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J. N. SHELTON and N. W. MOORE

Summary.

Control of the onset of oestrus in ewes by the daily intramuscular injection of progesterone had no effect on the survival of fertilized eggs transferred at the rate of two per ewe to the Fallopian tubes or the uterus. More ewes lambed and more lambs were born following transfer of eight (or more) cell eggs to the uterus than after transfer of eggs of less than eight cells to the Fallopian tubes.

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N. W. MOORE and J. N. SHELTON

Summary.

A factorial experiment is described in which fertilized sheep eggs were transferred at a rate of two per animal to 162 recipient ewes all of which were allowed to go to term.

The factors included and the results obtained were:

  1. Degree of synchronization 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<0·01) and number of eggs surviving to parturition (P<0·001), with maximum results in ewes in oestrus 12 hr before to 12 hr after their respective donors.
  2. Age of eggs. Eggs were collected 48 to 60, 60 to 72 and 72 to 84 hr after the donors were first observed in oestrus. There was a significant increase in the number of ewes which lambed (P<0·02) and lambs born (P<0·01) 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<0·05) and number of lambs born (P<0·01).

There were no significant interactions.

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J. N. SHELTON and N. W. MOORE

Summary.

When compared in ewes at three dose-rates horse anterior pituitary extract (hap) produced a different ovarian response to that of pregnant mare serum gonadotrophin (pmsg). Although there was no difference in number of ovulations, there was a greater number of persistent follicles after pmsg. Because persistent follicles are associated with a decreased percentage of fertilized ova, hap is more suited than pmsg to the production of large numbers of fertilized sheep ova.

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N. W. MOORE and J. N. SHELTON

Summary.

A trial of factorial design is described, in which was studied the response of the cyclic ewe to a horse anterior pituitary extract (hap) Three total-dose levels of hap (60, 90 and 135 mg) were administered over 2 or 3 days commencing on the 12th day of the oestrous cycle. The total dose was given as either two or three equal daily injections, or four or six equal twice-daily injections.

Highly significant (P<0·001) linear dose-response relationships were observed in the ovulatory response to both 2- and 3-day treatments. Treatment for 3 days was significantly better than treatment for 2 days in the proportion of ewes ovulating (P<0·05), ovulation rates (P<0·001) and fertilization of eggs shed (P< 0·001).

For both 2- and 3-day treatments the ovarian responses varied with the time elapsing between the final hap injection and the onset of oestrus. Maximum responses were observed in ewes in oestrus 24 to 48 hr after the cessation of treatment. Fertilization in ewes treated for 2 days, but not in those treated for 3 days, also varied with the time elapsing between the cessation of treatment and onset of oestrus.

The results showed hap to be a most effective preparation for the induction of multiple ovulation in the ewe.

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A. O. TROUNSON and N. W. MOORE

Summary.

The effect of `Protease' on the zona pellucida of fertilized and unfertilized sheep eggs was examined, and the effects of the treatment and of mechanical removal of part of the zona on the subsequent development of fertilized eggs was assessed in vitro and in vivo.

The zonae of follicular eggs were digested by `Protease' within 25 min of exposure. In ovulated eggs, the resistance of the zona to digestion decreased with age. Zonae of eggs collected 2 days after oestrus (Day 2) were extremely resistant whereas nearly all the Day-5 eggs lost their zonae within 10 min. Fertilization had little effect upon the susceptibility of zonae to digestion by `Protease'.

Neither age of egg nor method of treating the zona had any effect on subsequent development of fertilized Day-2 and Day-6 eggs in culture, but there were effects of both factors on the proportion of eggs which developed to normal Day-25 embryos. `Protease' treatment of Day-2 and Day-3 eggs failed to digest the zona completely and a high proportion of such eggs developed to normal embryos whereas only a few eggs of similar age, in which part of the zona was removed mechanically, developed normally. The zonae of Day-4 to Day-6 eggs were invariably removed by `Protease' and the method of treatment had no effect on the proportion of eggs which developed to normal embryos. Irrespective of method of treatment, more Day-6 than Day-4 eggs developed to normal embryos. Culture for 2 days following treatment did not increase the survival of Day-6 eggs when they were transferred to recipients.

Ovulation and ageing of eggs appear to be associated with changes in the zona pellucida which influence its susceptibility to digestion by proteolytic enzymes. In early cleavage stage eggs, the major rôle of the zona may be protection of the inner cell mass from the uterine environment.

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J. P. Ryan and N. W. Moore

Summary. Embryos collected from donor ewes 2 days after oestrus were transferred to the oviducts of entire cyclic (Group EC), unilaterally ovariectomized and cyclic (Group UO), entire anoestrous (Group EA), and bilaterally ovariectomized (Group BO) ewes, and 4 h, 1, 3 or 5 days after transfer the oviducts and uteri were flushed to recover embryos. Ewes in Group BO were untreated or treated with regimens of progesterone and oestradiol designed to simulate ovarian secretion before, around the time of, and after oestrus in entire ewes.

