

P-42 Effect of Various Concentrations ofCatalase in Extender on Cryopreservation of Buffalo Bull SpermatozoaL. Ali, A. U. Khan, andS. M. H. Andrabi*

Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan.*[email protected]

Catalase enzyme is usually distributedin mammalian seminal plasma, where it



decomposes hydrogen peroxide intowater and oxygen and enhances spermsurvivability. The objectives of thepresent study were to determine theeffect of catalase added at differentconcentrations (0, 100, 200 or 300IU/ml) in Tris citric acid (TCA) basedextender on visual motility,progressive motility (computerassisted sperm analyzer-CASA), motioncharacteristics (CASA), supra-vitalplasma membrane integrity (supra-vitalhypo-osmotic swelling test), acrosome(wet-mount) and DNA integrity(acridine orange assay) of buffalobull spermatozoa at post dilution (PD)and post thawing (PT) stages ofcryopreservation.Semen was collected from four adultNili-Ravi buffalo bulls withartificial vagina (420C). Qualifiedsemen samples from each bull weresubdivided into four aliquots fordilution with TCA extender containing0.0,100, 200 or 300 IU/ml catalase.Activity of catalase was 12660units/mg of dry weight. The experimentwas repeated three times and data wereanalyzed by using ANOVA (generallinear model). At PT, visual motility,CASA progressive motility, averagepath velocity and straight linevelocity were significantly higher (P< 0.05) for catalase fortifiedtreatment groups (61.25±2.62 %,21.05±2.23 %, 75.48±2.53 µm/s and62.01±2.55 µm/s, respectively) ascompared with control (46.25±0.72%,13.15±1.78 %, 62.72±4.80 µm/s and51.57±3.77 µm/s, respectively).Curvilinear velocity was significantlyhigher (P < 0.05) at PT with extendercontaining 300 IU/ml catalase ascompared with control (112.44±2.07 vs.93.75±6.67 µm/s). At PT stage of

cryopreservation, mean supra-vitalplasma membrane and acrosome integritywere significantly higher (P < 0.05)with extender containing 300 IU/mlcatalase as compared with control(34.25±6.55 vs. 20.59±5.54 % and68.84±3.50 vs. 49.96±2.56 %,respectively). Mean DNA integrity atPT was significantly higher forcatalase fortified treatment groups(97.18±0.11 %) as compared withcontrol (93.78±0.77 %). Therefore, itcan be concluded that addition ofcatalase particularly at aconcentration of 300IU/ml in TCA extender improved thepost-thaw motilities, motioncharacteristics, supra-vitalplasmalemma, acrosome and DNAintegrity of buffalo spermatozoa.

Key words: Buffalo sperm, Catalase,Cryopreservation,

CASA, DNA integrity

P-43 Effects of Asphaltum on Libido, SerumTestosterone, Hematology and Biochemical Metabolites in Lohi RamY. Saeed1*, M. Ahmad1 andT. G. Shafia2

1 Department of Theriogenology, 2Department ofPathology, Agriculture University, Faisalabad, Pakistan *[email protected]

Asphaltum, also known as Salajit, is apale-brown to blackish-brown exudationof variable consistency originatedfrom layers of mountains rocks ofHimalayan ranges. It is composed of220 mineral and metal substances usedin traditional Indian medical systems.This study was designed to investigatethe effects of asphaltum on libido,serum testosterone, hematology andsome biochemical metabolites in Lohi



rams in each group. Asphaltum wasgiven direct orally in capsule to therams of the groups A, B and C @ 0, 800and 1600 mg, respectively, daily for aperiod of seven weeks. Blood wascollected (once in a week) at thebeginning of the experiment and fromthe 5th week of administration ofasphaltum till the completion of thetrial and was analyzed for hematology,serum testosterone and biochemicalmetabolites. After seven weeks of thetreatment, the mean libido score,serum testosterone level, white bloodcells, mean corpuscular volume, meanplatelets volume, hematocrit,lymphocytes, granulocytes, minimuminhibitory dilution, albumin,globulin, total proteinsand totalcholesterol level were increasedsignificantly (P <0.001). Conversely,the mean values of red blood cells,red blood distribution width,platelets, hemoglobin, meancorpuscular hemoglobin concentration,lymphocytes percentage, granulocytespercentage and minimum inhibitorydilution percentage were nonsignificant. However, the mean valueof mean corpuscular hemoglobindecreased significantly. Conclusion:Asphaltum is an effective and potentlibido and serum testosterone enhancermedicine. It also alteredhematological parameter and serumbiochemical metabolites in rams.

