Female common shrews in their first pregnancy were collected near Oxford (UK) from the hybrid zone between the Oxford and Hermitage races. These races differ by Robertsonian chromosomal rearrangements and both Robertsonian heterozygotes and homozygotes are found in the zone. The three homozygotes examined in this study had significantly more oocytes than the six heterozygotes. Among the heterozygotes, the number of oocytes tended to be lower in more severely heterozygous individuals; one double heterozygote was particularly depleted. Although there were, on average, 41% fewer primordial follicles in the ovaries of the heterozygotes than those of the homozygotes, there was no significant difference in the numbers of growing follicles between the karyotypic classes. These data suggest that Robertsonian heterozygotes may have a shorter reproductive lifespan than do homozygotes, but the numbers of follicles being recruited for ovulation at a particular instance during the fertile period does not appear to differ between homozygotes and heterozygotes. Morphological differences between the follicles of homozygotes and heterozygotes were not detected. Only 0.12% of healthy growing follicles were biovular.
J. M. Wallace, J. J. Robinson and R. P. Aitken
Summary. After lambing in late November, oestrus and ovulation were induced by using a CIDR device and PMSG in early weaned (N = 13) or lactating (N = 14) Border Leicester × Scottish Blackface ewes between 23 and 29 days after parturition. Ewes were intrauterine inseminated under laparoscopic visualization 54–55 h after CIDR-device withdrawal and eggs recovered on Day 3 of the cycle. Ovum recovery and fertilization rates were higher in lactating than in early weaned ewes, with fertilization being achieved as early as 24 days post partum in both groups. Of the 7 early weaned and 11 lactating ewes yielding eggs, fertilization occurred in 4 and 7 ewes respectively. A total of 20 embryos were transferred to the normal uterine environment of 15 recipient ewes in which the interval from parturition was > 150 days. Pregnancies were successfully established in 9 recipient ewes, resulting in the birth of 10 viable lambs.
Prolactin concentrations were significantly higher (P < 0·001) in lactating than in early weaned ewes throughout the study. Nevertheless, normal luteal function (as assessed by daily progesterone concentrations) was exhibited by 12 of 14 lactating and 8 of 13 early weaned ewes. Two post-partum donors in which the corpora lutea completely failed to secrete progesterone yielded fertilized eggs which developed to term when transferred to a normal uterine environment.
The results show that sheep oocytes can be fertilized using laparoscopic intrauterine insemination as early as 24 days after parturition and that the resulting embryos are viable when recovered on Day 3 after oestrus and transferred to a normal uterine environment.
Keywords: post partum; fertilization; embryo viability; pregnancy; sheep
J. M. Wallace, R. P. Aitken and M. A. Cheyne
We examined the key events underlying maternal recognition of pregnancy and the prevention of luteolysis in early postpartum ewes by synchronously transferring single expanded blastocysts recovered from control ewes on day 11 of pregnancy into the uterus of either postpartum recipients that had been induced to ovulate 28 days after lambing (n = 12) or control recipients (n = 11). Conceptus development, uterine flush interferon (oTP-1) concentrations, endometrial oxytocin receptor concentrations and endometrial prostaglandin F2α (PGF2α) release in vitro were determined 5 days later (corresponding to day 16 of the ovarian cycle). By this stage, both conceptus mass and oTP-1 content of total uterine flush in the eight postpartum recipients that remained pregnant were significantly lower (P < 0.01) than in the eight pregnant control ewes (524 ± 116.6 versus 959 ± 80.6 mg and 968 ± 16.9 versus 1512 ± 106.2 ng oTP-1 for postpartum and control recipients, respectively). These effects were independent of ovulation rate and daily peripheral progesterone concentrations after blastocyst transfer, which were similar between groups. Endometrial oxytocin receptor density was variable in both groups when they were killed, and was generally higher in pregnant postpartum than in control recipients, and was significantly different (P < 0.05) when the values for the transfer but not the contralateral uterine horns were compared. Similarly, basal and oxytocin-stimulated endometrial PGF2α release during a 4 h incubation were higher (P < 0.01) in pregnant postpartum versus control recipients. Irrespective of treatment group and when expressed per uterine horn, conceptus mass was highly negatively correlated with number of oxytocin receptors and PGF2α release in vitro. The results of this study imply that suboptimal conceptus growth rates and secretion of oTP-1, resulting in an inability to regulate endometrial oxytocin receptor-mediated PGF2α secretion, may be central to pregnancy failure in the early postpartum ewe.
