(Received 29th July 1963)

EGG TRANSFER IN SHEEP EFFECT OF DEGREE OF SYNCHRONIZATION BETWEEN DONOR AND RECIPIENT, AGE OF EGG, AND SITE OF TRANSFER ON THE SURVIVAL OF TRANSFERRED EGGS N. W. MOORE and J. N. SHELTON Jf.S. W. and The McCaughey Memorial Institute, Jerilderie, Department of Animal Husbandry, University of Sydney, Sydney, Australia (Received 29th July 1963) Summary. A factorial experiment is described in which fertilized sheep eggs were transferred at a rate of two per animal to 162 recipient to term. ewes all of which were allowed to go The factors included and the results obtained were: (1) Degree ofsynchronization between donor and recipient. Transfers were made to recipients in oestrus 48 hr before to 48 hr after their respective donors. Highly significant effects were observed in the number of ewes which lambed (P<0\m.\01) and number of eggs surviving to parturition (P<0\m.\001), with maximum results in ewes in oestrus 12 hr before to 12 hr after their respective donors. (2) Age of eggs. Eggs were collected, and hr after the donors were first observed in oestrus. There was a significant increase in the number of ewes which lambed (P<0\m.\02) and lambs born (P<0\m.\01) with increase in age of the transferred eggs. (3) Site of transfer. Transfers were made to the Fallopian tubes or uterine horns. Tubal transfers were significantly more successful than uterine transfers in the number of ewes which lambed (P<0\m.\05) and number of lambs born (P<0\m.\01). There were no significant interactions. INTRODUCTION The potential value of egg transfer in farm animals, both for practical purposes and as a research tool, has been amply demonstrated. In sheep, Rowson & Adams (195) and Moore & Shelton (1962a) have shown that large numbers of lambs can be obtained from selected ewes, whilst Averill & Rowson (1958) and Moore & Rowson (1959, 1960) have used the technique to study the survival and development of fertilized sheep eggs. However, in this species precise information is lacking on many of the factors which affect the survival and development of transferred eggs. 145

146 JV. W. Moore and J. M. Shelton This paper presents the results of an experiment designed to study the effects on survival of age of egg transferred, degree of synchronization between the reproductive state of donors and recipients, and the site to which eggs are transferred. MATERIALS AND METHODS In the autumn of 1962, 324 fertilized eggs collected from mature Merino ewes, multiple ovulated with a horse anterior pituitary extract (Moore & Shelton, 1962b) and mated to Merino rams, were transferred at a rate of two per animal to 162 mature Merino recipient ewes. The experimental animals were run under field conditions. Table 1 experimental design Comparison Degree of synchronization* Time ofoestrus in recipi ents relative to donors (hr) Description Early Similar Late +48 +12 ^24 + 36 0-36 +24-12 -48 Factors Product Age of eggs Interval between onset of oestrus and egg re covery (hr) 2 Site of transfer Fallopian tubes Uterine horns Number of animals per cell 3; total animals 162. * Degree of synchronization: + Recipients first observed in oestrus before their respective donors. Recipients first observed in oestrus after their respective donors. Detection of oestrus 'Sire-sine' harnesses were attached to rams for the detection of oestrus for oestrus twice (Radford, Watson & Wood, 1960) and ewes were inspected daily at 6 a.m. and 6 p.m. Donor ewes were run with intact rams and recipients with vasectomized rams. Collection and transfer of eggs The procedures used for the collection and transfer of eggs were essentially those described by Hunter, Adams & Rowson (1955). Experimental design The experiment was of factorial design (Table 1). Degree of synchronization. Eggs were transferred to recipients first observed in oestrus 48 hr before to 48 hr after their respective donors. Potential recipients were discarded if they did not show at least one recent ovulation at the time of transfer.

Egg transfer in sheep 14 Age of eggs. Eggs were recovered in vivo by flushing with sheep serum the Fallopian tubes together with about 5 cm of the most distal portion of the uterine horns. Their age was classified by the time elapsing between onset of oestrus and egg recovery. Normal cleavage was taken as the criterion of fertilization and only cleaved eggs were transferred. Table 2 shows the distribu tion of cell stage relative to the time of recovery. tubes or uterine horns. Site of transfer. Eggs were transferred to the Fallopian There was an even distribution of cell stages between the two sites (Table 2). Table 2 cell stage of eggs in relation to their age, and distribution of cell stages according to site of transfer Age of Site of transfer No. eggs at various cell stages >8 transferred 25 22 26 29 14 13 10 26 26 43 48 * Interval between onset of oestrus and egg recovery (hr). Lambing All the recipients were allowed to go to term. During lambing they inspected were at least once daily when recently born lambs were identified. Analysis of results Standard 2 tests and analyses of variance were applied to the raw or appropriately transformed data. For simplicity of analysis the nine degrees of synchronization were grouped into three classes, namely 'early' (recipients 48, 36 and 24 hr before donors), 'similar' (12 hr before, 0 and 12 hr after) and 'late' (recipients 24, 36 and 48 hr after donors). RESULTS Eighty-three of the 162 recipients subsequently lambed producing (Table 3). 11 lambs Degree of synchronization There was a highly significant effect of degree of on synchronization the number of ewes which lambed (P<0-01) and the number of lambs born (P<0-001). Maximum survival occurred in recipients in oestrus 12 hr before to 12 hr after their respective donors (Table 3). Furthermore, the chances of