Key words: Asphaltum, Salajit, Lohi

rams. For this purpose, 12 adultclinically normal and healthy LohiRams were selected and kept for aperiod of 9 weeks under samemanagmental conditions. These ramswere divided randomly into threegroups, A, B and C with four

P-44 Juvenile In-Vitro Embryo Transfer from 4To 8-Week-Old Kundi Buffalo CalvesM. A. Kakar1,*, S. Jamali2, M. K. Baloch1 and M. T.M. Kiani3

1Department of Livestock & Dairy Development,Department Balochistan Quetta 87300 Pakistan2SKB Women University Brewery Road Quetta, Pakistan3 CASVAB, University of Balochistan Quetta 87300 Pakistan *[email protected]

The development of in vitro maturationand fertilization procedures, togetherwith the ability to harvest relativelylarge numbers of oocytes, has made itpossible to produce transferable gradeembryos from young animals. Theobjectives of the present studies wereto develop a robust and reliableprotocol to improve the production oftransferable grade embryos from 4- to8-week-old Kundi buffalo calves. Thisstudy was conducted on the rationalethat gonadotropin treatment protocolsused in the past failed to maximiseoocyte quality. Consequently, threeexperiments were conducted in KundiBuffalo calves (4– 8 week old) toexamine: (1) the method of FSHadministration (single versus multipletreatments), (2) the time of oocytecollection (48, 60 or 72 h after thefirst FSH treatment), (3) progesteronetreatment and (4) eCG treatment andits timing on oocyte yield, cleavagerate and blastocyst formation rate.Ovaries were exposed by mid-ventrallaparotomy conducted under generalanesthesia and all the oocytes werecollected in a single session from 12female calves. No factor significantlyinfluenced oocyte yield, but allsignificantly influenced the rates of



cleavage and/or blastocyst formationin one or more experiments. Thepreferred protocol consisted of theadministration of 4 x 40 mg of FSH, noprogesterone treatment and with 500 IUof eCG given at the time of the lastFSH treatment. The optimal time ofoocyte collection was 48 h after thefirst FSH treatment. A laboratoryevaluation, involving calves, resultedin the production of 39.0–89transferable embryos per donor calf.These JIVET preliminary results inKundi buffalo calves are encouragingfor improvement of its reproductionand production in Pakistan. At currentlevels of performance, the JIVET wouldbe an attractive means of rapidlymultiplying elite animals, althoughlarge-scale application is likely todepend on the identification ofsuitable gene markers and their use ingenetic selection programs.

Key words: JIVET, Buffalo, Embryo,Oocyte, In vitro

maturation, In vitro fertilization

P-45 Replacement for Commercial Dairy Farm:Intensive Breeding Program for Pre-pubertal Buffalo HeifersA. Husnain* and N. Ahmad


Efficient reproductive management isthe key for profitable dairyenterprise. Replacement heifers arethe back bone of the any commercialdairying. The objective of the studywas to evaluate the effect ofexogenous progesterone (P4) exposureon induction of cyclicity and

