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In Ireland, an attempt has been made to scale-up a laboratory procedure, which had yielded promising results, to meet the needs of extensive commercial exploitation of the technology of in vitro cattle embryo production. This paper examines some of the problems which have to be dealt with in bringing the embryo production technology to the point at which it becomes fully viable commercially. Much of the information included in the review is derived from on-going research and development work in the University; data are also derived from the work of. Ovamass Ltd., a company established in Ireland in 1987 to develop the technology of embryo production on a commercial basis.

An important aim of an oocyte recovery method is to maximize the number of oocytes per ovary which can be employed for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). In this study, primary bovine oocytes were collected by 2 methods: aspiration of visible follicles (2 to 6mm in diameter) or surface dissection in which the ovary surface is finely dissected. The oocytes were classified on the basis of cumulus cover and cytoplasmic appearance. The total number of oocytes and the yield of good-quality oocytes recovered per ovary by surface dissection and aspiration were 44.2 and 13.9 and 13.5 and 4.6 (P0.05), respectively. When a sample group of selected oocytes recovered by each method was measured, no significant difference was found in the mean diameter (144.11m vs 142.54m). A representative sample of good-quality oocytes recovered by each method was put through the IVM/IVF/IVC procedure: no significant difference in cleavage rate, cleavage index or blastocyst yield was found. However, when the blastocyst yield was compared on a per ovary basis, a significant difference was observed in favor of surface dissection (3.30+/-0.46 vs 0.96+/-0.16;P0.05). When unselected oocytes recovered by surface dissection of the ovaries were put through the standard embryo production system, an average of 15.4 blastocysts per dam was obtained.

Donor ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attemped seven days after progestagen with-drawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons.

Day 7 to 9 embryos were frozen by a rapid two-step method to −38°C before being plunged into liquid nitrogen. Glycerol was used as the cryoprotectant and, following thawing, the embryos were cultured for 12 – 24 hours in PBS + 15% heat-treated steer serum. In Experiment 1, embryos were frozen in 2.0 ml glass ampoules or 0.5 ml Cassou straws. Two levels of glycerol (1.0M and 1.4M) gave comparable in vitro survival rates ( 12 20 and 13 25 , respectively). A greater proportion of embryos developed in culture after freezing in straws. In Experiment 2, embryos were classified morphologically before and after freezing into 5 grades (1 = excellent; 2 = good; 3 = fair; 4 = poor; 5 = degenerate). Only embryos of grade 1, 2 and 3 were frozen. The post-thaw survival rates for embryos graded 1, 2 and 3 before freezing were 100% ( 11 11 ), 86% ( 24 28 ) and 83% ( 20 24 ), respectively. Furthermore, the porportion of surviving embryos estimated to be of poor quality (grade 4) was greater for embryos graded 3 before freezing ( 13 20 ) than for embryos graded 2 ( 6 24 ) or 1 ( 1 11 ). The percentage of embryos which developed normally after in vitro culture for each of the pre-freezing grades 1, 2 and 3 was 91% ( 10 11 ), 50% ( 14 28 ) and 29% ( 7 24 ), respectively. Of the total number of frozen-thawed embryos which developed in culture, 5 31 (16%) were of poor quality. The proportion of poor quality developing embryos was greater inembryos graded 3 before freezing ( 3 7 ) than those graded 2 ( 2 14 ). All of the embryos graded 1 before freezing and which developed in culture were of good quality. Results indicate that, if high post-thaw survival rates are to be obtained, stringent embryo selection processes will be required.

One hundred and ninety three beef heifers were superovulated using a PMSG-Oestradiol 17β-HCG regime beginning on day 16 of the oestrous cycle. Eggs were recovered 2–6 days post oestrus and classified morphologically. A mean of 8.3 fertilized eggs per donor was obtained, but 19% of these were considered non-viable. The percentage of abnormal embryos increased as ovarian stimulation increased and as the interval from estrus to recovery increased. Eggs classified as being degenerate showed no evidence of continued cleavage in the rabbit oviduct, although eggs with some forms of minor abnormality were capable of developing at the normal rate in the rabbit. We conclude that the uterine environment following high levels of superovulation can be hostile for egg development.

Thirty-eight adult ewes were superovulated following a 12 d progestagen treatment in association with either 135 mg crude horse anterior pituitary extract (HAP) or 30 mg soluble HAP with or without 1000 IU human chorionic gonadotrophin (hCG). Soluble HAP in combination with hCG significantly (P < 0.01) increased the ovulation rate as compared to HAP alone (14.5 vs 5.3, respectively). The number of oocytes recovered per ewe showing estrus following soluble HAP and HCG (6.9) was higher than after HAP alone (3.3), but this difference was not significant. A significantly higher (P < 0.05) ovulation rate and ovarian response were obtained following crude HAP than soluble HAP (14.0 vs 6.3 and 15.3 vs 9.4, respectively). There was a tendency for the proportion of follicles which ovulated to be higher in the crude HAP group. The mean number of oocytes or the proportion of oocytes recovered was significantly higher (P < 0.05) in the crude HAP group than in the soluble HAP group.