١ An attempt at alleviating heat stress infertility in male rabbits with some antioxidants El-Tohamy M.M., Kotp,M.S.Z., El-Nattat,W.S. Amira, H.M.&Soliman,S.I. Department of Animal Reproduction,National Research Centre,Cairo Faculty of Veterinary Medicine,Beni Suef University ABSTRACT The present experiment was conducted to study the effect of summer heat stress and the selective antioxidants on spermogram, serum and seminal plasma biochemical and endocrinal parameters, and oxidative/antioxidative status of NZW rabbits buck. Forty-eight sexually mature NZW male rabbits were randomly divided into six equal groups. The study was performed in two experimental periods (winter and summer), each period lasting 12 weeks. In winter, one group was kept as a winter control group and receives basal diet only. Other five groups reared in summer and serve as heat-stressed groups. Heat-stressed five groups were fed on basal diet and the first group kept as control positive and given distal water. The second group was given ascorbic acid 40 mg/kg BW/day. The third group was given zinc methionine 10 mg/kg BW/day. The forth group was given co enzyme Q 10 10 mg/kg bw/day. Finally, the fifth group was given l-carnitine 40 mg/kg/day. The climatic data have continuously recorded among the experimental period and the weekly averages of temperature–humidity index (THI) was calculated. Semen samples have collected weekly. Three ejaculates were collected for each buck. One served as row semen sample for spermogram. The other two consecutive ejaculates were pooled for seminal plasma separation, which stored at -80 °C ultra freezer, until further analysis. The season's averages of temperature–humidity indices (THI) were 18.52±0.22 in winter and 34.38±0.46 in summer, indicating absence of heat stress in winter and exposure to very severe heat stress in summer. Summer heat stress reversely affected both qualitative and quantitative traits of spermogram but bucks remain within fertile limit. Of antioxidants used in present study, zinc and l-carnitine were found to be the most beneficial antioxidants in the relief of spermogram heat stress-induced effects. In contrast to serum testosterone and cortisol, which showed no changes in summer, seminal plasma testosterone and cortisol showed significant increase in summer group compared to winter group. Only ascorbic acid restored seminal plasma cortisol to the winter level. In the present study, serum and seminal plasma oxidative/antioxidant status seem to take an identical (same, equivalent) pattern in response to heat stress. TBARS showed PDF created with pdfFactory Pro trial version www.pdffactory.com
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An attempt at alleviating heat stress infertility in male rabbits with some antioxidants
El-Tohamy M.M., Kotp,M.S.Z., El-Nattat,W.S. Amira, H.M.&Soliman,S.I. Department of Animal Reproduction,National Research Centre,Cairo Faculty of Veterinary Medicine,Beni Suef University ABSTRACT The present experiment was conducted to study the effect of summer heat stress and the selective antioxidants on spermogram, serum and seminal plasma biochemical and endocrinal parameters, and oxidative/antioxidative status of NZW rabbits buck.
Forty-eight sexually mature NZW male rabbits were randomly divided into six equal groups. The study was performed in two experimental periods (winter and summer), each period lasting 12 weeks. In winter, one group was kept as a winter control group and receives basal diet only. Other five groups reared in summer and serve as heat-stressed groups. Heat-stressed five groups were fed on basal diet and the first group kept as control positive and given distal water. The second group was given ascorbic acid 40 mg/kg BW/day. The third group was given zinc methionine 10 mg/kg BW/day. The forth group was given co enzyme Q10 10 mg/kg bw/day. Finally, the fifth group was given l-carnitine 40 mg/kg/day.
The climatic data have continuously recorded among the experimental period
and the weekly averages of temperature–humidity index (THI) was calculated. Semen samples have collected weekly. Three ejaculates were collected for
each buck. One served as row semen sample for spermogram. The other two consecutive ejaculates were pooled for seminal plasma separation, which stored at -80 °C ultra freezer, until further analysis.
The season's averages of temperature–humidity indices (THI) were
18.52±0.22 in winter and 34.38±0.46 in summer, indicating absence of heat stress in winter and exposure to very severe heat stress in summer.
Summer heat stress reversely affected both qualitative and quantitative traits
of spermogram but bucks remain within fertile limit. Of antioxidants used in present study, zinc and l-carnitine were found to be the most beneficial antioxidants in the relief of spermogram heat stress-induced effects.
In contrast to serum testosterone and cortisol, which showed no changes in
summer, seminal plasma testosterone and cortisol showed significant increase in summer group compared to winter group. Only ascorbic acid restored seminal plasma cortisol to the winter level.
In the present study, serum and seminal plasma oxidative/antioxidant status seem to
take an identical (same, equivalent) pattern in response to heat stress. TBARS showed
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Total sperm output (106/ejaculate) TSO calculated by multiplying semen ejaculate
volume by semen concentration.
Packed sperm volume (%) PSV has recorded using capillary tubes and
microhematocrit centrifuge adjusted at 12000 rpm for 15 min and read by
microhematocrit reader. Semen sample must gently shaken before tube filling.-2.