pregnancy rate in pre-pubertal buffaloheifers. This study was conducted atcommercial dairy farm in DistrictKasur-Pakistan during the breedingseason (September to November).Buffalo heifers (n=29; 32 ± 1.2 mo;303 ± 8.3 kg; mean ± SEM) withoutluteal tissues on ovaries wereconsidered pre-pubertal. These weredivided randomly in two groups;control and treated. In control group(without P4 exposure, n=13); notreatment was given rather bullexposure. In treatment group (with P4exposure, n=16); heifers were treatedwith CIDR (1.38 g progesterone PfizerCo, USA) synchronization protocol andtimed insemination 48 and 60 h afterdevice removal. Bull was introducedfrom 15 to 30 days post insemination.1st pregnancy test was performed at 40days post AI with ultrasound. Theheifers found pregnant at scan wereseparated. Heifers without CL wereresynchronized with CIDR protocol asbefore and pregnancy determined at 40days post AI. Pregnancy in controlgroup was determined on 40 and 80 dayssimilar to treated heifers. Thepregnancy rate (total number ofpregnant heifers/treated) was comparedthrough Chisquare in SPSS software.The overall pregnant heifers at 80days were 63% (10/16) in treated and16% (2/13) in control groups. It isconcluded that the progesteroneexposure can effectively induce thecyclicity in prepubertal buffaloheifers and provide the morereplacement for dairy herds.

Key words: Progesterone, Breeding, Pre-pubertal,

Buffalo heifer



P-46 Effect of Clomiphene Citrate and HumanChorionic Gonadotropin (hCG) on Ovulation Induction in Pre- Pubertal Sahiwal HeifersS. A. A. Bukhari 1, S. Ali 1, I. Ahmad1,Z. I. Qureshi1,M. Zubair1*andU. Rehman 1

1Department of Theriogenology, University ofAgriculture, Faisalabad, Pakistan *[email protected]

The aim of this pilot study was toevaluate the ovulation inducing effectof clomiphene citrate along with hCGin prepubertal Sahiwal heifers.Clomiphene citrate (an antiestrogen)causes gonadotropin secretion frompituitary gland by removing thenegative feedback effect of estrogen.This causes more FSH secretion whichultimately stimulates folliculargrowth and upon hCG administration(luteinizing hormone activity)ovulation occurs. Twelve prepubertalSahiwal heifers were divided intoexperimental and control groups, withsix animals in each group. Thegrouping of animals was made on thebasis of plane ovary. The status ofovarian puberty was checked throughrectal palpation and history ofprevious estrus signs. Experimentalheifers were given clomiphene citrateat 300mg/heifer for 9 days orally,while heifers in control group weregiven placebo tablets. The dose ofclomiphene citrate was selected on thebasis of previous literature. On day10, hCG (IVF-C 5000 iu (hCG) LG LifeSciences, Korea) was given to allheifers of both groups at2500IU/heifer through intravenousroute. Two base line blood sampleswere collected before start of the

treatment at each rectal palpationexamination for selection of animals.Subsequently, during and aftertreatment three blood samples per week(on alternate days) for 4 weeks werecollected for measuring theprogesterone (P4) in serum. Theconcentration of progesterone wasmeasured though radioimmunology assay.For the comparison of variables,student t-test was applied. The P4concentrations in five heifers (83.3%)in the experimental group with (P4 > 1ng/ml) and one heifer (16.7%) in thecontrol group along with corpusleuteum on ovary showed ovulation. Oneheifer in control group ovulated thatmight be due to hCG. Mean progesteroneconcentration was significantly higher(P <0.05) in treated heifers thancontrol heifers. In conclusion,administration of clomiphene citrateand hCG can induce ovulation inductionin pre-pubertal Sahiwal heifers.

Key words: Clomiphene citrate,Ovulation induction,

Sahiwal heifers

P-47 Duration of CIDR Placement’s Effect onEstrus Induction in Dairy Holstein CowsC. Öztürk1*, Ş. Güngör1, M. N. Bucak1

and M. B. Ataman1

1Department of Reproduction and Artificial Insemination,Faculty of Veterinary Medicine, Selcuk University,Konya, Turkey*[email protected]

