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Summary. Administration of charcoal-treated bovine follicular fluid to Damline ewes twice daily (i.v.) from Days 1 to 11 of the luteal phase (Day 0 = oestrus) resulted in a delay in the onset of oestrous behaviour and a significant increase in ovulation rate following cloprostenol-induced luteolysis on Day 12. During follicular fluid treatment plasma levels of FSH in samples withdrawn just before injection of follicular fluid at 09:00 h (i.e. 16 h after previous injection of follicular fluid) were initially suppressed, but by Day 8 of treatment had returned to those of controls. However, the injection of follicular fluid at 09:00 h on Day 8 still caused a significant suppression of FSH as measured during a 6-h sampling period. Basal LH levels were higher throughout treatment due to a significant increase in amplitude and frequency of pulsatile secretion. After cloprostenol-induced luteal regression at the end of treatment on Day 12, plasma levels of FSH increased 4-fold over those of controls and remained higher until the preovulatory LH surge. While LH concentrations were initially higher relative to those of controls, there was no significant difference in the amount of LH released immediately before or during the preovulatory surge. These results suggest that the increase in ovulation rate observed during treatment with bovine follicular fluid is associated with the change in the pattern of gonadotrophin secretion in the luteal and follicular phases of the cycle.

Summary. Treatment of Damline ewes with twice daily i.v. injections of bovine follicular fluid during the luteal phase for 10, 6 or 2 days before prostaglandin-induced luteolysis resulted in an increase in ovulation rate. This was associated with a large rebound increase in plasma concentrations of FSH after the last injection of bovine follicular fluid. While conception rate was not affected by bovine follicular fluid treatment, a higher percentage embryonic loss was observed between Days 3 and 34 of pregnancy in the 10-day treatment group only compared to controls. This reflected the increase in ovulation rate above the optimum for embryonic survival in this breed. The present results suggest that the increase in ovulation rate induced by bovine follicular fluid treatment in the luteal phase of the cycle before mating would result in a significant increase in the number of lambs born.

Summary. In Exp. 1, 40 ewes were used in a 2 × 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P < 0·01) decrease in egg recovery of over 40% compared with cervically inseminated ewes.

In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria.

In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P < 0·05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery.

From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.

Keywords: superovulation; intrauterine insemination; fertilization; ovum recovery; ewe

Summary. In Exp. 1 the effect of lactation versus early weaning on luteal function was examined in seasonally anoestrous Finn Dorset ewes that were induced to ovulate at 21 (N = 14) or 35 (N = 14) days post partum by using a CIDR device and PMSG. Prolactin concentrations were significantly higher (P < 0·001) in lactating compared with early weaned ewes throughout the study. The proportion of lactating ewes with inadequate luteal function (as assessed by daily progesterone concentrations) in the 21-day group was 0·43 (3 or 7) compared with 0·67 (4 of 6) for those weaned within 2 days after parturition. Corresponding values for the 35-day group were 0 (0 of 4) and 0·14 (1 of 7) respectively. There was no evidence of abnormal luteal function in standard ewes (N = 8) for which the interval from parturition was > 150 days.

In Exp. 2 we examined whether pregnancy can be successfully established during the breeding season following transfer of embryos into lactating or early weaned ewes in the early post-partum period. Embryos were donated from Border Leicester × Scottish Blackface ewes for which the interval from previous parturition was > 150 days. These embryos were transferred synchronously on Day 5 after behavioural oestrus to recipient ewes with the same breeding history as the donors (standard ewes, N = 15) or to lactating or early weaned recipients that had been induced to ovulate on Day 21 (N = 16) or 35 (N = 24) post partum. In the 21-day group inadequate luteal function was observed in 2 of 7 (0·28) lactating and 4 of 9 (0·44) early weaned ewes compared with corresponding values of 1 of 13 (0·08) and 2 of 11 (0·18) in the 35-day post-partum group. Luteal function was normal in all standard ewes. The proportion of successful pregnancies in the standard ewes was 0·80 (12 of 15) compared with 0 in lactating and early weaned ewes in the 21-day group and 0·08 (1 of 13) and 0·36 (4 of 11) respectively in the 35-day group.

