151.
Effect Of Cholesterol Loaded Cyclodextrin (Clc) Addition On Post Thaw Quality Of Jack Semen
by Muhammad Rafi Ullah (2009-VA-54) | Dr. Mushtaq Ahmad | Dr. Muhammad Zahid Tahir | Dr. Muhammad Hassan Saleem.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation 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. Availability: Items available for loan: UVAS Library [Call number: 2553-T] (1).
152.
Effect Of Day Of Estrous Cycle On Superovulatory Response And Embryo Quality By Using Fsh In Balb/C Mice
by Umair Ashfaq (2009-VA-138) | Dr. Amjad Riaz | Dr. Ali Husnain | Dr. M. Hassan Mushtaq.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The inconsistent superovulatory response to gonadotropins is a challenge in biological experiments using mice as a research model. In mice, limited superovulatory response to gonadotropin may be related to the initiation of gonadotropin treatment as suggested in domestic animals. Therefore, the objective of the study was to evaluate the effect of stage of estrous cycle on the superovulatory response and embryo quality after treating with FSH or eCG in in BALB/c mice.
In experiment 1, relative ratios of vaginal epithelial cells and leukocytes were used to characterize the stages of estrous cycle in mice (n=10). In experiment 2, effect of stage of estrous cycle on no. of oocytes/cleaved embryos at 36 h post hCG, no. of cells in blastocysts at 84 h post hCG and no. of somites in embryos at dpc 9.5 were determined by treating mice (n=62) with eCG or FSH at each stage of estrous cycle. A total of twenty eight mice (n= 7 per stage) were treated with single dose of eCG (5 IU; IP). A total of thirty four mice (n=7-10 mice per stage) were treated with FSH (2.5 IU per does; SC) in four equal doses at every 12 h. All mice were given hCG (5IU, I.P) 48h after eCG or 12h after last injection of FSH.
Results revealed that the relative ratios of parabasal cells, cornfield cells and leukocytes were 84, 13, 3% in proestrus, 2, 95, 3% in estrus, 6, 37, 57% in metestrus, and 4, 5, 91% in diestrus, respectively. The mean interval between two consecutive estrus stages was 4.9 ± 0.3 days. Despite the inconsistent transitional pattern among the stages, the duration of proestrus, estrus, metestrus and diestrus was 24.0±2.7, 28.6±3.7, 17.3±2.9 and 52.6 ±3.7 h, respectively.
In FSH-treated mice, stage of estrous cycle did not affect the total no. of oocytes/embryos. However, in eCG-treated mice, proestrus and estrus had higher (P< 0.05) number of oocytes/embryos. Similarly, the number of cleaved embryos were not different (P> 0.05) among
Summary
47
stages in FSH-treated mice while eCG-treated mice had higher (P< 0.05) no. of cleaved embryos in proestrus. Differential staining 84 hours post hCG revealed that proestrus stage of eCG treatment has blastocysts of highest total number of cells, number of trophectoderm cells and number of inner cell mass cells. At 9.5 dpc there was no significant difference in number of somites in embryos of all groups.
In conclusion, superovulatoion by eCG at proestrus yielded higher number of good quality oocytes/embryos as compared to FSH. Availability: Items available for loan: UVAS Library [Call number: 2575-T] (1).
153.
Comparison Of Commercial Triladyl Extender With A Tris-Citric-Egg-Yolk (TCEY) Extender On Post-Thaw Semen Quality Of Nili Ravi Buffalo
by Muhammad Asad Ullah Khan | Prof. Dr. Mian Abdul Sattar | Prof. Dr. Nasim Ahmad | Prof. Dr. Mansur ud Din Ahmad.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Cryopreservation of semen is the most important step for its usage in artificial insemination. Freezing of semen leads to a remarkable reduction in post-thaw semen quality. Therefore, selection of a better semen extender has always been considered priority that could serve as a good cryoprotectant.. Our semen production units (SPUs) have been using Tris based egg yolk semen extender since long time. Some modern SPUs like CEBG are using commercially available semen extenders for better post-thaw semen quality.
After collection pooled semen divided into two equal aliquots in separate sterilized test tubes and kept in water bath at 37 ºC. Semen was diluted with each of extender (TCEY and Triladyl) on the basis of sperm concentration (40x106sperm/ ml). Diluted semen was placed bottles and placed in safety cabinet cooled to 4 ºC over and equilibrated for 4 hrs. After equilibration semen was filled in 0.5 ml French straws (20x106sperm/ 0.5 ml). All semen straws placed in automatic freezer 4cm above liquid nitrogen surface in vapors for 10 minutes. Liquid Nitrogen vapors used in automatic programmable freezer to reduce temperature from 4 ºC to -180 ºC and then plunged into liquid nitrogen -196 ºC for freezing and was stored until analyzed. The experiment was repeated for seven times (replicates = 07)
CASA sperm motility parameter and kinematics were analyzed at Center of Excellence for Bovine Genetics (CEBG) Renala khurd District Okara. For further analysis frozen semen straws were brought to the Department of Theriogenology UVAS, Lahore. Effects of Triladyl and TCEY on post-thaw semen quality of the Nili Ravi buffalo semen were compared.
Summary
54
In Triladyl group, significantly (P<0.05) higher post-thaw motility (PTM %), Plasma membrane integrity (PMI, %),) DNA integrity (%), Live percentage was found. However, no significant (P<0.05) difference was found regarding NAR results between both groups. Sperm abnormalities were found significantly lower in Triladyl group as compared to TCEY group.
In overall assessment regarding and post-thaw CASA motility parameters, CASA motility, (PROG %), rapid (RAP %), medium (MED%), and slow (Slow, %) and sperm motility kinematics (VAP μm/sec), (VSL μm/sec), (VCL μm/sec), (ALH μm), (BCF HZ), (STR%) and (LIN%) Triladyl was found better than TCEY.
This was concluded that use of commercial semen extender Triladyl resulted in significantly better post-thaw semen quality as compared to Tris citric egg yolk (TCEY) extender. Availability: Items available for loan: UVAS Library [Call number: 2581-T] (1).