The aim of this study was to evaluatethe effect of duration of CIDR deviceplacement in vagina on estrus



induction, corpus luteum status,plasma progesterone levels, folliclediameter, and pregnancy rate (PR) inHolstein dairy cows. Cows (n = 66)were randomly assigned by age, calvingdate and body condition score. InGroup 1, CIDR was inserted on day 0and cows were assigned to CIDR (n =20) protocol starting with theinjection of GnRH (10 µg buserelin,i.m.; Oviren). On day 7, CIDR wasremoved and PGF2α (10 mg,i.m.;Dinolytic)was applied (CIDR7). InGroup 2, the same protocol was usedexcept for CIDR (n = 23) removal onday 9 (CIDR9). In Group 3, cows wereassigned to CIDR (n = 23) from day 0to 14, and PGF2α(10 mg) was applied on day 30(CIDR14). Artificial insemination wasperformed at predetermined fixed times(56 h, CIDR7 and CIDR9; 72 h, CIDR 14)and all cows were administered GnRH atFTAI. Ultrasonography (ibex pro, 7.5MHz linear probe) was performed forfollicular diameter during FTAI. CLstatus was also evaluated byultrasonography on day 0 and 7(CIDR7), day 0 and 9 (CIDR9), and day0 and 14 (CIDR14), respectively. Bloodsamples were collected via jugularvein on the day of CIDR removal. P4analysis was performed viaElectrochemiluminescence immunoassay(ECLIA) method and ROCHE E170 device.Pregnancy rate was determined twicevia transrectal ultrasonographybetween 40 and 60 days after FTAI.Follicle diameter was larger in CIDR7and CIDR14 compared to CIDR9 (P<0.05)(ANOVA, SPSS 21). There was no effectof treatments on the diameter ofcorpus luteum and progesteroneconcentration among groups (P>0.05)(ANOVA, SPSS 21). Pregnancy rates(70%, 52% and 39%) were positively

correlated with plasma progesteronelevels (4.10 ng/ml,4.83 ng/ml and2.74ng/ml) in CIDR7, CIDR9, CIDR14respectively (SPSS 21).In this study,results suggest that CIDR7 (PR 70%, P44.10 ng/ml)would be a more effectiveprotocol for fertility of Holsteindairy cows.

Key words: CIDR based, Estrus induction, Fixed-time

artificial insemination,Pregnancy rate

P-48 Impact of Intensity of Biostimulation onOestrus Incidence, Conception Rate inPostpartum Nili-Ravi Buffaloes during Peak Breeding SeasonN. Zaidi1* and M. Anwar2

1PARC Institute of Advanced Studies in Agriculture NARC,Park Road, Islamabad, 44000, Pakistan,2Animal SciencesInstitute, National Agricultural Research Centre, ParkRoad, Islamabad, 44000. Pakistan *[email protected]

Biostimulation (male effect) plays animportant role in improvingreproductive efficiency incattle.There is some evidence toindicate that duration of dailypheromonal stimuli influences the timerequired for cows to respond to thebiostimulatory effect of bulls.Thestudy was planned to assess the impactof intensity of biostimulation onresumption of ovarian cyclicity andfertility in postpartum suckledbuffalo kept under standard farmcondition. The buffaloes given either24 h physical exposure to mature



fertile bull (BE), or partial, fenceline exposure (BPFE) and no bullexposure (BN) for 60 days (n = 8 pergroup). Animals had calved(38.23±5.38d) before the experiment.Over 60 d study period, blood sampleswere collected once a week from 5animals per group for Progesterone(P4). Changes in plasmaP4concentrations were used ascriterion for resumption of ovarianactivity. An increase in P4 levels>1ng/ml in two consecutive samples wasindication of ovarian activity.Oestrus detection was done twice dailyin BE and BPFE by bull and manually inBN. Animals showing one or more behaviouraloestrus signs were considered in heat.. Theresults showed that a higher number ofanimals of BE showed one or morebehavioral oestrus signs than BN group(100% vs. 62.5%; p<0.05,respectively). Number of buffaloshowed standing heat in BE and BN werefound as 100.0 and 50.0%,respectively. However BPFE (75.0%)group was not different (p>0.05) fromBE or BN groups. Interval from thestart of the treatment to resumptionof ovarian activity was shorter(p<0.05) for BE (11±2.9d) and BPFE(20±2.6d) than for BN group (39±3.8d).Conception rate was higher (p<0.05) inBE (87.5%) than BN group (25 %) butnot difference (p>0.05) with BPFE(50%). It was concluded thatresumption of ovarian activity andconception rate in response tobiostimulatory effect of bull dependedupon exposure intensity in buffalo toa bull as these parameters wereimproved in animals having 24 hphysical exposure for 60 daysexperiment period.Key words: Buffalo,Biostimulation,

