Bovine embryos obtained from donors six to nine days after oestrus were transferred non-surgically at a rate of one per recipient using a sterile insemination instrument, protected from contamination by the vagina with a plastic sheath. The percentage of recipients pregnant increased with the age of embryo transferred and for day 6 and 7 embryos was 33% compared to 58% for day 9 and 8 embryos. This difference approached statistical significance. Bacterial contamination of the instrument on withdrawal after transfer was not related to the success or failure of pregnancy. Maintenance of pregnancy to term and calving appeared to be normal. It is suggested that this method could be used for the routine transfer of eight and nine day embryos.

Agricultural Research Council Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge (Received 24th March 1975) Successful techniques of freezing and thawing have recently been developed for embryos of the mouse (Whittingham et al., 1972; Wilmut, 1972a) and rabbit (Bank & Maurer, 1974; Whittingham & Adams, 1974). The proportion of embryos which survived was very high and normal young have been born for both species. Experiments on freezing and thawing the embryos of farm animals have been more limited, and although some success has been achieved with cow (Wilmut & Rowson, 1973b) and sheep (Willadsen et al., 1974) embryos high survival rates have not yet been obtained. When 8- to 16-celled cow embryos were subjected to a variety of methods of freezing and thawing similar to those which have been successful in the mouse and rabbit, only 2/111 developed normally when transferred to a rabbit oviduct for 48 to

The blood groups are described of three sheep chimaeras produced by injection of blastomere cells into fertilized eggs of different parental origin. Evidence is presented that these chimaeras had a blood type which could only have been derived from both sets of parental genes. Two antigenically distinct red cell populations were identified in all three sheep and chimaerism of serum transferrin and albumin types was also present in two of them. One sheep had a mixture of red cell potassium types and another of haemoglobin types. Soluble R and O blood group substances were present together in the saliva of all three sheep and on the red cells of two of them. The relative proportions of the two red cell populations changed with age of the sheep, the blastomere-derived population becoming less. In one sheep one transferrin type also declined. A fourth sheep studied was not a blood chimaera but was apparently composed of blood derived entirely from that of the transplanted blastomere cells. All four sheep were phenotypically male but studies of cultured leucocytes showed that female cells were present in two of the chimaeras (34 per cent and 89 per cent respectively).

SUMMARY The androgenic effects of testosterone and dihydrotestosterone on the bovine seminal vesicles have been evaluated by (i) introducing the steroid directly into one seminal vesicle in situ, while retaining the other vesicle as a control or for the introduction of another steroid, or (ii) injecting the steroid subcutaneously into a whole animal, and examining the seminal vesicles after autopsy. When introduced directly into a seminal vesicle dihydrotestosterone oenanthate stimulated growth and the formation of fructose and citric acid, but the magnitude of the effect was not greater than that of a similar dose of testosterone oenanthate. When twin calves were injected subcutaneously, one with dihydrotestosterone propionate, and the other with the same dose of testosterone propionate, only the latter responded with increased output of fructose, citric acid and α-mannosidase in electro-ejaculated semen. But the seminal vesicles grew to approximately the same size in both twins. Parenterally administered bovine growth hormone produced a sudden but transient burst of secretory activity in the seminal vesicles, resulting in the appearance of fructose and citric acid in electro-ejaculated semen.

This paper describes the development of conceptuses obtained from animals in which the onset of estrus was known to within about 8 hours. Also the viability of conceptuses of similar age but variable sizes was studied after transfer to recipient ewes. There were 177 donor ewes superovulated with 800-1000 I.U. pregnant mares serum on Day 12 of the cycle. Conceptuses were removed by operation between Days 5 and 9 in 86 and between Days 12 and 14 in 91 with Day 0 the onset of estrus although ovulation normally takes place 24 hours later. 521 developing conceptuses and 81 unfertilized eggs were recovered. (47 of the unfertilized eggs were from ewes showing an exaggerated response to gonadotrophin with more than 20 ovulations per animal. ) 97 morulae were recovered 95 between the ages of 112 to 128 hours postestrum 2 at 160 hours. There were 30 in the 16-32 cell stage and 67 in the 32-64 cell stage. The development of a central cavity determined the blastocyst stage ovserved in 190 recovered from 128 to 224 hours after estrus. Some were still within the intact zona pellucida. Others were larger with no zona pellucida. By 208 hours postestrum the blastocysts showed a prominent embryonic disc. In the third phase of development many were damaged by recovery methods but 123 were obtained undamaged. At about Day 12 the conceptus begins to elongate and within 24 hours changes to an elongated chorionic sac with the embryo clearly discernable. The enlongating process continues rapidly but the embryonic disc enlarges only slightly. On Day 12 some degree of damage to embryonic membranes was tolerated when conceptuses were transferred to suitable recipients. When of the same age transferred conceptuses did equally well irrespective of different sizes but had all remained within the donor uterus many would have degenerated and died due to the competition. The marked growth of the conceptus from Day 12 to Day 14 postertrus commences when the corpus luteum becomes dependent on the presence of the conceptus for its continued maintenance. Other experiments have shown that the sheep conceptus can develop until Day 11 even in rabbit oviducts but does not survive longer there.