There were no differences in the proportions of transferred embryos that were recovered, or in their location (oviduct or uterus), between the two sides of Group UO ewes and they were similar to recovery rates and locations of embryos in Group EC ewes. At 3 days after transfer, 62% and 50%, respectively, of embryos recovered from ewes in Groups EC and UO were in the uterus and by 5 days the percentages had risen to 89% and 75%, respectively.

With all treatment regimens fewer of the transferred embryos were recovered from Group BO ewes than from Group EC ewes and few were located in the uterus. In Group BO ewes low recovery rates, and failure of embryos to enter the uterus, appeared to be due to deficiencies in the treatment regimens rather than to effects of ovariectomy.

Most embryos recovered from treated ewes in Group BO and those in Groups EC and UO showed apparently normal development (86% and 79%, respectively), while 65% and 75%, respectively, recovered from untreated Group BO and Group EA ewes had developed normally. Only 9 of 163 embryos recovered from the untreated Group BO and EA ewes were located in the uterus and 8 of the 9 had failed to develop normally. Clearly, the steroid hormone requirements for development in the oviducts are not critical, but this is not so for the uterus.

Keywords: ewe; embryo; oviduct; transport; development

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R. J. Bilton and N. W. Moore

Several laboratories have reported the birth of young from frozen–thawed mouse embryos transferred to recipient females (Whittingham, Leibo & Mazur, 1972; Wilmut, 1972) and frozen mouse embryos have been successfully transported from North America to the United Kingdom (Whittingham & Whitten, 1974). Of the domestic species, continued development in vivo and in vitro has been achieved with cattle (Wilmut & Rowson, 1973; Bilton & Moore, 1976a), sheep (Moore & Bilton, 1976; Willadsen, Polge, Rowson, & Moor, 1976) and goat (Bilton & Moore, 1976b) embryos stored in liquid N2, and the potential value of frozen storage for the transport of embryos has been noted.

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B. G. Miller and N. W. Moore

Summary. The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus.

The rate of protein synthesis and tissue RNA: DNA and protein: DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2– 4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4–6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues.

The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4–7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.

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R. K. Munro and N. W. Moore

Summary. The effects of treatment with intravaginal progesterone (PRID) oestradiol benzoate and cloprostenol at various stages of the oestrous cycle were examined in 2 experiments. In Exp. 1, PRIDs were inserted for 14 days commencing on Day 3, 10 or 17 of the oestrous cycle and half the animals in each group received 5 mg oestradiol benzoate at PRID insertion. Plasma samples were collected daily from the time of PRID insertion for 16 days, then every 4 days for a further 24 days. Samples were analysed for plasma progesterone concentration. In Exp. 2, heifers were treated on Day 10 as in Exp. 1, but half of each treatment group received 750 μg cloprostenol at PRID insertion. In Exp. 1, treatment with PRID alone appeared to inhibit endogenous progesterone production when treatment began on Day 3, but not when treatment began on Days 10 or 17. Treatment with oestradiol benzoate at the time of PRID insertion depressed progesterone levels, from about 2–5 days after injection. When treatment began on Day 10, the fall in progesterone levels after oestradiol benzoate was preceded by a marked rise in levels. In Exp. 2, treatment with cloprostenol markedly reduced peripheral concentrations of progesterone and also removed the effect of oestradiol. We suggest that oestradiol benzoate had a luteolytic effect, depressing luteal progesterone production when given on Days 3, 10 or 17 and that when given on Day 10 there was a transitory luteotrophic effect which preceded its luteolytic action.

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I. D. KILLEEN and N. W. MOORE

Summary.

Two experiments are described in which were studied the morphological appearance and the subsequent development of sheep ova fertilized by uterine insemination. Ewes were naturally mated, or inseminated with freshly ejaculated semen, either whole or following removal of the seminal plasma, or with semen recovered from the uteri of naturally mated ewes ('uterine semen'). Fertilized eggs were collected after insemination or mating and were either stained and examined or transferred to recipient ewes.

Uterine insemination with whole or fractionated semen gave very high rates of fertilization. 'Uterine semen' gave a low fertilization rate due to poor quality samples. The proportion of eggs fertilized by uterine insemination which developed into lambs or embryos was similar to that of eggs collected from ewes naturally mated. Treatment of semen before insemination had no effect upon subsequent development of fertilized eggs.

More than 50 % of the fertilized eggs contained anucleate particles, but neither the method of mating (uterine insemination or natural mating) nor treatment of semen before insemination had any major effect upon the incidence of such eggs. The presence of anucleate particles did not preclude subsequent normal development.

It is concluded that the low lambing percentages recorded following uterine insemination are not due to fertilization failure or abnormalities of fertilization, but to surgical interference with the tract resulting in expulsion or rapid transport of fertilized eggs.