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Effect Of Cholesterol Loaded Cyclodextrin (Clc) Addition On Post Thaw Quality Of Jack Semen

By: Muhammad Rafi Ullah (2009-VA-54) | Dr. Mushtaq Ahmad.
Contributor(s): Dr. Muhammad Zahid Tahir | Dr. Muhammad Hassan Saleem.
Material type: materialTypeLabelBookPublisher: 2016Description: 45p.Subject(s): Department of TheriogenologyDDC classification: 2553-T Dissertation note: Improvement of post-thaw quality of Donkey (Equusasinus) semen is essential to augment the in-vivo and in-vitro fertilization rate. Whenever we think about the techniques for long term storage of the germ cells cryopreservation appears most useful but it causes lethal and sub-lethal damage to the sperm. Sperm cryosurvival rates are not optimal for most species including donkey because of its plasma membrane composition. Egg yolk is an important component of most of the equine freezing extenders. But another factor regarding this important component is that its higher concentrations can have some deleterious effects such as oxidase activity of dead spermatozoa, bacterial/ xenobiotic contamination, reduced respiration and motility of the spermatozoa, which ultimately results in lowering fertility rate. That’s why, cold shock resistance is more in those species semen possessing higher membrane cholesterol to phospholipids ratio as compare to those species semen having less cholesterol to phospholipids ratio. Indeed it may be a useful strategy to improve cryopreservation protocols for jack sperm. Addition of CLC may also be a substitute for egg yolk in semen extender always considering that the cryo-protective effect of EY is partially due to its high cholesterol content. Semen was collected in artificial vagina at 42ºC from two mature donkeys (n=2) five times (replicates=10) which were maintained at Ravi campus Pattoki. Semen samples possessing >60% motility were used in this study. Each ejaculate was divided into 6 aliquots and CLC was added into these aliquots according to desired concentrations (0 mg, 1.5 mg and 3 mg/120 million sperms) then kept at 25ºC for 15 minutes. After incubation semen aliquots were diluted in 1:1with centrifugation media and centrifuged at 600g for 15 minutes to separate seminal plasma after bead formation. Beads of the semen were resuspended in the centrifugation media having different concentrations of egg yolk for different CLC concentration groups in such a way that 100 million / ml sperms concentration was achieved. When this final dilution was done semen was shifted at 4 ºC for 2 hour CHAPTER 6 SUMMARY Summary 37 cooling then further 2 hours for equilibration at 4º C. Then semen was packed in 0.5 ml plastic straws. These straws were suspended on liquid nitrogen vapors upto7 mints then plunging these straws in the liquid nitrogen in freezing box and then shifting these straws to the liquid nitrogen container by placing them in the goblets and stored there until post thawing was done. After post thawing semen was analyzed for motility, Acrosomal and plasma membrane integrity, live ratios DNA integrity and MDA levels. Post-thaw parameters were analyzed by PROC MIXED as factorial ANOVA using SAS enterprise Guide Version 4.2. And it was observed that. And it was observed that both CLC 1.5 and 3 mg/120 million sperms with full egg yolk did not improved (p˃0.05) the post thaw quality except in malondialdehyde levels in which 3 mg dose with full yolk significantly (p˂0.05) decreased the level of malondialdehyde. While CLC with lower levels of egg yolk maintained the quality same as the control or improved significantly in some parameters.CLC1.5 HEY and CLC3 NEY both compete the control in motility, PMI by HOST and total viability parameter..DNA integrity increased with the decrement of egg yolk as CLC1.5 HEY, CLC3 HEY and CLC3 NEY are significantly (p˂0.05) better than the remaining three groups.MDA level is significantly lower in CLC1.5 HEY and CLC3 HEY groups comparable to control means that with partial removal of egg yolk and supplementation of CLC malondialdehyde levels are decreased significantly as compare to control. Thus CLC substitutes the egg yolk completely when 3mg of CLC /120 million sperms is added in kenney’s extender for jack semen cryopreservation.
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Thesis Thesis UVAS Library
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Veterinary Science 2553-T (Browse shelf) Available 2553-T
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Improvement of post-thaw quality of Donkey (Equusasinus) semen is essential to augment the in-vivo and in-vitro fertilization rate. Whenever we think about the techniques for long term storage of the germ cells cryopreservation appears most useful but it causes lethal and sub-lethal damage to the sperm. Sperm cryosurvival rates are not optimal for most species including donkey because of its plasma membrane composition. Egg yolk is an important component of most of the equine freezing extenders. But another factor regarding this important component is that its higher concentrations can have some deleterious effects such as oxidase activity of dead spermatozoa, bacterial/ xenobiotic contamination, reduced respiration and motility of the spermatozoa, which ultimately results in lowering fertility rate. That’s why, cold shock resistance is more in those species semen possessing higher membrane cholesterol to phospholipids ratio as compare to those species semen having less cholesterol to phospholipids ratio. Indeed it may be a useful strategy to improve cryopreservation protocols for jack sperm. Addition of CLC may also be a substitute for egg yolk in semen extender always considering that the cryo-protective effect of EY is partially due to its high cholesterol content. Semen was collected in artificial vagina at 42ºC from two mature donkeys (n=2) five times (replicates=10) which were maintained at Ravi campus Pattoki. Semen samples possessing >60% motility were used in this study. Each ejaculate was divided into 6 aliquots and CLC was added into these aliquots according to desired concentrations (0 mg, 1.5 mg and 3 mg/120 million sperms) then kept at 25ºC for 15 minutes. After incubation semen aliquots were diluted in 1:1with centrifugation media and centrifuged at 600g for 15 minutes to separate seminal plasma after bead formation. Beads of the semen were resuspended in the centrifugation media having different concentrations of egg yolk for different CLC concentration groups in such a way that 100 million / ml sperms concentration was achieved. When this final dilution was done semen was shifted at 4 ºC for 2 hour
CHAPTER 6
SUMMARY
Summary
37
cooling then further 2 hours for equilibration at 4º C. Then semen was packed in 0.5 ml plastic straws. These straws were suspended on liquid nitrogen vapors upto7 mints then plunging these straws in the liquid nitrogen in freezing box and then shifting these straws to the liquid nitrogen container by placing them in the goblets and stored there until post thawing was done. After post thawing semen was analyzed for motility, Acrosomal and plasma membrane integrity, live ratios DNA integrity and MDA levels. Post-thaw parameters were analyzed by PROC MIXED as factorial ANOVA using SAS enterprise Guide Version 4.2. And it was observed that. And it was observed that both CLC 1.5 and 3 mg/120 million sperms with full egg yolk did not improved (p˃0.05) the post thaw quality except in malondialdehyde levels in which 3 mg dose with full yolk significantly (p˂0.05) decreased the level of malondialdehyde. While CLC with lower levels of egg yolk maintained the quality same as the control or improved significantly in some parameters.CLC1.5 HEY and CLC3 NEY both compete the control in motility, PMI by HOST and total viability parameter..DNA integrity increased with the decrement of egg yolk as CLC1.5 HEY, CLC3 HEY and CLC3 NEY are significantly (p˂0.05) better than the remaining three groups.MDA level is significantly lower in CLC1.5 HEY and CLC3 HEY groups comparable to control means that with partial removal of egg yolk and supplementation of CLC malondialdehyde levels are decreased significantly as compare to control. Thus CLC substitutes the egg yolk completely when 3mg of CLC /120 million sperms is added in kenney’s extender for jack semen cryopreservation.

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