Progesterone, Fertility rate

P-49 Estrus Synchronization does not ImproveFertility in Chronically Repeat Breeding Sahiwal CowsM. Hassan, A. Razzaq, A. Husnain, A. A. Channa and N. Ahmad*


Repeat breeding can be caused by anumber of factors, including subfertile bulls, endocrine problems,malnutrition, sub-clinical infectionof reproductive tract and poormanagement. The objective of thepresent study was to determineefficacy of three major estrussynchronization protocols (CIDR,Ovsynch and Single PG) as reproductivemanagement tool in terms of estrusresponse, timing of ovulation,ovulation rate and pregnancy rate inchronically repeat breeding Sahiwalcows. Seventeen adult Sahiwal cowshaving 350-400 Kg body weight with BCS(3.5±0.0), parity ranges from 4 to 6and had earlier received on an averageeight inseminations but did notconceive, before the start ofsynchronization were selected. Cowswere randomly divided into one of thefollowing treatment groups: 1) CIDR(n=6), cows were inserted with anintra-vaginal device containing 1.38 gprogesterone for seven days andadministered PGF2α on day 6 and fixedtimeinsemination was performed at 48 and72 h of CIDR removal. 2) Ovsynch(n=7), cows received an injection ofGnRH on day 0, PGF2α on day 7, secondinjection ofGnRH on day 9 and timed AI at 12 and24 h after last injection and 3) PG



(n=4), cows received a singletreatment of PGF2α on random stage ofestrous cycle and inseminated at fixedtime (72 and 84 h after PGF2αadministration) with frozen thawedsemen from bulls of known fertility.Estrus detection was performed byvisual observation and teasing wasperformed twice daily for at least 30min. Size of the largest follicle wasmeasured from PG injection toovulation by repeated ultrasonographyafter 12 hourly intervals. Pregnancydiagnosis was conducted usingultrasonography around 35 days afterinsemination. Data for timing ofovulation and size of the ovulatoryfollicle was analyzed by using One wayANOVA and for estrus responseovulation and pregnancy rate wasanalyzed by using Z-test forpopulation proportion. Resultsrevealed that estrus response wassignificantly higher (P=0.046) (100%;6/6) in CIDR group as compared to PG(50%; 2/4), but not in Ovsynch (85%;6/7). Size of ovulatory folliclebefore ovulation was significantly (P< 0.05) different13.2±1.97, 13.6±0.17 and 9.4±0.00 mmamong all groups ie., CIDR, Ovsynchand PG, respectively. Mean ovulationtime with reference to prostaglandininjection was significantly higher(P=0.032) among CIDR (87±11.49 h) andOvsynch (66±12.00 h) than PG group(90±0.00 h). Ovulation rate did notdiffer and was 66 (4/6), 71 (5/7), 25%(1/4) in CIDR, Ovsynch and PG groupsrespectively. Pregnancy rate was 0(0/6), 20 (1/7) and 0% (0/4) in threegroups. It is concluded that estrussynchronization does not enhancepregnancy rate of chronically repeatbreeding Sahiwal cows.