Qualitative Parameters
Mass motility MM were estimated immediately after semen collection, by visual
examination under low-power magnification (100x) using a warm plate light
microscope with heated stage and take a grade ranged from 0 to 9 according to
Petitjean (1965) notation scale showed in table (2).
Table (2): Notation scale used to measure mass motility Grade Note Description
0 No spermatozoa or motionless spermatozoa (spz) 1 Few stirring spz without notable movement 2 Important number of stirring spz without any movement 3 Few spz moving slowly 4 Few motionless spz, few stirring spz without any movement, few
mobile spz 5 Same as 4, but higher proportion of mobile spz. Rather high, but not
homogeneous motility in the observed field 6 Nearly all spz moving. High and homogeneous motility 7 Same as 6, wavelike movements begin 8 Same as 7, with slow wave movements 9 Strong waves; whirlwind appearance
Individual progressive motility (%) IM assessed within 5 min after collection using a
37 ºC warm plate light microscope at 200x and 400x magnification. Semen sample
was diluted at a rate of 1:4 with a commercial rabbit semen extender GALAB (IMV,
France) and the result was expressed as the percentage of spermatozoa (nearest to 5
%) exhibiting progressive rectilinear movement.
Sperm motility index SMI calculated by multiplying motility grade by individual
motility.
Total motile sperm (106/ejaculate) TMS calculated by multiplying percentage of
motile sperm and total sperm output.
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The enzyme activity in the antibody-bound fraction is inversely proportional
to the serum native antigen concentration. By utilizing several different serum
references of known antigen concentration, a dose response curve can generated, from
which the antigen (Cortisol) concentration of unknown can be ascertained Burtis and
Ashweed, 1994 ).
IV. Statistical analysis
All data were subjected to statistical analysis including the calculation of the mean (M), standard error of mean (SE) and F-test (one way ANOVA) at a confidence limit of 95% (P<0.05) according to the method of Armitage (1971) using practicing statistical analysis program (SPSS, Edition 11). Duncan’s multiple range test was used for testing pairs for Comparisons among means at probability 5%.
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There was non significant decrease in individual motility percentage noticed in summer control
bucks compared to winter group. Ascorbic and co enzyme Q10 supplementations significantly decreased
IM, while L-carnitine supplemented group showed non significant increase in individual motility
percentage, in comparison with summer control group.
Motility index:
Motility index values were recorded to be decreased significantly in summer heat stressed bucks
compared to winter ones. Among different antioxidants supplementations, zinc and l-carnitine
supplemented groups restored motility index to winter group values.
Alive spermatozoa:
Summer control bucks noticed to significantly decreased life sperm percentage than winter
group. Ascorbic supplemented bucks showed non significant increase in life sperm percentage, while
zinc and l-carnitine supplemented groups restored this parameter to winter group values.
Spermatozoa abnormalities:
There was non significant increase in sperm abnormalities percentage in summer control group
compared to winter group. All antioxidant supplemented groups showed non significant decrease in
sperm abnormalities except co enzyme Q10 supplemented bucks, which showed non significant increase
in sperm abnormalities in comparison with summer control group percentage. Spermatozoa concentration:
There was no difference in sperm concentration between summer and winter control groups.
While only l-carnitine supplemented bucks showed significant increase in sperm concentration
compared to summer control group.
Packed sperm volume:
No significant difference in packed semen volume was noticed in all experimental groups.
Total sperm output:
There was marked significant decrease in TSO in summer heat stressed bucks compared to bucks
reared in winter. Ascorbic and zinc supplemented bucks showed significant increase in TSO in
comparison with summer control group. Total motile sperm:
Notable significant decrease in TMS was recorded in summer control group compared to winter group. Zinc supplemented bucks significantly increased TMS, while ascorbic and l-carnitine supplemented Bucks showed non significant increase in TMS, in comparison with summer control group. Total functional sperm fraction:
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Summer control group showed significant decrease in TFSF compared to winter group.
However, zinc, ascorbic and l-carnitine supplemented groups showed increase in TFSF in comparison
with summer control group, which was significant in zinc supplemented group. Table (4): Overall means (±SE) of reproductive efficiency (sexual activity and spermogram) of rabbit bucks as affected by different
seasons and antioxidant supplementations (N = 84).
Means within the same row followed by the different superscripts are significantly different at p ≤ 0.05. NS = non significant TSO = Total sperm output TMS = Total motile sperm TFSF = Total functional sperm fraction SE = Standard error
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Overall means of serum and seminal plasma malondialdehyde levels, total antioxidant capacity
and catalase activities in winter and summer (control and antioxidants supplemented) groups were
shown in table (6)
Serum Oxidative and Antioxidant Status
Lipid peroxides:
There was a significant increase in serum TBARS levels in summer control group compared to
winter group. All antioxidants supplemented bucks showed no change in serum TBARS levels in
comparison with summer heat stressed bucks.
