Birth of Live Calves by in Vitro Embryo Production of Slaughtered Cows in a Commercial Herd in South Africa

INTRODUCTION The rescue of genetic material from valuable animals that die or need to be culled is a service that is currently available for certain species in many parts of the world


INTRODUCTION
The rescue of genetic material from valuable animals that die or need to be culled is a service that is currently available for certain species in many parts of the world 6,11 .In vitro fertilisation (IVF), a technology that uses eggs and sperm to produce embryos in a Petri dish, is particularly well-developed for cattle.Bovine IVF and embryo transfer have become standard procedures in which multiple offspring may be produced from a single dam.However, results are highly variable, with many donors yielding no offspring while others may yield several 10 .On average, 2-3 viable embryos can be produced from each donor, which, after transfer to a surrogate mother, will result in the birth of 1-2 live calves 6,7 .Although bovine IVF has been commercially available to farmers in many countries, this has occurred only recently in South Africa.This report describes the success of IVF and embryo transfer in a commercial herd of South African Bovelder beef cattle, with the birth of live calves.

Oocyte recovery and embryo production
Six Bovelder cows due for slaughter with no special prior treatment were selected for the IVF trial.The ovaries were collected at an abattoir within 30 minutes of slaughter and were delivered to the laboratory within 2 hours in phosphate buffered saline at approximately 30 °C.The ovaries were collected on Day 1 relative to IVF (Fig. 1).
Oocytes were retrieved by superficially slicing the cortex of the ovaries with a razor blade into tissue culture medium (TCM) 199 (all chemicals obtained from Sigma unless otherwise indicated) containing 0.3 % bovine serum albumin and 25 µg/m gentamicin.Oocytes were located under a stereo microscope and washed in TCM199 to remove ovarian debris.Those with at least one complete layer of compact granulosa cells were selected for further culture 3 .Selected oocytes were matured for 22 h under mineral oil in 50 µ droplets of TCM199 supplemented with 25 µg/m gentamicin, 100 µM 2-mercaptoethylamine, 25 mM HEPES and 10 % steer serum 12,19 .The steer serum was collected from a steer housed at the Faculty of Veterinary Science, University of Pretoria.All incubations occurred at 39 °C in 5 % carbon dioxide in air with 100 % humidity.
Sperm and cumulus cells were washed from the presumptive zygotes approximately 20 hours after the addition of sperm (Day 1, Fig. 1).For the embryo culture period, embryos were co-cultured with granulosa cells derived from the Bovelder follicles in 50 µ droplets of TCM199 supplemented with 10 % steer serum and 25 µg/m gentamicin under oil 8 .The number of zygotes cleaved to at least 2 cells was counted on Day 2 of culture (44 h after the addition of sperm) as an indication of the success of fertilisation.
On Day 7 after fertilisation (Fig. 1), all blastocysts were counted and graded.The best 11 embryos, all grade one 22 , were selected for fresh embryo transfer.Embryos were loaded into 0.25 m straws (IMV, Taurus, South Africa) in the laboratory and transported to the farm at room temperature.One embryo was nonsurgically transferred per recipient.
Additional blastocysts that appeared by Day 10 were also counted, to give the total number of blastocysts produced.

Recipient management
Thirteen Bovelder recipients were synchronised with an EAZI-BREED CIDR ® (Solvay, AH) for 10 days (Days -12 to -2) in combination with an intramuscular injection of 500 µg cloprostenol (Estrumate ® , Schering-Plough AH, South Africa) on Day -4 (Fig. 1).Once the CIDR ® devices were removed, field staff observed the recipients for signs of oestrus at 12-hourly intervals.All 13 cows were in heat on Day 1 of the IVF programme.The embryos were transferred on Day 7 of the programme (6 days after oestrus).Of the 13 synchronised cows (all first calvers), 11 were chosen as recipients.All the selected recipients had a corpus luteum, which was, judged by transrectal palpation, fully developed.Pregnancy was diagnosed 42 days after fertilisation (or 35 days after embryo transfer) by transrectal ultrasonography.At birth the date, sex and mass of each calf was recorded.

Statistical analysis
t-tests were performed on the gestation lengths and birth masss of calves born.Variation is reported as ± one standard deviation.

Embryo production
Table 1 summarises the results of the oocyte recovery and embryo production.
Ovaries from 6 mature Bovelder cows were recovered within 30 minutes of slaughter.One of the ovaries from cow number 4 was not recovered, thus the results reflect the use of only 1 of her ovaries, or 11 ovaries in total.Using a razor blade to lightly slice the ovarian surface, 199 reasonable quality oocytes were obtained.This corresponds to an average of 18.1 oocytes per ovary (range: 5-44) or 36.2 per cow.
The overall percentage of initial cleavage to at least 2 cells was 47 % (93/199), with a range of 20-58 %.By early Day 7, 27 blastocysts had been produced from the 11 ovaries.Of these, 24 were of transferable quality 22 .The best 11, all Grade 1, were chosen for embryo transfer (results described below).No blastocysts were produced by Day 7 from 2 of the donors, numbers 2 and 4. For all donors, a mean of 4.5 ± 1.8 blastocysts had developed by Day 7 (range 0-10).By Day 10, a total of 43 blastocysts (7.8 per donor) had been produced from 199 oocytes (22 %).

