Effect of different cryodiluents on Nguni bull semen viability and in vitro fertilizing capacity
Mohapi, Maliengoane Rebecca
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This study was aimed at evaluating the effects of different extenders and cryoprotectants on the quality of Nguni bull semen after cryopreservation, and to evaluate the performance of frozen-thawed Nguni bull semen in IVF. The study was conducted at ARC-Animal Improvement Institute in Pretoria, in conjunction with the University of the Free State from April to October 2008. Three Nguni bulls (average age of 5 years) were used, and the semen collected from each bull, twice a week, using an electroejaculator. The semen quality parameters were evaluated prior and post freezing. The parameters evaluated included sperm motility rate and percentage live sperm, semen pH and semen concentration. The semen samples collected were divided into three equal portions following every collection and allocated to three groups - based on the semen extender used. One portion was extended with egg yolk citrate, the other extended with egg yolk Tris, while the other sample was left undiluted and served as a control. Following the addition of the extenders, the semen samples were incubated for a period of 9h. Evaluation of semen quality parameters was done at 3h intervals, within that incubation period of 9 h. The egg yolk Tris extender exhibited a reduction in performance in terms of the sperm motility rate and the percentage live sperm, compared to the egg yolk citrate extender after 6 and 9h of incubation respectively. Thus the diluted semen was not further used in the second experiment. The semen samples extended with the egg yolk citrate diluents were incubated at different temperature regimes (50C and 250C) for a period of 12h, to evaluate the effect of temperature on the sperm quality of the diluted semen. In the second trial, the semen sample that was diluted with egg yolk citrate was further divided into three portions - in order to add three different cryoprotectants, namely glycerol, dimethyl sulfoxide and ethylene glycol. The percentage live sperm and the sperm motility rate of the semen sample following addition of the cryoprotectants were also evaluated after 2h of incubation but prior to freezing. The semen samples were then loaded into 0.5ml semen straws, which were sealed with polyvinyl alcohol. The semen straws were then placed in a programmable freezer for 40 minutes for semen cooling from an initial temperature of 50C, to a temperature of -1300C. After freezing the straws were removed from the programmable freezer and placed in liquid nitrogen vapour in a styro-foam box, for 5 minutes to cool the semen straws from -1300C to -1960C, after which the straws were plunged directly into liquid nitrogen (-1960C) tank, for storage until thawing. A total of 1560 bovine oocytes were retrieved by aspiration from 127 ovaries collected from Strydfontein abattoir in Pretoria. The oocytes were then matured in vitro in bovine maturation media (consisting of TCM 199, FSH, LH and estradiol), for a period of 24h. After 24h of incubation, the matured bovine oocytes with expanded layers of cumulus cells were washed in a BO-IVF solution and fertilized in vitro using frozen-thawed Nguni bull semen from the first trial, while the others were fertilized with fresh Nguni bull semen, used as a control. For IVF, mature oocytes were incubated with semen for 18h. Thereafter, the presumptive zygotes were vortexed in TCM 199 for 90 seconds in order to remove the cumulus cells. After that, the presumptive zygotes from each treatment were randomly allocated into two different culture media namely, KSOM and SOF. The control group, that is the fresh semen group (n=481), 242 zygotes were allocated to KSOM culture media, while 239 zygotes were allocated to SOF culture media containing BSA. The treatment group, that is the frozen-thawed semen (n=559), 280 zygotes were allocated to the KSOM culture media, while 279 zygotes were allocated to SOF- BSA culture media. The presumptive zygotes were then allowed to develop (incubated) for 7 days until reaching the blastocyst stage. On day 2 following IVC (onset of IVC = day 0), cleavage rate was evaluated, the presumptive zygotes at 2-4 cell stage and those at 8 cell stage were evaluated