22/11/2006 Evaluation of the Quality of Deep-Frozen Semen and Selection of an Effective Method of Goat Sperm Preparation for In Vitro Fertilization By.

22/11/2006

Evaluation of the Quality of Deep-Frozen Semen and Selection of an Effective Method of Goat Sperm

Preparation for In Vitro Fertilization

By

W.P.D.K. FERNANDO

Introduction Objectives Semen Quality Analysis Selection of a Sperm Preparation method Results & Discussion Conclusion Acknowledgements End

CONTENT

INTRODUCTION

Production & Productivity of Local Goat

Demand for kids, meat & milk HIGH & ESCALATING

Supply ?

Reasons Lack of good quality breeding materials Selling of breedable or pregnant she goats for meat

Cont. INTRODUCTION

WHAT IS NEEDED Large amount of better breeding materials Sooner the possible At a lower cost*

Potentials Many sustainable farming systems AI & ET

Good but limited IN VITRO techniques have a higher potential

IVM IVF IVC

The success of the in vitro techniques depends on…Semen factors Semen quality (motility, abnormalities, live/dead ratio, etc.)

fresh or cryopreserved ? how long preserved ? semen of which breed ?

Sperm preparation why Sperm preparation ? which method ?

Classical sperm swim-up method Percoll gradient method Ficoll medium method Method of centrifugation on a discontinuous density gradient and

etc)

For Sri Lanka …

OBJECTIVES

To evaluate whether the quality of deep frozen semen is affected by the breed &/or preserved period

To select an effective method of sperm preparation for IVF in goat

To facilitate the implementation of IVF technology for breed improvement in local goat.

EX:1 EVALUATION OF SEMEN QUALITY 58 Cryopreserved semen straws of four cattle

Breeds Friesian Australian Milking Zeebu (FrAMZ) Jersey (Jr) Friesian (Fr) Friesian Sahiwal (FrSw) were selected

Classified into 5 age categories according to the semen straw preserved year > 20 years 19 – 15 14 – 10 9 – 5 <5

Quality Analysis 1

1.1 Motility Visual detection

1.2 Dead Sperm Percentage Eosin/Nigrosin staining method

1.3 Abnormalities William’s staining method

Ex1

EX:2 SELECTION OF A SPERM PREPARATION METHOD

Two sperm preparation methods were compared

Classical Sperm Swim-up Method A Simplified Sperm Swim-up Method

Ex2

Quality Analysis 22.1 Cleanliness of the semen

2.2 Motility Before & after preparation Visual Detection

2.3 Concentration Haemocytometer

Ex2

RESULTS & DISCUSSION

Means in a column with the same letter are not significantly different.Alpha = 0.05

Breed

Mean Motility

Age(yrs) Mean Motility

FrAMZ 24.50a >20 26.54a

Jy 39.06a 15-19 25.30a

Fr 41.88a 10-14 48.75a

FrSw 44.00a 5-9 40.89a

<5 45.25a

1.1 Sperm Motility

Table 1: Sperm Motility of different breeds and age categories

Ex1

Breed Mean

Death%

Age

(Yrs)

Mean

Death%

FrAMZ 65.63a >20 64.77a

Jy 54.56a 15-19 69.20a

Fr 56.12a 10-14 48.13a

FrSw 53.60a 5-9 56.16a

<5 46.38a

Table 2: Dead sperm percentages of different breeds and age categories

Means in a column with the same letter are not significantly different.Alpha = 0.05

1.2 Dead Sperm PercentageEx1

Correlation of sperm motility and Dead Sperm Percentage

A highly significant negative (Pearson) correlation (r = -0.882, P<0.01) between Sperm Motility and Dead Sperm Percentage.

Ex1

Table 3 - Sperm abnormalities according to breed:

Means in a column with the same letter are not significantly different.Alpha = 0.05

BreedAbnormalities

Head Mid Tail

FrAMZ 18.1a 12.1ab 17.9a

Jy 14.9a 8.4b 11.1b

Fr 18.3a 13.4a 10.4b

FrSw 17.5a 14.8a 8.9b

1.3 AbnormalitiesEx1

Table 4 - Sperm abnormalities according to storage time:

Means in a column with the same letter are not significantly different.Alpha = 0.05

Age Category

Abnormalities

Head Mid Tail

>20 16.8ab 9.5a 12.7a

15-19 17.2ab 9.4a 18.2b

10-14 13.6b 8.4a 8.9a

5-09 17.4ab 14.5b 11.2a

<5 20.8a 16.5b 11.1a

1.3 AbnormalitiesEx1

2.1 Cleanliness of prepared sperms Both methods resulted with slightly particulated

semen

Method

Swmup Sswmup

Initial motility 71a 72a

Final motility 90b 78a

Means with a same letter are notsignificantly different.Alpha = 0.05

2.2 Sperm Motility of both preparation methods

Ex2

Table 5 - Sperm concentration after sperm preparation

Method

Swmup Sswmup

Sperm Concentration

(x 106)4.5a 7.5a

swmup=swim-up methodsswmup=simplified swim-up methodMeans with the same letter are not significantly different.Alpha = 0.05

Ex2

CONCLUSION & SUGGESTIONS There is no effect of storage time and breed on motility

and dead sperm percentage but they affect on abnormalities. Other factors affecting motility & Dead Sperm Percentage need

to be evaluated

The simplified swim up method is as effective as the classical swim up method, but the former is more convenient and more economical. But this selection should be broadened to evaluate the

fertilization rate

ACKNOWLEDGEMENTS

Prof. D.P.S.T.G. Attanayake, Head, Dept. of Biotechnology, FAPMSL

is deeply acknowledged for his valuable encouragement & guidance.

Mr.D.V.S. de S. Gamage Head, Division of Animal Breeding,VRI Mr. A.S.B.Rathnayake Mr. P.A.B.S. Kumara,

Division of Animal Breeding, VRI are also deeply acknowledged for their valuable assistance during the research.

Mr.W.K.S. Amarasinghe and the staff Central Artificial Insemination Station (CAIS), Kundasale.

Dilip Fernando

IntroductionObjectives Ex 1Ex 2Results & DiscussionConclusion

Dilip Fernando

Abnormalities

Semen smear on glass slide Air dried and fixed in flame. Absolute alcohol for 4 min and air dried Washed d. water & with 96% ethanol Stained Carbolfuchsin-eosin solution (william’s

stain) for 10 min Washed with water Located on the stage of LMC

AI & ET vs IVF

we should not stay here we need develop more gene technologies spawning here and there thus we

thought of a far future IVF cant be used for gene manipulation which will be key

in future Immediately dead animals’ semen/oocytes can be used Even oocytes of new born she goats can be matured and

used in IVF