Evaluation of cryopreserved ram semen following fertilization in vitro

[摘要] Three experiments were done at the University of Free State in Bloemfontain in 2014 and in2015. Experiment 1 was carried to evaluate the effect of breed (Dorper and SAMM) on freshram semen parameters before cryopreservation. Experiment 2 was carried to evaluate theeffects of breed difference, different extenders and different freezing protocols on the viabilityof frozen ram semen during different incubation periods after thawing. Experiment 3 wascarried to evaluate effects of breed, extender and freezing protocol on in vitro fertilizing capacity of frozen-thawed ram semen. Experiments 1 and 2 were done in 2014 during thebreeding season while experiment 2 was done in 2015 from mid of January until the end ofApril.For the first trial, the semen was collected with the aid of artificial vagina from two breeds oframs. The ejaculates were evaluated for the following parameters: semen volume, semencolour, sperm motility, sperm concentration, percentage live/dead sperm and spermmorphology. The parameters were then compared against the breeds to assess the breed effect.The semen volume and sperm motility showed a significant difference between the two breeds(P<0.05) in that South African Mutton Merino (SAMM) semen recorded better motility andhigher semen volume than Dorper semen for the entire experiment. There was no significantdifference between breeds for semen colour, sperm concentration, percentage live and deadsperm and sperm morphology.For the second trial, the study to evaluate the effect of different breeds, extenders and freezingprotocols on the sperm motility of ram semen including survival at different incubation periodsafter thawing was conducted during the natural breeding season. Semen was collected fromtwo breeds of rams (South African Marten Merino (SAMM) and Dorper). After fresh semenevaluation and selection of good quality ejaculates, semen was pooled for each breed anddivided into two then randomly allocated to Tris-egg yolk and Na-Citrate egg yolk basedextenders. Semen samples were further divided into two more groups and allocated randomlyto two different freezing protocols (freezing up to -20°C using the programmable freezer andthen by liquid nitrogen vapour prior to storage vs freezing to -80°C prior to storage).Microscopic evaluation of the semen for sperm motility was performed 24 hrs followingfreezing. The semen frozen by the use of liquid nitrogen vapour prior to storage recorded bettersperm motility than semen frozen to -80°C prior to storage. The viability of the frozen ramsemen was inversely proportional with incubation time increase after thawing. Only thefreezing protocols and incubation periods exhibited a significant difference in sperm motilitythroughout the experiment. Statistically, the difference breeds and the different semenextenders recorded no significant difference. However, when Tukey's grouping was used(studentised range), the Tris-egg yolk based extender showed better sperm motility comparedto the Na-Citrate egg yolk based extender. The results suggest that the breed does not haveimpact on semen freezing and the Tris-egg yolk based extender is better suited as a diluent forram semen cryopreservation. Results also indicate that freezing with the aid of liquid nitrogen vapour prior to storage will lead to a better fertility if insemination is done immediately afterthawing or alternatively the laparoscopic method will remain an alternative.In the last experiment, ovaries were collected during spring and at the beginning of summer atthe Bloemfontein abattoir from unknown, untreated sheep and transported to the laboratory insterile saline water (37°C) in the flask. Cumulus oocytes complexes (COC's) were recoveredfrom the ovarian follicles by aspiration method, washed and stained in brilliant crysel blue forselection of good quality oocytes. The COC were then washed and matured in vitro for 24 hrsbefore in vitro fertilization. The frozen semen (frozen by different extenders and differentfreezing protocols and from different breeds) was thawed and then centrifuged twice at 1500rpm for 8minutes at 38°C. Then 50 μl of the prepared semen was placed in the IVF aliquotcontaining the mature oocytes and incubated for 18 hrs at 38°C (5% carbon dioxide and 90%relative humidity). After 18 hrs of oocyte-sperm incubation, presumptive zygotes wereremoved from the IVF aliquots into a 1.5 ml eppendorf tube containing 100 μl of pre-incubatedM199 + FBS and vortexed for 1.5 min to strip off the cumulus cells. After vortexing, zygoteswere washed 3 times in both M199 + FBS and pre-incubated culture media (SOF-BSA)droplets after which the zygotes were allocated to groups of 20 per aliquot which were coveredwith mineral oil and placed in gas chamber with 3 gases, then incubated at 39°C for 7 days inhumidified incubator (5% oxygen, 5% carbon dioxide and 90% nitrogen). During thisincubation time, the culture medium was changed at 48 hrs after fertilization to SOF-FBS byaspirating medium from the aliquots and replacing the medium with the fresh pre-incubatedmedium with the aid of a pipette, during which the cleavage rate (2 to 8 cells) was examinedand embryos were then separated according to number of cell division. The media was changedagain during day 5. Microscopic evaluations were performed during day 6, and day 7 for thedevelopment into morula and blastocyst respectively and results were recorded into the excelsheet for data analysis.The semen frozen by with the aid of liquid nitrogen vapour prior to storage recorded bettercleavage compared to semen frozen to -80°C prior to storage. Only the freezing protocols andsemen extenders exhibited a significant difference in post in vitro fertilization throughout theentire in vitro fertilization experiment. Statistically the different breeds had no significantdifference in post in vitro fertilization. The results suggests that, in general frozen-thawed ramsemen irrespective of the breed can perform well in the in vitro fertilization procedures used ifthe Tris-egg-yolk based extender is used as diluents in combination with exposure to liquidnitrogen vapour prior to storage as a freezing protocol. Further research following thawing is warranted to confirm fertility results in vivo. The results thus also indicate that the breed hasno effect on the in vitro results of frozen-thawed ram semen.

[发布机构] University of the Free State