Key words: Repeat breeding,Synchronization, Sahiwalcows, CIDR, Ovsynch,Prostaglandin

P-50 Predominance of Bovine Trichomoniasis in Multan District, Adjacent Areas, PakistanS. Murtaza1*, M. A. Raza2, M. S. Akhtar1, M. M. Ayaz1, M. A. A. Farooq1,M. Ali1andF. Ali3

1Faculty of Veterinary Sciences, Bahauddin ZakariyaUniversity Multan, 2Higher College of Technology,Al-ain United Arab Emarat (UAE), 3College ofVetreinaryScienses, Islamia University Bahawalpur *[email protected]

Economic impact of Trichomoniasis issevere as calf crop can be abridged upto 40-50% in bovines’ infection. It isthe most common venereal disease whichcauses early embryonic loss, abortion,pyometra and overall reducesreproductive efficiency. For thispurpose, a study was designed to ruleout the predominance of Trichomoniasisin cattle and buffaloes in MultanDistrict and adjacent areas.Initially, epidemiological Survey wasmade and samples (preputial flush n=50and vaginal secretions n=50) werecollected. Due to limited number ofInpouchTM TF kits (Bio Med USA), finallya total of eighty samples (preputialflush n= 40 and vaginal secretions n=40) were inoculated into InpouchTM TF(TF=Trichomonas fetus) mediavertically in wide mouth jar andincubated at 37oC for six days. Out ofeighty cultured samples, only fourshowed turbidity and observed undermicroscope at ×400 first and then at×1000 for the presence or absence offlagellated protozoan. Results



revealed that among 80 cultures, fourbecome turbid and confirmed positiveunder microscopic examination. Out ofeighty (cattle n=69 & buffaloes n=11),the entire four positive cultures wereof cattle. Based upon these results,it can be speculated that 5%populations of animals in the studyarea were positive. Alternatively, ithas need of further broad scaleinvestigation through culture as wellas more advance diagnostic techniqueslike Elisa and PCR.

Key words:Predominance,Bovines,Trichomoniasis,

Multan, PakistanP-51 Effects of Ebselen on Ram Sperm Parameters during Liquid StorageKenan Çoyan1*, Mustafa Numan Bucak2, Şükrü Güngör2, Nuri Başpinar3, Şükrü Dursun4and Pınarİli5

1 Department of Hıstology and Embryology, Faculty ofMedicine, Pamukkale University, Denizli, Turkey,2Department of Reproduction and Artificial Insemination,Faculty of Veterinary Medicine, Selcuk University,Konya, Turkey, 3Department of Biochemistry, FacultyofVeterinary Medicine, Selcuk University, Konya, Turkey, 4 Ministry of Food, Agriculture and Livestock,Bahri Dağdas International Agricultural Research Institute,Konya, Turkey, 5Denizli Health Services Vocational HighSchool, Pamukkale University, Denizli, Turkey *[email protected]

The aim of this study was toinvestigate the effects three levelsof ebselen, on ram sperm parametersduring liquid storage up to 72 h at 5

°C. Five Merino rams (2-3 years ofage) were housed at Selcuk UniversityFaculty of Veterinary MedicineResearch and Application Farm, Konya,and maintained under uniform feedingand housing conditions. A total numberof 30 ejaculates (10 ejaculates foreach ram) were collected from the ramswith the aid of an artificial vaginatwice a week, according to AI standardprocedures. The ejaculates containingspermatozoa with >80% forwardprogressive motility andconcentrations higher than 2x109

spermatozoa/ml were used in the study.Eight ejaculates for each ram wereincluded in the study. Ejaculates werepooled at 37°C and pooled ejaculatewas divided into four equal aliquotsand diluted (37°C) with the Tris baseextender, containing ebselen at threedoses (10, 20 and 40 µM) and noadditives (control), at a finalconcentration of approximately 2 x 108

sperm. Subjective sperm motility wasdetermined at phase-contrastmicroscope, viability (SYBR-14/PI),andmitochondrial activity (JC-1/PI)were determined at fluorescentmicroscopy and DNA integrity wasevaluated by COMET test after 0, 24,48 and 72 h of storage at 5°C.Thestudy was replicated eight times.Results were expressed as themean±SEM. Data for sperm motility,viability, mitochondrial activity andDNA integrity were analyzed byanalysis of variance, followed byTukey’s post-hoc test to determinesignificant differences betweengroups. Differences with values ofP<0.05 were considered to bestatistically significant. Statisticalanalyses were performed by using SPSS11.5 package program. The extendersupplemented with 10 µM ebselen



