Effect of different cryodiluents on Nguni bull semen viability and in vitro fertilizing capacity

[摘要] This study was aimed at evaluating the effects of different extenders andcryoprotectants on the quality of Nguni bull semen after cryopreservation, andto evaluate the performance of frozen-thawed Nguni bull semen in IVF. Thestudy was conducted at ARC-Animal Improvement Institute in Pretoria, inconjunction with the University of the Free State from April to October 2008.Three Nguni bulls (average age of 5 years) were used, and the semencollected from each bull, twice a week, using an electroejaculator. The semenquality parameters were evaluated prior and post freezing. The parametersevaluated included sperm motility rate and percentage live sperm, semen pHand semen concentration. The semen samples collected were divided intothree equal portions following every collection and allocated to three groups -based on the semen extender used. One portion was extended with egg yolkcitrate, 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, thesemen samples were incubated for a period of 9h. Evaluation of semenquality parameters was done at 3h intervals, within that incubation period of 9h. The egg yolk Tris extender exhibited a reduction in performance in terms ofthe sperm motility rate and the percentage live sperm, compared to the eggyolk citrate extender after 6 and 9h of incubation respectively. Thus the dilutedsemen was not further used in the second experiment. The semen samplesextended with the egg yolk citrate diluents were incubated at differenttemperature regimes (50C and 250C) for a period of 12h, to evaluate the effectof temperature on the sperm quality of the diluted semen.In the second trial, the semen sample that was diluted with egg yolk citratewas further divided into three portions - in order to add three differentcryoprotectants, namely glycerol, dimethyl sulfoxide and ethylene glycol. Thepercentage live sperm and the sperm motility rate of the semen samplefollowing addition of the cryoprotectants were also evaluated after 2h ofincubation but prior to freezing. The semen samples were then loaded into0.5ml semen straws, which were sealed with polyvinyl alcohol. The semenstraws were then placed in a programmable freezer for 40 minutes for semencooling from an initial temperature of 50C, to a temperature of -1300C. Afterfreezing the straws were removed from the programmable freezer and placedin liquid nitrogen vapour in a styro-foam box, for 5 minutes to cool the semenstraws from -1300C to -1960C, after which the straws were plunged directlyinto liquid nitrogen (-1960C) tank, for storage until thawing.A total of 1560 bovine oocytes were retrieved by aspiration from 127 ovariescollected from Strydfontein abattoir in Pretoria. The oocytes were thenmatured in vitro in bovine maturation media (consisting of TCM 199, FSH, LHand estradiol), for a period of 24h. After 24h of incubation, the matured bovineoocytes with expanded layers of cumulus cells were washed in a BO-IVFsolution and fertilized in vitro using frozen-thawed Nguni bull semen from thefirst trial, while the others were fertilized with fresh Nguni bull semen, used asa control.For IVF, mature oocytes were incubated with semen for 18h. Thereafter, thepresumptive zygotes were vortexed in TCM 199 for 90 seconds in order toremove the cumulus cells. After that, the presumptive zygotes from eachtreatment 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 wereallocated to SOF culture media containing BSA. The treatment group, that isthe frozen-thawed semen (n=559), 280 zygotes were allocated to the KSOMculture media, while 279 zygotes were allocated to SOF- BSA culture media.The presumptive zygotes were then allowed to develop (incubated) for 7 daysuntil reaching the blastocyst stage. On day 2 following IVC (onset of IVC =day 0), cleavage rate was evaluated, the presumptive zygotes at 2-4 cellstage and those at 8 cell stage were evaluated under a contrast microscopeand development recorded. Thereafter, on days 2 and 5 of culture the culturemedia were changed. KSOM-step 1 was changed to KSOM-step 2, whileSOF-BSA was changed SOF-FBS. On the 7th day the expanded blastocystswere 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 periodof 6h of incubation, no significant differences were recorded between thetreatment groups, in terms of the semen pH. The semen pH was found to beacidic, however it became neutral after 9h of incubation in the semen samplethat was diluted with egg yolk citrate extender and incubated at 50C. Thepercentage live sperm was similar for semen extended with egg yolk citrateand egg yolk Tris extenders incubated at 50C up to a period of 6h ofincubation. Thereafter the percentage live sperm decreased in the semensample 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 to3h of incubation at 50C. After 6 and 9h of incubation (50C), there was a drasticdecline in the sperm motility rate of the semen samples extended with an eggyolk Tris extender. The percentage live sperm and pH values differed significantly (P<0.05) following addition of a cryoprotectant. The semensample in which glycerol was used (75±5.3%) exhibited a significantly(P<0.05) higher sperm survival rate, compared to the semen sample in whichethylene glycol was used (55±8.5%). Semen sample in which glycerol wasused as a cryoprotectant (6.6±0.2) exhibited a significantly (P<0.05) higherpH, compared to the semen sample in which dimethyl sulfoxide was used as acryoprotectant (6.3±0.3).The semen samples diluted at 50C exhibited a significantly (P<0.05) highersperm concentration immediately following dilution, compared to samplesdiluted at 250C. The sperm motility rate immediately following dilution wassimilar 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 foundto be similar between the treatment groups, up to 9h of incubation. However,after 12h incubation the semen sample incubated at 50C exhibited asignificantly (P<0.05) higher percentage live sperm, compared to the sampleincubated at 250C (46±21% vs 35±31%). The interaction between incubationtemperature and the semen extender used did not affect all the measuredsperm quality parameters.In vitro fertilization of cow oocytes with the frozen-thawed bull semenexhibited a significantly (P<0.05) higher percentage of presumptive zygotes atthe 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 offrozen-thawed semen (14.2±11.9%). Culturing of the presumptive zygoteswith KSOM media (23.2±17.5%) exhibited a significantly (P<0.05) higherpercentage 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 dilutingNguni 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 detrimentaleffect on the percentage live sperm and the sperm motility rate. Nguni bullsemen can be best frozen using glycerol as a cryoprotectant. Frozen-thawedNguni bull semen can be successfully used in IVEP since it resulted in higherpercentage of the presumptive zygotes at the 2-4 cell stage and also attainedday 7 blastocysts. Frozen-thawed Nguni bull semen can also be usedsuccessfully within 60 minutes following thawing incubated at 50C.Nevertheless, fresh Nguni bull semen can still be used successfully for IVFpurposes since it resulted in a higher percentage of day 7 blastocysts,compared to frozen-thawed semen. KSOM medium proved to be a better IVCmedium for bovine semen than SOF medium in terms of the percentage ofday 7 blastocysts obtained.

[发布机构] University of the Free State