Evaluation of cryopreserved ram semen following fertilization in vitro
Mohlomi, Masilo Henoke
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Three experiments were done at the University of Free State in Bloemfontain in 2014 and in 2015. Experiment 1 was carried to evaluate the effect of breed (Dorper and SAMM) on fresh ram semen parameters before cryopreservation. Experiment 2 was carried to evaluate the effects of breed difference, different extenders and different freezing protocols on the viability of frozen ram semen during different incubation periods after thawing. Experiment 3 was carried 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 the breeding season while experiment 2 was done in 2015 from mid of January until the end of April. For the first trial, the semen was collected with the aid of artificial vagina from two breeds of rams. The ejaculates were evaluated for the following parameters: semen volume, semen colour, sperm motility, sperm concentration, percentage live/dead sperm and sperm morphology. 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 and higher semen volume than Dorper semen for the entire experiment. There was no significant difference between breeds for semen colour, sperm concentration, percentage live and dead sperm and sperm morphology. For the second trial, the study to evaluate the effect of different breeds, extenders and freezing protocols on the sperm motility of ram semen including survival at different incubation periods after thawing was conducted during the natural breeding season. Semen was collected from two breeds of rams (South African Marten Merino (SAMM) and Dorper). After fresh semen evaluation and selection of good quality ejaculates, semen was pooled for each breed and divided into two then randomly allocated to Tris-egg yolk and Na-Citrate egg yolk based extenders. Semen samples were further divided into two more groups and allocated randomly to two different freezing protocols (freezing up to -20°C using the programmable freezer and then 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 following freezing. The semen frozen by the use of liquid nitrogen vapour prior to storage recorded better sperm motility than semen frozen to -80°C prior to storage. The viability of the frozen ram semen was inversely proportional with incubation time increase after thawing. Only the freezing protocols and incubation periods exhibited a significant difference in sperm motility throughout the experiment. Statistically, the difference breeds and the different semen extenders recorded no significant difference. However, when Tukey’s grouping was used (studentised range), the Tris-egg yolk based extender showed better sperm motility compared to the Na-Citrate egg yolk based extender. The results suggest that the breed does not have impact on semen freezing and the Tris-egg yolk based extender is better suited as a diluent for ram 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 after thawing or alternatively the laparoscopic method will remain an alternative. In the last experiment, ovaries were collected during spring and at the beginning of summer at the Bloemfontein abattoir from unknown, untreated sheep and transported to the laboratory in sterile saline water (37°C) in the flask. Cumulus oocytes complexes (COC’s) were recovered from the ovarian follicles by aspiration method, washed and stained in brilliant crysel blue for selection of good quality oocytes. The COC were then washed and matured in vitro for 24 hrs before in vitro fertilization. The frozen semen (frozen by different extenders and different freezing protocols and from different breeds) was thawed and then centrifuged twice at 1500 rpm for 8minutes at 38°C. Then 50 μl of the prepared semen was placed in the IVF aliquot containing 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 were removed from the IVF aliquots into a 1.5 ml eppendorf tube containing 100 μl of pre-incubated M199 + FBS and vortexed for 1.5 min to strip off the cumulus cells. After vortexing, zygotes were 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 covered with mineral oil and placed in gas chamber with 3 gases, then incubated at 39°C for 7 days in humidified incubator (5% oxygen, 5% carbon dioxide and 90% nitrogen). During this incubation time, the culture medium was changed at 48 hrs after fertilization to SOF-FBS by aspirating medium from the aliquots and replacing the medium with the fresh pre-incubated medium with the aid of a pipette, during which the cleavage rate (2 to 8 cells) was examined and embryos were then separated according to number of cell division. The media was changed again during day 5. Microscopic evaluations were performed during day 6, and day 7 for the development into morula and blastocyst respectively and results were recorded into the excel sheet for data analysis. The semen frozen by with the aid of liquid nitrogen vapour prior to storage recorded better cleavage compared to semen frozen to -80°C prior to storage. Only the freezing protocols and semen extenders exhibited a significant difference in post in vitro fertilization throughout the entire in vitro fertilization experiment. Statistically the different breeds had no significant difference in post in vitro fertilization. The results suggests that, in general frozen-thawed ram semen irrespective of the breed can perform well in the in vitro fertilization procedures used if the Tris-egg-yolk based extender is used as diluents in combination with exposure to liquid nitrogen 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 has no effect on the in vitro results of frozen-thawed ram semen.