(93.39±1.80) resulted in higherviability, in comparison to thecontrol group (85.57±1.27) for 0 h ofstorage (P<0.05). Sperm motility wasbetter in 40 µM ebselen (82.66±1.20)than control (78.33±1.05) and also 10µM ebselen group (65.33±1.14) higherDNA integrity than control(58.50±3.10) group for 24 h of storage(P<0.05). Ebselen 10 µM group motility(73.33±1.05; 69.16±1.05) and DNAintegrity (58.00±0.44; 54.33±0.88) wasbetter than control group respectivelyat 48 and 72 hour of storage (P<0.05).This study highlights the protectiveeffect of ebselen on sperm motility,viability, and DNA integrity duringthe liquid storage of ram semen.

Key words: Ram sperm, Ebselen,Liquid storage,

Fluorescent staining, DNAintegrity



AUTHOR INDEXAbbas, S. 13,Abdullah, M. 23,25Adams, G. P. 1Afridi, S. 24Afridi, S. S. 16,Aftab, M. 23Ahamd, E. 8,Ahirwar, K. K.16,Ahmad, F. 25Ahmad, I. 31, 33.38Ahmad, M. 4, 10,,15, 18, 22, 25,27, 30, 31, 31,36Ahmad, N. 2, 6,13 15, 18,22, 23, 25, 25,30, 31, 37, 39Ahmad, S. 28, 29,29, 30Ahmad, T. 26,Ahmad, Z. 31Ahmed, H. 12, 34,35, 35Ahmed, M. 10 ,18, 25, 27Akhtar, M. S. 4,33, 40Akhtar, S. 21, 26Akhtar, T. 14,Akhter, S. 14Aksoy, M. 8,Aleem, M. 3, 23Ali, A. 23, 30Ali, F. 40Ali, I. 21Ali, L. 36Ali, M. 40Ali, M. A. 29, 29

Ali, S. 38Andrabi, S. M. H.5, 12, 19,20, 34, 35, 35,36Anwar, M. 39Anwar, S. 31Anzar, M. 4Arslan, M. 6Ashraf, Z.18, Aslam,A. 14,Ataman, M. B. 38Avais, M. 14, 22Awan, F. 30,32 Ayaz, M.M. 40Azam, A. 14,Azam, B. E. 31Baloch, M. K.37 Baruselli,P. S. 1Başpinar, N.12, 17, 40Bhatti, J. A. 25,Bhuyan, A. M. 30Binyameen, M.31 Brar, P. S.3, 16 Bucak,M. N. 12. 17,38, 40Bukhari, S. A. A.38Chandolia, R. K.27Channa, A. A. 18,25, 39Çoyan, K. 12, 17,40Devkota, B. 6Dhami, D. S.10 Durrani,

A. Z. 18, 32Dursun, Ş.12, 17, 40Farooq, M. A. A.40Farooq, M. Z.30 Farooq, U.24,Farooqi, S. H. 32Fayyaz, M. H. 27,Fazili, M. 33Gandotra, V. K.16,Ghufranullah 21Ghuman, S. S. 10,16Gofur, Md. R. 30Gohar, A. 16, 19,20, 20, 26Güngör, Ş. 12,17, 38, 40Hameed, A. 19,Haq, E. 14,Hashmi, A. S. 23,Hassan, F. 29, 29Hassan, M. 13, 39Hassan, U. 28,Husna, A. 14,Husnain, A. 13,24, 33, 37,39Hussain, I. 33Hussain, M. 19,Hussain, N. 15,

Hussain, T. 23Ihsanullah 24,Ijaz, A. 23,Ijaz, M. 10,14, 32, 32İli Pınar 40Inayat, S. 33Iqbal, S. 17, 25