The incidence of inadequate luteal function is therefore independent of the suckling stimulus and is higher in ewes induced to ovulate on Day 21 than Day 35 post partum during breeding and non-breeding seasons. For early post-partum recipient ewes with normal luteal function it is suggested that the high incidence of pregnancy failure after transfer of embryos may be due to embryo mortality caused by an inappropriate uterine environment or the inability of the embryo to sustain its luteotrophic signal.

Keywords: post partum; corpus luteum; embryo transfer; pregnancy; uterus; ewe

Low birthweight is a risk factor for later adverse health. Here the impact of placentally mediated prenatal growth restriction followed by postnatal nutrient abundance on growth, glucose metabolism and body composition was assessed in both sexes at key stages from birth to mid-adult life. Singleton-bearing adolescent dams were fed control or high nutrient intakes to induce normal or growth-restricted pregnancies respectively. Restricted lambs had ~40% reduced birthweight. Fractional growth rates were higher in restricted lambs of both sexes predominantly during suckling/juvenile phases. Thereafter, rates and patterns of growth differed by sex. Absolute catch-up was not achieved and restricted offspring had modestly reduced weight and stature at mid-adulthood necropsy (~109 weeks). Dual-energy X-ray absorptiometry revealed lower bone mineral density in restricted vs normal lambs at 11, 41, 64 and 107 weeks, with males > females from 41 weeks onwards. Body fat percentage was higher in females vs males throughout, in restricted vs normal lambs at weaning (both sexes) and in restricted vs normal females at mid-adulthood. Insulin secretion after glucose challenge was greater in restricted vs normal of both sexes at 7 weeks and in restricted males at 32 weeks. In both sexes, fasting glucose concentrations were greater in restricted offspring across the life course, while glucose area under the curve after challenge was higher in restricted offspring at 32, 60, 85 and 106 weeks, indicative of persistent glucose intolerance. Therefore, prenatal growth restriction has negative consequences for body composition and metabolism throughout the life course with the effects modulated by sex differences in postnatal growth rates, fat deposition and bone mass accrual.

The influence of maternal obesity during oocyte development and its putative interaction with nutrient reserves at conception on pregnancy outcome were examined in an adolescent sheep model. Donor ewes were nutritionally managed to achieve contrasting adiposity (control (CD)/obese (ObD)) for 6 weeks prior to superovulation and inseminated by a non-obese sire. Morulae from 6 CD and 7 ObD were transferred in singleton into adolescent recipients of identical age but differing adiposity, classified as relatively fat or thin respectively. Thereafter, all were overnourished to promote rapid growth/adiposity (2 × 2 design, 13/14 pregnancies/group). A fifth recipient group of intermediate adiposity received embryos from another 5 CD, was offered a moderate intake to maintain adiposity throughout gestation and acted as controls for normal pregnancy outcome (optimally treated control (OTC), 19 pregnancies). Donor obesity did not influence ovulation, fertilisation or recovery rates or impact embryo morphology. Gestation length and colostrum yield were unaffected by donor or recipient adiposity and were reduced relative to OTC. Total fetal cotyledon and lamb birth weights were independent of initial donor adiposity but reduced in relatively thin vs relatively fat recipients and lower than those in the OTC group. In spite of high placental efficiency, the incidence of fetal growth restriction was greatest in the thin recipients. Thus, maternal adiposity at conception, but not pre-conception maternal obesity, modestly influences the feto-placental growth trajectory, whereas comparison with the OTC indicates that high gestational intakes to promote rapid maternal growth remain the dominant negative influence on pregnancy outcome in young adolescents. These findings inform dietary advice for pregnant adolescent girls.

