Evaluation of cryopreserved ram semen following fertilization in vitro
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Date
2015-07
Authors
Mohlomi, Masilo Henoke
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
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.
Description
Keywords
Dissertation (M.Sc.Agric. (Animal, Wildlife and Grassland Sciences))--University of the Free State, 2015, Ruminants -- Reproduction, Ruminants -- Productivity, Artificial insemination, Dorper (Sheep), Frozen semen