Iqbal, U. 30, 32Iqbal, Z. 25,Islam, M. H. 11,21Jahan, S. 12, 35Jamali, S. 37Javed, K. 23, 25,30Kakar, M. A. 37Kausar, R. 19,Khan A. 16, 19,20, 20Khan, A. G. 26,Khan, A. U. 31,36Khan, H. 16, 19,20, 20, 24,26Khan, I. 21,24 Khan, I.K. 23,Khan, I. M.16,19, 20, 20Khan, J. A. 14Khan, M. I. 18,Khan, M. S. 14,25 Khan, R. U.16, 19, 20, 20.26Khanum, S. A. 19Kiani, M. T. M.37Kumar, K. 28,Lal, D. 28Lashari, M. H. 33Lodhi, L. A. 29,29, 33Mahmood, T. 30Mahsud, T. 24,Malik, A. R. 22,Malik, M. I. 7Masoood, A. 31

Absracts of International Symposium on Dairy Animal Reproduction (ISDAR), March 30-31, 2015, Lahore Pakistan


Mehmood, A. 30Mehmood, K. 32Mehmood, M. U. 3,10, 22Mirza, M. A. 18Mohan, C. 28,Mubeen, M. 32Mudgal, V. 28,Muhammad, J. 22Muhammad, S. A.33Munir, T. 23,Murtaza, S. 40Mushtaq, A. 15,Mustafa, B. 28,Naseer, Z. 8Naveed, M. I. 24,Nawaz, H. 28Nawaz, M. 30Öztürk, C. 38Pandey, A. K. 28,Pathak, D. 16,Qureshi, M. S. 7,16, 19, 10,20, 21, 24, 26Qureshi, N. A. 19

Qureshi, Z. I.31, 38Rabbani, I. 17,Rabbani, M. 22,Rafiq, M. S.32 Rahil, T.9Rahman, M. 21Rahman, M. M. 11Rashid, M. A. 7,Raza, M. A. 40Raza, S. 22Razzaq, A. 22, 39Rehman, A. 10,25. 31. 33Rehman, H. 16,19, 20, 20,22, 26Rehman, H. K. 34,35Rehman, S. 24,Rehman, U. 38Riaz, A. 8, 10,21, 24, 35Riaz, U. 24,Sabir, A. J. 32

Saddique, U. 16,19, 20, 20,26Saeed, Y. 36Saleem, A. H. 30Sarder, M. J. D.11, 21Sarkar, M. A. H.21Sarkar, S.K. 28,Sattar, A. 10,21, 25, 27Sattar, M. A. 33Saxena, N. 28,Shabbir, M. Z. 14Shafia, TG 36Shahab, M. 6Shahid, B. 18Shahzad, M. 10,25Shahzad, Q. 14,Sharma, M. L. 28,Sheikh, A. A. 22,Singh, G. 27Singh, J. 1Singh, J. K. 28,Singh, N. 16,

Singh, O. 16,Sultan, S.11 Sultan,S. 26Tahir, M. Z. 15Tarin, A. G. 22,Tariq, M. 17, 28,29, 29Tariq, M. M. 28Tehseen, F. 35Ullah, M. R. 15,Ullah, N. 9, 14Ullah, S. 33Waheed, A. 28,29, 29Warriach, H. M. 2Yaqub, W. 14,Younas, M. 16,19, 20, 20,26Younis, M. 22Yousaf, M. S. 17,Zaidi, N. 39Zakirullah 16,19, 20, 20Zaneb, H. 17Zubair, M. 38

SCIENTIFIC REVIEW COMMITTEEISDAR, 2015, Lahore Pakistan

Ahmad, E. Turkey

Ahmad, M. Pakistan

Ahmad, N. Pakistan

Ahmad, N. Pakistan

Andrabi, S.M.H. Pakis


tanBrar, P. IndiaDevkota, B. NepalGhuman, S. IndiaHusnain, A. Pakis

tanKhan, M.I.R. Pakis

tanRashid, A. Pakis

tanRiaz, H. Pakis

tanSattar, A. Pakis

tanTahir, M.Z. Pakis

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Recent Advances in Nutrition- Reproduction Interactions in Dairy Animals

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