The competition for nutrients when pregnancy coincides with continuing growth in biologically immature adolescent girls increases their risk of preterm delivery and low birthweight and is partly replicated in the overnourished adolescent sheep paradigm. Although overfeeding to promote rapid maternal growth robustly leads to a reduction in average birthweight relative to slow-growing control-fed adolescents of equivalent age, the extent of prenatal compromise is variable. This retrospective analysis of a large cohort of identically managed pregnancies determined whether maternal anthropometry predicts the severity of fetal growth-restriction (FGR) in growing adolescents. Singleton pregnancies were established by embryo transfer in adolescents subsequently control-fed (n = 96) or overnourished. The latter pregnancies were classified as non-FGR (n = 116) or FGR (n = 96) if lamb birthweight was above or below the optimally fed control mean minus 2SD. A similar approach categorised placental growth-restriction (PlGR) and preterm delivery. Gestation length, placental mass and lamb birthweight were FGR < non-FGR < control (post hoc P < 0.01). Relative to the non-FGR group, overnourished dams with FGR were marginally leaner and lighter at conception (P = 0.023/P = 0.014) and had greater gestational weight gain (GWG) during the first-third of pregnancy (P < 0.001). GWG during this early period was also higher in PlGR compared with non-PlGR, and in very preterm vs term deliveries (P < 0.01). Likewise maternal leptin concentrations (fat accrual biomarker) were FGR > non-FGR by day 60, and changes in leptin throughout pregnancy predicted attenuated fetal cotyledon mass and birthweight (P = 0.01 to <0.001). The anthropometric antecedents of FGR in still-growing adolescent sheep originate in early pregnancy coincident with early placental development.

To establish the basis for altered placental development and function previously observed at late gestation, fetoplacental growth and placental vascular development were measured at three stages of gestation in a nutritional paradigm of compromised pregnancy. Singleton pregnancies to a single sire were established and thereafter adolescent ewes were offered an optimal control (C) or a high (H) dietary intake. At day 50, the H group had elevated maternal insulin and amniotic glucose, whereas mass of the fetus and placenta were unaltered. At day 90, the H group exhibited elevated maternal insulin, IGF1 and glucose; fetal weight and glucose concentrations in H were increased relative to C, but placental weight was independent of nutrition. By day 130, total placentome weight in the H group was reduced by 46% and was associated with lower fetal glucose and a 20% reduction in fetal weight. As pregnancy progressed from day 50 to 130, the parameters of vascular development in the maternal and fetal components of the placenta increased. In the fetal cotyledon, high dietary intakes were associated with impaired vascular development at day 50 and an increase in capillary number at day 90. At day 130, all vascular indices were independent of nutrition. Thus, high dietary intakes to promote rapid maternal growth influence capillary development in the fetal portion of the placenta during early to mid-pregnancy and may underlie the subsequent reduction in placental mass and hence fetal nutrient supply observed during the final third of gestation.

Overnourishing pregnant adolescent sheep promotes maternal growth but reduces placental mass, lamb birth weight and circulating progesterone. This study aimed to determine whether altered progesterone reflected transcript abundance for StAR (cholesterol transporter) and the steroidogenic enzymes (Cyp11A1, Hsd3b and Cyp17). Circulating and placental expression of ovine placental lactogen (oPL) was also investigated. Adolescent ewes with singleton pregnancies were fed high (H) or moderate (M) nutrient intake diets to restrict or support placental growth. Experiment 1: peripheral progesterone and oPL concentrations were measured in H (n=7) and M (n=6) animals across gestation (days 7–140). Experiment 2: progesterone was measured to mid- (day 81; M: n=11, H: n=13) or late gestation (day 130; M: n=21, H: n=22), placental oPL, StAR and steroidogenic enzymes were measured by qPCR and oPL protein by immunohistochemistry. Experiment 1: in H vs M animals, term placental (P<0.05), total cotyledon (P<0.01) and foetal size (P<0.05) were reduced. Circulating oPL and progesterone were reduced at mid- (P<0.001, P<0.01) and late gestation (P<0.01, P<0.05) and oPL detection was delayed (P<0.01). Experiment 2: placental oPL was not altered by nutrition. In day 81 H animals, progesterone levels were reduced (P<0.001) but not related to placental or foetal size. Moreover, placental steroidogenic enzymes were unaffected. Day 130 progesterone (P<0.001) and Cyp11A1 (P<0.05) were reduced in H animals with intrauterine growth restriction (H+IUGR). Reduced mid-gestation peripheral oPL and progesterone may reflect altered placental differentiation and/or increased hepatic clearance respectively. Restricted placental growth and reduced biosynthesis may account for reduced progesterone in day 130 H+IUGR ewes.