Eggs were recovered in vivo in a modified Tyrode's solution from 29 donor gilts and transferred to 27 unmated recipients. Ovulation time of donors and recipients was synchronized. Eggs from B donors were transferred to the left oviduct or upper uterine horn, and eggs from W donors were transferred to the right side in 18 gilts. When the uteri and fetal contents were examined at about 90 days of gestation, migration of one or more eggs from one horn to the other had occurred in all (14) of the pregnant animals. Mixing of embryos from the two different sides occurred in 9 of 11 gilts in which both B and W fetuses survived to examination. Eggs were transferred to one uterine horn only in nine gilts. Five of these nine were pregnant when examined at 21 days of gestation. Two had embryos in both uterine horns and three had embryos in only one horn. Of 263 eggs transferred 110 (42%) developed into fetuses. Of 184 eggs transferred to recipient gilts which were pregnant at examination, 60% had developed into fetuses. Egg transfer can be used effectively in studies on implantation and other aspects of swine reproductive physiology.

Summary. Two experiments were carried out involving the exportation by air from Great Britain of a total of three rabbit does carrying in their oviducts fertilized sheep ova for transfer to recipient ewes in South Africa. In October, 1960, ova were recovered in Cambridge from Border Leicester ewes which had been mated to a ram of the same breed. The ova were transported in two does to Pietermaritzburg, where they were transferred to Dorper ewes. In the second experiment early in December, 1960, fertilized ova were recovered from Welsh Mountain ewes (after they had been mated to Welsh Mountain rams) and finally transferred to German Merino ewes in South Africa. Following storage periods in the rabbits ranging (in the two experiments) from 101 to 128 hr, seventeen sheep blastocysts or late morulae were recovered in Pietermaritzburg and sixteen were transferred to a total of ten recipient ewes. Six pregnancies resulted and four live lambs were born to foster mothers in South Africa. The techniques are described in detail.

Fertilized ova have been transferred from Border Leicester to Welsh Mountain ewes, and vice versa. The oestrous cycles of the ewes were synchronized by progesterone treatment, and donor ewes were super-ovulated with PMS. 68–90 hr. after the onset of oestrus in the donors, thirty-seven ova, representing 58% of the ovulations, were recovered in vivo from thirteen ewes. Nineteen ova were transferred to eighteen recipient ewes and a total of eight pure-bred lambs were born to ewes of the opposite breed.Transfers resulted in pregnancy when oestrus in the recipients commenced from 16 hr. before to 20 hr. after that in the donors; two of the ova which developed to term were 4-cell, five were 8-cell and one was a 16-cell ovum at the time of transfer. Four out of seven and four out of eleven transfers, using blood serum and Ringer's solution respectively, resulted in the birth of lambs. Prior to transfer to the recipients seven of the ova which developed to term were exposed for 30–45 min. at room temperature, whilst one developed after exposure for 70 min.

SUCCESSFUL transfer of ova by surgical techniques in sheep has been reported by Warwick and Berry1 and Lopyrin, Loginova and Karpov2. In our experiments, transfers of ova have been carried out between the Border Leicester (mature weight 140–180 lb.) and Welsh Mountain (mature weight 80–100 lb.) breeds in order to evaluate the maternal influence on the size of lambs at birth and at subsequent ages.

DURING the last few years we have been investigating the possibility -of using moderate or inferior cows as incubators for the development of genetically high-grade germinal material. Preliminary investigations in rabbits proved highly successful with most of the transplanted ova being successfully carried to term, and in this species it has been possible to store fertilized ova at reduced temperatures for sever#l days and then to transplant them successfully. When attempts were made to repeat these findings in the cow they consistently failed despite the fact that many workers all over the world were engaged on the problem. There was, however, a certain difference in technique, for whereas in the rabbit the ovum transfer had been an aseptic surgical operation, in the cow the ova were placed in the uterus by means ofa pipette passed through the cervical canal, and complete asepsis was therefore extremely difficult.

1. A comparison has been made between the effect of various forms of P.M.S. on the cow's ovaries by giving 3000 i.u. of: (a) processed P.M.S., (b) fresh whole P.M.S., (c) freeze-dried P.M.S.; the average follicle numbers produced being 13·9, 20·1, and 14·5 respectively.

Follicles in relatively large numbers (average 26) have been consistently produced by the injection of 3600 and 4500 i.u. of whole pregnant mares' serum, and to a slightly lesser degree (average 14), by the injection of similar amounts of commercial processed pregnant mares' serum. Ovulation after this treatment has been spontaneous, but to a lesser degree when using the processed material (5·4% ovulations) than when using the whole plasma (24% ovulations). The percentage of ovulations after this treatment has been increased by the intravenous injection of chorionic gonadotrophin at a dosage of 2000 i.u. (22% for processed material and 42% for whole serum). Where a large corpus luteum was present in the ovary during the time of treatment, the percentage ovulations was 52 as compared with only 14 in those cases in which no corpus luteum was present. Injections of 20 mg. progesterone daily for 4 days after removal of the corpus luteum, and after the p.m.s. injections, had the same effect on the ovulation rate (55% ovulations) as the presence of a large corpus luteum. Ova produced by either processed or whole serum can be fertilized fairly readily in the absence of a corpus luteum, but in its presence or after daily injections of progesterone, no fertilization takes place. In the presence of a corpus luteum or after injections of progesterone, the ova travel down the Fallopian tube at a greatly increased rate, but were in some cases slowed up by the injection of oestrogens.