148 JV. W. Moore and J. jv. Shelton survival of eggs which were out of phase seemed unrelated to whether they were 'older' or 'younger' than the relative stage of development of the repro ductive tract of the recipient (Tables 4 and 5). Age of egg transferred The older the egg, the greater was the chance of survival (ewes which lambed, P<0-02; lambs born, P<0-01; Table 3). Table 3 results-main effects; number of recipient ewes which lambed and number of lambs born Main effect Degree of synchronization (hr)* +48 'Early' +36 + 24 + 12 'Similar' 0-12 'Late' -24-36 -48 linear quadratic cubic Age of eggs (hr) linear quadratic Site of transfer s No. ewes which lambed 18) 8 14 12 11 9 8 <001 ) 23 25 35 <002 81) 48 35 <005 162) 83 No. lambs born - 36) 8 9 13 19 18 16 13 12 9 < 0-001 108) 31 35 51 <001 162) 2 45 <0-01 324) 11 * For analysis, comparison was made of 'early', 'similar' and 'late' classes. For the 'early' class, the recipients were in oestrus before the donors; for the 'late' class, after. There were no significant interactions. Site of transfer Tubai transfers were more successful than uterine transfers in the number of ewes which lambed (P<0-05) and number of lambs born (i3<0-01 ; Table 3). Interactions There were no significant interactions.

Egg transfer in sheep 149 Table 4 NUMBER OF EWES WHICH LAMBED AND LAMBS BORN IN RELATION TO AGE OF REPRODUCTIVE TRACT RELATIVE TO THAT OF TRANSFERRED EGGS Ewes which lambed 9) No. lambs born 18) Site of transfer Age of eggs (hr) Cell stage 2 to 6 4 to 8 6 to >8 Older + 48 to +24 hr Age of reproductive tract relative to eggs Similar + 12 to -12 hr Tounger -24 to -48 hr Older +48 to + 24 Ar 13 19 16 1 2 to 6 4 to 8 6 to >8 Age of reproductive tract relative to eggs Similar + 12 to -12 Ar 13 23 8 14 30 Tounger -24/0-48 Ar 11 25 Table 5 percentage of transferred eggs surviving in ewes which lambed Site of transfer Age of egg* (hr) Eggs surviving (%) Synchronization (hr) +48 to +24 + 12 to -12-24 to -48 6 80 50 80 6 0 88 9 3 5 65 9 5 5 63 62 50 64 64 50 50 62 60 60 0 2 64 56 64 Table of 2 Source d.f Synchronization Age of egg Site of transfer Interaction 0-05 0-12 0-6 2-45 2 2 1 12 3-29 1

150 JV. W. Moore and J. N. Shelton Analysis of egg survival Eighty-three ewes which lambed received 166 eggs of which 11 (0-5%) survived to lambs. Survival in these ewes was unaffected by degree of synchroni zation, age of eggs or site of transfer (Table 5). Table 6 presents a summary of egg losses. A total of 63-9% was lost, the majority (48-8%) as a result of loss of whole pregnancies. Table 6 summary of losses of eggs A. losses Recipient ewes (total) Eggs transferred Eggs lost in ewes which failed to lamb lost in ewes which lambed Eggs No. 162 324 158 49 eggs lost 20 eggs surviving 11 B. Losses in ewes which lambed Recipient ewes which lambed Eggs transferred to ewes which lambed Lambs born Eggs lost 83 166 11 49 100-0 48-8 151 63-9 36-1 1000 0-5 29-5 DISCUSSION that the three factors The absence of any significant interactions suggests studied, degree of synchronization between egg and tract, age of egg, and site of transfer act independently of one another. Maximum survival occurred in recipients first observed in oestrus within 12 hr of their respective donors. However, recipients did show some tolerance to eggs up to 2 days 'older' or 'younger' than themselves, with no evidence that either the older or younger reproductive tract a presented more suitable transfer site. In this respect the sheep is similar to the rabbit, somewhat similar to the rat, but dissimilar to the mouse. In the rabbit, Chang (1950) recorded maximum survival in recipients exactly synchronized with their donors, while Noyes & Dickman (1960, 1961) found that rat eggs of the same age or 1 day older than the uterus were more likely to survive than those 1 day younger. Maximum survival in the mouse was obtained by transferring 3 -day eggs to 2 -day recipients; it decreased with exactly synchronized transfers of both 2\- and 3J-day eggs, whereas virtually no eggs of 2\ days survived when transferred to 3J-day recipients (McLaren & Michie, 1956). Egg survival increased significantly with age of egg transferred. Whether younger eggs are more susceptible to the collection and transfer procedures, or to transfer itself, is not clear. However, it has been postulated (Robinson, 1951) and limited evidence has been presented (Averill & Rowson, 1958) that the uterus does not provide an environment suitable for the survival and develop ment of eggs of two and four cells.

