In vitro culture of in vivo-produced sheep, goat and cattle embryos
Date
2005-03
Authors
Barry, Daniel Malan
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
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 ...
Description
Thesis (PhD(Agric) (Animal Sciences))--University of Stellenbosch, 2005.
Keywords
In vitro fertilization, Sheep, Goats, Cattle, Embryos, Dissertations -- Animal sciences, Theses -- Animal sciences, Dissertations -- Agriculture, Theses -- Agriculture