Two trials were conducted at Agricultural Research Council (Irene â Pretoria),
between March (autumn) and October (spring), 2008. The first trial evaluated the
effect of the oocyte harvesting technique on the quantity and quality of ovine
oocytes recovered, as well as the effect of the culture media used on embryonic
development. The second trial evaluated the effect of breed and semen
cryopreservation on the embryonic development following in vitro fertilization
(IVF).
For the first trial, ovine ovaries were collected from slaughtered animals at the
Boekenhout abattoir near Pretoria. All ovaries were immediately transported to
the laboratory for further processing and use in the trials. On reaching the laboratory, ovaries were processed, and oocytes were harvested, either by
slicing (140 ovaries), or follicular aspiration (167 ovaries). The oocytes collected
were matured, fertilized with fresh ram semen and cultured in order to produce
embryos. The average total number of ovine oocytes recovered per ovary, and
the total number of oocytes collected were higher (P<0.05) when using the slicing
(5.2±0.4 oocytes/ovary and 75.3±12.9 total oocytes) technique, compared to the
aspiration technique (2.6±0.5 oocytes/ovary and total of 40.3±9.1 oocytes). The
number of acceptable quality (intact cumulus layers) oocytes was also higher
(P<0.05) following the slicing (46.7±10.3 oocytes), compared to aspiration
(10.3±2.0 oocytes) technique. However, the number of poor quality oocytes did
not differ between the 2 oocyte harvesting techniques. The acceptable oocytes
were then matured in vitro for 24h and no difference (P > 0.05) in oocyte
maturation rate between the oocytes recovered using the slicing or aspiration
technique was recorded.
A comparison of the 3 culture media (Potassium simplex optimization medium -
KSOM, Synthetic oviductal fluid - SOF and Charles Rosenkrans medium - CR1)
used for maintaining subsequent embryonic development was then evaluated.
All oocytes were further matured, using the maturation medium - TCM 199
containing FSH, LH and E2, supplemented with 10% FBS. After maturation, the
oocytes were fertilized (using fresh ram semen) and incubated for a period of
18h. At the end of 18h fertilization period, oocytes were vortexed in an Eppendorf
tube containing 100μl M199 + 10% FBS for 1.5min. The vortexing was performed
to remove the cumulus cells surrounding the zygote. A total of 1405 presumptive
zygotes were randomly allocated to the 3 different culture media (481, 461 and
463 zygotes in KSOM, SOF and CR1, respectively). The zygotes were then
cultured for a period of 7 days. No significant difference between all 3 culture
media was recorded regarding cleavage rates, showing that culture media had
no effect on the subsequent cleavage. However, the percentages of embryos
decreased with an advancement of the embryonic developmental stages.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-10172011-134553 |
| Date | 17 October 2011 |
| Creators | Mahoete, Nts'emelo |
| Contributors | Dr TL Nedambale, Prof JPC Greyling, Dr KC Lehloenya |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-10172011-134553/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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