In vitro culture of in vivo-produced sheep, goat and cattle embryos

Barry, Daniel Malan (2005-03)

Thesis (PhD(Agric) (Animal Sciences))--University of Stellenbosch, 2005.


As most researchers have foreseen, and many breeders have hoped, the in vivo and in vitro production of livestock embryos and the birth of subsequent offspring never really replaced artificial insemination during the past 30 years. This was, to a large extent, due to very variable and unreliable numbers of embryos produced using these two methods. The present study was therefore undertaken to investigate certain aspects of assisted reproductive techniques (ART) to try and solve some of these difficulties. Problems addressed were the management of follicular development on the ovary by controlling the dominant follicle, and investigating alternative and more cost-effective culture media and conditions for embryo culture. A method to control the development of the dominant follicle in a cohort of follicles as well as the waves of follicular development in the ovaries of sheep, goats and cows with an estrogenic product was investigated. Estradiol cypionate (ECP) was used for this purpose, injected intramuscularly after the insertion of the progesterone or progestagen implant. ECP has a negative feedback effect on the secretion of pituitary FSH, and therefore follicular development. The animals of the three different species were randomly divided into two groups each, the ECP-group receiving the estradiol cypionate injection, and the control group receiving a saline injection. Five days after the ECP injection a program of follicular multi-stimulation with FSH hormone was initiated. The females of the different species were bred by either natural service (goats) or inseminated by laparoscopy (sheep) or trans-cervically (cows) to fertilize the ovulated ova. Embryos and unfertilized ova were collected surgically at the 8 to 16- cell stage 3 to 4 d after breeding in the sheep and goats, and trans-cervically in the cows. Significantly more CL formed, and a total number of ova and embryos, as well as transferable embryos, were collected from the ECP-group of sheep ewes and goat does compared to the control group that received no ECP (p<0.01). There was, however, no difference in the average number of unfertilized ova that were collected in the two sheep or goat groups. In the cows the number of CL counted, the total number of embryos and ova and of transferable embryos collected, were significantly greater (p<0.05) in the group that were injected with ECP compared to the group that received no ECP. The control group also had a significantly larger number of unfertilized ova than the ECP-group (p<0.05). It could therefore be concluded that more reliable numbers of embryos can be produced in vivo if the development of the dominant follicle as well as the subordinate follicles is controlled with estradiol cypionate. Since more than half a century ago, attempts have been made to culture cells and embryos outside the body (in vitro or ex vivo). This was done with different culture media and in various "incubators". Chapter 2 deals with two different culture media used: a standard TCM-199 culture medium and first trimester amniotic fluid (BAF) collected sterilely from pregnant cows after slaughter. Two different culture conditions were also investigated, the standard laboratory CO2 incubator versus culturing bovine embryos in the vagina of a goat doe. Two experiments were done: Firstly the permeability of different receptacles to CO2 gas was analyzed for possible culture in the vagina. Four-well plates and straws were used to incubate TCM-199 and BAF for a period of 120 h in the presence or absence of 5% CO2 gas. The pH values were measured every 24 h and recorded. In the second experiment pre-compacted morula stage bovine embryos were incubated in the above culture media in sealed 0.25 mL straws in a standard laboratory incubator and in the vagina of a goat doe. Evaluation was done on (1) stage of development and (2) number of blastomeres after 96 h of culture. In experiment one it was shown that the CO2 gas diffused out of the 4-well plate as well as the straws in the absence of CO2 gas, while in the presence of CO2, the pH of both media stabilized between 7.3 and 7.5. This meant that the semen straws were permeable to CO2 gas and could therefore be used as receptacles for culturing early stage bovine embryos. In the second experiment no statistical differences (p>0.05) were found in the number of Grade 1 pre-compacted bovine embryos that developed to the blastocyst stage, or the hatched blastocyst stage, neither for the culture medium used, or the method of culturing in the two incubators. Neither was there any difference (p>0.05) in the number of blastomeres that developed at the blastocyst stage between the two types of incubators used. Embryos tended to develop more blastomeres when cultured in BAF than when cultured in TCM-199 in both the standard laboratory incubator and when using the vagina of a goat doe as an incubator (p<0.05). After the collection of in vivo produced livestock embryos, they are evaluated under high magnification (minimum of 80X) with the aid of an inverted or stereo microscope. The Grade 1 embryos will give the best conception results when transferred to synchronized recipient female animals, while the Grade 3 embryos will give the worst results. The aim of the next experiment was to culture all three quality grades of in vivo produced pre-compacted morula-stage embryos of sheep, goats and cows in two different culture media and then compare the development of the embryos by evaluating the number of embryos reaching the hatched blastocyst stage. The results have shown that there were no significant differences between the development of the Grade 1 and the Grade 2 embryos from any of the three species when either cultured in TCM- 199 or heat inactivated early pregnancy-stage (<60 d) bovine amniotic fluid (BAF) were used as culture media. Significantly more in vivo produced Grade 3 pre-compacted morula-stage sheep, goat and cow embryos, however, developed to the hatched blastocyst stage when cultured in BAF with 10% FBS and antibiotics, compared to culture in TCM-199 with 10% FBS and antibiotics (p<0.05). The effect of co-culture on the survival of caprine embryos post transfer to a synchronized recipient female goat was also assessed. A total of 120 Kashmir embryos at the blastocyst stage were divided into three groups after thawing and reconstitution in four steps in glycerol and sucrose medium. The first group of embryos (G1, n=40) was individually transferred semi laparoscopically in D-PBS with 10% FBS and antibiotics to the ipsilateral horn of the CL over a period of 3 d. The second group of caprine blastocysts (G2, n=40) was similarly transferred in TCM-199 with FBS and antibiotics. The third group of frozen-thawed caprine blastocyst-stage embryos (G3, n=40) were first co-cultured for ~24 h in TCM-199 with serum and antibiotics in groups of up to five embryos inside a ~50 mm length of a semen straw in a cylindrical sponge in the anterior part of the vagina of a goat doe in her luteal phase. After the culture period these embryos were transferred in a similar way in TCM-199 without the co-culture as in G1 and G2. Ultrasound scanning showed that significantly more of the blastocyst embryos that were cocultured in the vagina (G3) before transfer developed to a pregnancy compared with the embryos transferred in D-PBS (G1). The co-culture Group 3 blastocyst-stage caprine embryos produced significantly more offspring than the non-cultured embryos transferred in both D-PBS (G1) and TCM-199 (G2) (p<0.05). The maturation of bovine oocytes to allow the oocyte to resume meiosis, is the first step in in vitro fertilization to produce IVMFC embryos. The composition of the maturation medium plays an important role in the success achieved with maturation. An investigation was therefore launched to evaluate the maturation ability of first trimester bovine amniotic fluid (BAF) to mature prophase I oocytes collected from abattoir ovaries to metaphase II oocytes, compared to a standard maturation medium such as TCM-199. In the first experiment three groups of ~100 oocytes each were matured in TCM-199 with estrus cow serum (ECS). The first group of oocytes was matured in a 50 μL micro-drop in an incubator, while the other two groups were matured in semen straws, one group in an incubator and the other group in the vagina of a goat doe in di-estrus. Six further groups of ~100 oocytes each, with BAF as maturation medium, three groups with ECS and three without ECS, were matured in the same receptacles and under the same conditions as with the TCM-199. No significant differences in number of oocytes reaching the metaphase II stage could be found for any of the nine treatment groups. In the ...

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