Egg transfer in sheep 151 Tubai transfers were more successful than were uterine transfers. This appears to disagree with the observations of Moore & Rowson (1960) and Moore & Shelton (1962a). They reported the survival of eggs of less than eight cells transferred to the tubes to be significantly less than that of eggs of eight cells or more transferred to the uterus. They suggested that the difference was due to site of transfer. However, site of transfer and age of eggs were confounded. It is now clear that both factors are important. There are two possible explanations for the superiority of tubai transfers. First, further time spent in the tubes would result in an advance in the stage of development of eggs by the time they reached the uterus. Second, the tubai environment may condition the eggs for reception by the uterus. It is apparent that precise relationships between the age of eggs and their position within the reproductive tract must be satisfied if maximum survival is to occur. A general decrease in fertility is a characteristic of almost all work on the hormonal control of breeding in the ewe (Robinson, 1959) and in some instances is due to the premature appearance of eggs in the uterus. Both Robinson (1962) and Shelton (unpublished data) have evidence suggesting rapid tubai transport of eggs following progesterone treatment of the cyclic ewe. Egg losses had a strong tendency to fall on litters as a whole. That is, success or failure tended to be an all-or-none phenomenon. By analogy with the detailed analysis by Brambell (1948) on pre-natal losses in the wild rabbit, it can be concluded that when either the stage of the cycles of donor and recipient are out of phase, or the eggs are in the early stages of cleavage, or are placed directly into the uterus, the maternal environment in a high proportion of cases is so unsatisfactory as to result in total loss of the pregnancy. On the other hand, when the maternal environment is suitable, as in the eighty-three ewes which lambed, egg losses are at random and are not excessive. In order to obtain maximum survival of transferred eggs in the sheep, three conditions should be satisfied. First, eggs should be collected 2 hr or more after the onset of oestrus, second, transfers should be made to the Fallopian tubes, and third, recipients should be in oestrus no more than 12 hr before or after the donors. When these conditions are satisfied some 5% of recipients should subsequently lamb. ACKNOWLEDGMENTS This project was financed in part by the Sheep of Australia to whom grateful acknowledgment Mr B. J. Carlin provided and Wool Research Committe«is made. valuable technical assistance. REFERENCES Averill, R. L. W. & Rowson, L. E. A. (1958) Ovum transfer in sheep. J. Endocrin. 16, 326. Brambell, F. W. R. (1948) Prenatal mortality in mammals. Biol. Rev. 23, 30. Chang, M. C. (1950) Development and fate of transferred rabbit ova or blastocysts in relation to the ovulation time of recipients. J. exp. ool. 114, 19. Hunter, G. L., Adams, C. E. & Rowson, L. E. A. (1955) Inter-breed ovum transfer in sheep. J. agrie. Sci. 46, 143.

152 JV. W. Moore and J. JV. Shelton McLaren, A. & Michie, D. (1956) Studies on the transfer of fertilized mouse eggs to uterine fostermothers. I. Factors affecting the implantation and survival of native and transferred eggs. J. exp. Biol. 33, 394. Moore, N. W. & Rowson, L. E. A. (1959) Maintenance of pregnancy in ovariectomized ewes by means of progesterone. Nature, Lond. 184, 1410. Moore, N. W. & Rowson, L. E. A. (1960) Egg transfer in sheep. Factors affecting the survival and development of transferred eggs. J. Reprod. Fértil. 1, 332. Moore, N. W. & Shelton, J. N. (1962a) Oestrous and ovarian response of the ewe to a horse anterior pituitary extract. Nature, Lond. 194, 1283. Moore, N. W. & Shelton, J. N. (1962b) The application of the technique of egg transfer to sheep breeding. Aust. J. agrie. Res. 13, 18. Noyes, R. W. & Dickman, Z. (1960) Relationship ofovular age to endometrial development. J. Reprod. Fértil. 1, 186. Noyes, R. W. & Dickman,. (1961) Survival of ova transferred into the oviduct of the rat. Fértil. Steril. 12, 6. Radford, H. M., Watson, R. H. & Wood, G. F. (1960) A crayon and associated harness for detection of mating under field conditions. Aust. vet. J. 36, 5. Robinson, T. J. (1951) The control of fertility in sheep. Part II. The augmentation of fertility by gonadotrophin treatment of the ewe in the normal breeding season. J. agrie. Sci. 41, 6. Robinson, T. J. (1959) Reproduction in domestic animals, vol. 1, chap. 9. Ed. H. H. Cole & P. T. Cupps. Academic Press, New York. Robinson, T. J. (1962) The time of ovulation and efficiency of fertilization following progesterone and pregnant mare serum treatment in the cyclic ewe. J. agrie. Sci. 5, 129. Rowson, L. E. A. & Adams, C. E. (195) An egg transfer experiment in sheep. Vet. Ree. 69, 849.