Total antioxidant capacity:
Summer heat stressed bucks significantly decreased serum TAC level than winter ones. As in
serum MDA, all antioxidants supplemented groups showed no change in serum TAC levels in
comparison with summer control group.
Catalase:
There was a significant decrease in serum catalase activity in summer heat stressed bucks
compared to winter group. Ascorbic, co enzyme Q10 and l-carnitine supplemented bucks showed
significant increase in serum catalase activities, while zinc supplemented bucks showed non significant
increase in serum catalase activity, in comparison with summer control bucks. Table (6) Overall means (±SE) of serum and seminal plasma oxidative and antioxidant status of rabbit bucks as affected by different
seasons and antioxidant supplementations (N = 36). Seasons Winter Summer
Groups Parameters Control Control Ascorbic Zinc
Co enzyme Q10
L-carnitine
P ≤
TBARS (nmol/ml)
2.14a ±0.09
3.68b ±0.10
3.48b ±0.18
3.54b ±0.12
3.57b ±0.13
3.47b ±0.13 0.000
TAC (mmol/L)
1.23b ±0.10
0.68a ±0.02
0.76a ±0.06
0.73a ±0.03
0.82a ±0.05
0.80a ±0.05 0.000 Serum
Catalase (U/ml)
19.36d ±0.75
13.76a ±0.23
15.14bc ±0.39
14.33ab ±0.36
15.90c ±0.33
15.61bc ±0.46 0.000
TBARS (nmol/ml)
1.21a ±0.09
1.93d ±0.04
1.62bc ±0.09
1.82cd ±0.08
1.64bc ±0.07
1.58b ±0.05 0.000
TAC (mmol/L)
1.71c ±0.05
1.12ab ±0.03
1.07a ±0.04
1.07a ±0.07
1.26b ±0.09
1.24b ±0.04 0.000 Seminal
plasma
Catalase (U/ml)
24.84c ±0.66
13.55a ±0.27
15.11b ±0.62
15.03b ±0.39
16.26b ±0.59
16.09b ±0.49 0.000
Means within the same row followed by the different superscripts are significantly different at p ≤ 0.05.
TBARS = Thiobarbituric acid-reactive substances TAC = Total antioxidant capacity
SE = Standard error
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compared to winter bucks. Ascorbic and zinc supplemented bucks significantly decreased seminal
plasma cortisol levels. On contrary, l-carnitine supplemented group showed non significant decrease in
cortisol levels, in comparison with summer control group. Table (7) Overall means (±SE) of serum and seminal plasma testosterone and cortisol of rabbit bucks as affected by
different seasons and antioxidant supplementations (N = 36).
Season Winter Summer
Groups Parameters Control contro
l Ascorbi
c Zinc Co
enzyme Q10
L-carnitin
e
P ≤
Testosterone
(ng/ml) 3.03a ±0.16
3.01a ±0.16
2.86a ±0.18
2.93a ±0.18
2.49a ±0.14
2.73a ±0.18 NS
Serum
Cortisol (µg/dl)
3.84a ±0.24
3.21a ±0.25
3.86a ±0.11
3.80a ±0.21
4.65b ±0.38
3.79a ±0.12
0.004
Testosterone
(ng/ml) 1.87a ±0.21
3.18b ±0.33
4.98c ±0.18
4.34c ±0.37
2.87b ±0.42
2.99b ±0.23
0.000
Seminal
Plasma Cortisol (µg/dl)
1.08a ±0.06
2.45d ±0.20
1.46ab ±0.16
1.70bc
±0.07
2.23d ±0.16
2.06cd ±0.11
0.000
Means within the same row followed by the different superscripts are significantly different at p ≤ 0.05. NS = non significant
SE = Standard error
In present study, the results obtained in the chemistry of seminal plasma components were in
harmony with those of spermogram characteristics. The changes in seminal traits due to variations in
climate may be due to differences in food intake and other factors such as fertility, number of ejaculates
and sexual desire.
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compared to winter bucks. This finding is in contrast to the result reported by Marai et al. (2002).
Ascorbic and zinc supplementations significantly decreased seminal plasma cortisol levels, while l-
carnitine supplemented group showed non significant decrease in cortisol level in comparison with
summer control group.
Conclusion In regard to summer heat stress consequence (effect), the present study shows that NZW rabbits
buck have continuous and acceptable spermatogenic activity and apparently normal body physiology
during summer as well as during winter. Contrary to our expectation, the examined semen traits of NZW
rabbits buck are not much inferior in summer compared to that obtained in winter. However, apparently
(relatively) seasonal variations in semen characteristics are detected.
To sum up, it should be said that (separate) protective administrations of zinc and l-carnitine
cause significant improvement in rabbit sperm characteristics, meanwhile, ascorbic acid and co enzyme
Q10 administrations have no considerable enhancement in such concern.
Taking the current results into consideration, it can be concluded that an adequate reproductive
performance in NZW rabbits buck can be achieved in summer and from male side of breeding, it is not
obligatory to stop breeding in summer months, but such some extraordinary (protecting) managements
should be attained.
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