under a contrast microscope and development recorded. Thereafter, on days 2 and 5 of culture the culture media were changed. KSOM-step 1 was changed to KSOM-step 2, while SOF-BSA was changed SOF-FBS. On the 7th day the expanded blastocysts were evaluated and recorded. Extended semen exhibited a significantly (P<0.05) lower sperm concentration, than undiluted semen. The semen pH values were significantly (P<0.05) different at 0 to 3h of incubation between the treatment groups. After a period of 6h of incubation, no significant differences were recorded between the treatment groups, in terms of the semen pH. The semen pH was found to be acidic, however it became neutral after 9h of incubation in the semen sample that was diluted with egg yolk citrate extender and incubated at 50C. The percentage live sperm was similar for semen extended with egg yolk citrate and egg yolk Tris extenders incubated at 50C up to a period of 6h of incubation. Thereafter the percentage live sperm decreased in the semen sample extended with egg yolk Tris diluents, after a period of 9h of incubation (50C). The sperm motility rate was similar between the treatment groups up to 3h of incubation at 50C. After 6 and 9h of incubation (50C), there was a drastic decline in the sperm motility rate of the semen samples extended with an egg yolk Tris extender. The percentage live sperm and pH values differed significantly (P<0.05) following addition of a cryoprotectant. The semen sample in which glycerol was used (75±5.3%) exhibited a significantly (P<0.05) higher sperm survival rate, compared to the semen sample in which ethylene glycol was used (55±8.5%). Semen sample in which glycerol was used as a cryoprotectant (6.6±0.2) exhibited a significantly (P<0.05) higher pH, compared to the semen sample in which dimethyl sulfoxide was used as a cryoprotectant (6.3±0.3). The semen samples diluted at 50C exhibited a significantly (P<0.05) higher sperm concentration immediately following dilution, compared to samples diluted at 250C. The sperm motility rate immediately following dilution was similar between the treatment groups. However, the sperm motility rates at 3, 6, 9 and 12h of incubation were significantly (P<0.05) different (67±10% vs. 55±24%; 60±12% vs. 47±25%; 47±20% vs. 38±25% and 40±20% vs. 28±27%) at 50C and 250C respectively. The percentage live sperm was found to be similar between the treatment groups, up to 9h of incubation. However, after 12h incubation the semen sample incubated at 50C exhibited a significantly (P<0.05) higher percentage live sperm, compared to the sample incubated at 250C (46±21% vs 35±31%). The interaction between incubation temperature and the semen extender used did not affect all the measured sperm quality parameters. In vitro fertilization of cow oocytes with the frozen-thawed bull semen exhibited a significantly (P<0.05) higher percentage of presumptive zygotes at the 2-4 cell stage, compared to the use of fresh semen (32.1±13.0% vs. 24.3±12.8%). IVF with fresh semen (23.2±16.5%) resulted in a significantly (P<0.05) higher percentage of day 7 blastocysts, compared to the use of frozen-thawed semen (14.2±11.9%). Culturing of the presumptive zygotes with KSOM media (23.2±17.5%) exhibited a significantly (P<0.05) higher percentage of day 7 blastocysts, than culturing with SOF media (14.2±10.4%) in vitro. In conclusion, egg yolk citrate proved to be the best extender for diluting Nguni bull semen. Fresh Nguni bull semen diluted with egg yolk citrate can probably be incubated up to a period of 9h at 50C, without any detrimental effect on the percentage live sperm and the sperm motility rate. Nguni bull semen can be best frozen using glycerol as a cryoprotectant. Frozen-thawed Nguni bull semen can be successfully used in IVEP since it resulted in higher percentage of the presumptive zygotes at the 2-4 cell stage and also attained day 7 blastocysts. Frozen-thawed Nguni bull semen can also be used successfully within 60 minutes following thawing incubated at 50C. Nevertheless, fresh Nguni bull semen can still be used successfully for IVF purposes since it resulted in a higher percentage of day 7 blastocysts, compared to frozen-thawed semen. KSOM medium proved to be a better IVC medium for bovine semen than SOF medium in terms of the percentage of day 7 blastocysts obtained.