Embryo transfer
The results of the embryo transfers are summarised in Table 2 and the details of the calves born in Table 3.A single embryo was transferred into each of 11 synchronised, 1st-calver Bovelder recipients.Seven of the 11 transferred embryos resulted in pregnancy on Day 42, a pregnancy rate of 64 % (Table 2).All 7 cows diagnosed as pregnant produced a live calf (2 heifers and 5 bull calves).No assistance was necessary for the calvings.The average mass of the male and female calves in this study were 38.8 ± 6.2 and 34.0 ± 7.1 kg respectively and the males were not significantly different from the herd average of 36.76 (P > 0.05).The male and female calves were born at mean gestation periods of 291.6 ± 3.7 and 293.5 ± 7.8 days respectively, the males having a significantly longer gestation period than the breed average of 285.2 days (P < 0.05).Because there were only 2 female calves, the power of the statistical tests was too low to provide valid tests of differences of the calves in the present study from herd averages, 32.84 kg birth mass and 283.8 days gestation.

DISCUSSION
The post-mortem production of offspring from an animal that dies suddenly or is culled is a valuable method for rescuing desirable genetic material.In the bovine, intensive research over the last 15 years has enabled the practical, commercial production of offspring by in vitro fertilisation (IVF) 4,7 .However, this technology is new in the domestic animal sector of South Africa, and in this study the success of these procedures under South African conditions using Bovelder donors and recipients was examined.
In general, embryo production and subsequent pregnancy rates from the Bovelder cows were on par with interna- The number of cleaved oocytes was counted approximately 44 hours after fertilisation and represents all embryos that had at least 2 cells.The number of blastocysts produced by Day 7 and Day 10 were also counted.tional standards.As expected, the number of good-quality oocytes retrieved per donor was variable, but the average of approximately 36 was within normal expectations 5,13 .The initial cleavage to 2 cells of 47 % was lower than the 70 % that is normally expected 16 , but less stringent oocyte quality selection would at least partially account for this decrease.Although not typically cultured by research laboratories, oocytes with only a single layer of cumulus cells were used in the present study in order to maximise the potential for blastocyst production per donor, as some of these oocytes are capable of forming blastocysts 3 .The overall percentage of blastocysts (22 %) was comparable to results published internationally despite the selection of poorer quality oocytes 13,23 .Again, there was considerable variation among donors (0-38 % of total oocytes cultured), as has been reported in other studies 2 .
The number of transferable blastocysts per donor, 4.4, is equal to that reported in other studies.Mylne et al. 14 produced 3 blastocysts per cow, while Stringfellow et al. 18 reported 5 per cow.
The pregnancy rate of 64 % after embryo transfer corresponds with international standards.With good-quality embryos and recipients, pregnancy rates of 60-70 % can be obtained 7,9 .In this study, calves were produced from 4 of the 6 donors, again indicating the variability that can be seen in this type of system.Three of these had 2 offspring each, while the 4th had only 1.Because of the limited number of recipients available, not all good-quality Day 7 embryos were transferred, therefore on a per donor basis more calves could have been obtained with additional recipients.The lack of recipients would not have affected the 2 cows that did not produce blastocysts by Day 7. Similar to results obtained here, an Italian study reported 1-3 calves produced per donor 6 .
One concern regarding IVF production of embryos is that foetuses produced might be oversized, causing calving problems 20,21 .In this study, the average birth mass of the male calves was not significantly different from the herd average and no calving problems were observed.Among calves born as a result of over 1000 bovine embryo transfers, no problems due to large calves occurred, but increased incidence of hydroallantois was reported 10 .No hydroallantois was observed during the pregnancies of the cows that gave birth to the 7 calves in the present study.However, the gestation mass of the male calves (291.6 ± 3.7) was significantly longer than the breed average of 285.2 days (P < 0.05).Because the average gestation length of calves from the sire used in this study is not available, it cannot be determined whether the prolonged gestation period is a sire, IVF or other effect.
Besides aiding stock farmers, the development of in vitro fertilisation and embryo transfer technology in South Africa may contribute to the preservation of genetic diversity of endangered or threatened wildlife species by salvaging the oocytes and sperm from culled animals, or animals that have died unexpectedly.Such embryos can then be frozen indefinitely and transferred into a recipient at a convenient time and place anywhere in the world.This can be achieved either by using vaccines such as the RB51 strain that elicit a different response from natural infection, or by using serological tests that can distinguish the antibodies provoked by pathogenic Brucella from vaccine-induced and crossreacting antibodies.The last section consists of 2 papers presented at the 19th Conference of the OIE Regional Commission for Europe in September 2000.The first is a comprehensive overview of swine vesicular disease, which can cause severe production losses, more in terms of the control measures applied than the actual effect of the disease.Its main importance lies in the fact that it is clinically impossible to distinguish from foot-and-mouth disease.Controversy exists as to whether this disease should be retained as a List A disease, but in general countries have been in favour of risking the losses incurred by eradication procedures rather than risking confusion and consequent delayed diagnosis of foot-and-mouth disease.The last paper concentrates on how to limit erosive diseases that are not OIE-listed diseases but merit control owing to their devastating effects on productivity.The approach is valid and should be taken seriously, even if the outbreaks of foot and mouth disease that have occurred subsequent to the publication of these reports have demonstrated again why List A diseases should never be ignored simply because they may only appear at long intervals!This publication is of considerable value to all veterinarians.