J. M. Wallace, R. P. Aitken and M. A. Cheyne
A highly controlled model to investigate nutrient partitioning and the control of fetal growth in the rapidly growing adolescent sheep is described. Embryos recovered from superovulated adult ewes inseminated by a single sire were transferred in singleton to the uterus of prepubertal adolescent recipients induced to ovulate at 21 weeks of age (liveweight 44.4 ± 0.38 kg). After embryo transfer, the adolescent recipients were individually offered a high (n = 28) or low (n = 20) quantity of a complete diet calculated to achieve rapid (RMG) or normal (NMG) maternal growth rates. After day 100 of gestation the feed intake of the NMG group was adjusted weekly to meet the increasing nutrient demands of the gravid uterus. The proportion of adolescent recipients initially conceiving was significantly (P < 0.01) influenced by maternal nutrient intake and was lower in the RMG (0.57) than in the NMG (0.85) group. For adolescent dams that maintained their pregnancies, liveweight gain during the first 95 days of gestation was significantly (P < 0.001) higher in the RMG compared with the NMG group (234 ± 9.5 and 75 ± 5.0 g day−1, respectively). Rapid maternal growth rates were associated with a significant reduction in both fetal and placental weights as determined when the animals were killed on day 95 of gestation (n = 3 per group) or at term. For the RMG (n = 8) and NMG (n = 11) groups, respectively, mean lamb birthweights at term were 2.74 ± 0.25 and 4.34 ± 0.27 kg (P < 0.001), while term placental weights were 263 ± 16.8 and 438 ± 44.6 g (P < 0.002). The number of fetal cotyledons per placenta and mean fetal cotyledon weight were significantly lower in RMG compared with NMG ewes (P < 0.05). Irrespective of treatment group, lamb birthweight was highly positively correlated with placental weight and both parameters were negatively correlated with maternal liveweight gain during the first 100 days of gestation. The incidence of non-infectious spontaneous abortion at 125 ± 1.3 days of gestation was higher (P < 0.001) in the RMG (4 of 12) than in the NMG (1 of 12) group. Similarly, duration of gestation for those ewes delivering live young was shorter (P < 0.01) in the RMG compared with the NMG group (140 ± 0.94 versus 143 ± 0.28 days). Colostrum yield at parturition was positively related to placental weight and significantly lower (P < 0.001) in the RMG than in the NMG group (35 ± 12.1 and 247 ± 36.2 g, respectively). Neonatal survival rates at 72 h after parturition were reduced (P < 0.05) in the RMG (38%) compared with the NMG group (91%). These data suggest that in rapidly growing adolescent ewes, the established anabolic drive to maternal tissue synthesis is maintained at the expense of the gradually evolving nutrient requirements of the gravid uterus. This results in a major restriction in placental growth and a highly significant decrease in birthweight.
M. J. Harris, M. E. Wallace and E. P. Evans
Summary. The spontaneous appearance of a Robertsonian translocation in a laboratory colony of genetically wild Peru—Coppock mice gave the opportunity to study potential meiotic nondisjunction soon after the formation of the new chromosome and also in a hitherto untested combination of genotype and environment. Metaphase II scores from the progenitor male had indicated a nondisjunction rate of approximately 10%, a figure that was confirmed by the finding of an estimated 12–16% total trisomic and probable monosomic zygotes in chromosomal studies of Day 9 embryos from heterozygous females. The chromosome studies also showed the presence of a significant excess of normal embryos that were heterozygous for the Robertsonian chromosome.
Jacqueline M. Wallace, J. J. Robinson and R. P. Aitken
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
J. J. Robinson, Jacqueline M. Wallace and R. P. Aitken
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
L. M. Mitchell, M. E. King, R. P. Aitken, F. E. Gebbie and J. M. Wallace
The objective of this study was to determine the relative importance of seasonal changes in ovulation rate, fertilization rate and embryo survival as the cause of reduced lambing rates in ewes mated in February compared with those mated in November. The study was conducted at 57°N using mature Mule ewes and Suffolk rams. Sixty ewes were allocated equally to five groups: unbred (UB) or mated at a natural oestrus during November (N) or February (F) by natural (N) or cervical artificial (A) insemination. Groups were maintained separately at pasture supplemented with hay. A raddled vasectomized or non-vasectomized ram was present with UB, NN and NA groups from 26 October 1995 to 1 January 1996 and with UB, FN and FA groups from 25 January 1996 to 31 March 1996. Ewes marked by the ram were recorded twice a day, and those in groups NN, NA, FN and FA were inseminated at their second behavioural oestrus. For all ewes, blood samples were obtained once a day from introduction of the vasectomized rams until 30 days after mating (groups NN, NA, FN and FA) or 20 days after the first oestrus (group UB), and ovulation rate was measured by laparoscopy 7 days after the first oestrus. For ewes in groups NN, NA, FN and FA, ovulation rate was measured again after the second oestrus and ova were recovered from six ewes per group for assessment of fertilization before autotransfer. Pregnancy and lambing rates were recorded at term. Mean (± se) dates of the first recorded oestrus for ewes in groups NN, NA and UB, and FN, FA and UB were 4 ± 1.1 November and 4 ± 0.9 February, respectively, and intervals between the first and second oestrus were 16 ± 0.2 and 17 ± 0.3 days (P < 0.01), respectively. Ovulation rates were 2.6 ± 0.08 and 2.0 ± 0.05 (P < 0.001), and peripheral progesterone concentrations during the luteal phase were 8.5 ± 0.25 and 7.6 ± 0.31 ng ml−1 (P < 0.05), for November and February, respectively. The difference in peripheral progesterone concentration was not solely attributable to the difference in ovulation rate. There was no significant effect of month or method of insemination, or of embryo recovery and autotransfer procedures on pregnancy rates and the proportion of ewes that became pregnant were NN 0.92, NA 0.83, FN 0.67 and FA 0.75. For ewes undergoing embryo recovery and autotransfer, ova recovered per corpus luteum were 1.00, 0.93, 1.00 and 0.92, fertilized ova per ovum recovered were 0.69, 0.92, 1.00 and 0.83, and lambs born per corpus luteum were 0.62, 0.79, 0.78 and 0.58 for NN, NA, FN and FA groups, respectively. There were no significant seasonal effects on fertilization rate or embryo survival. It is concluded that a seasonal decline in ovulation rate is the primary cause of reduced lambing rates in ewes mated in February compared with those mated in November. Pregnancy rates were high after mating in both periods and were not enhanced by the use of cervical insemination.
J. J. Robinson, J. M. Wallace, R. P. Aitken and A. S. McNeilly
Thirty-two Scottish Blackface ewes that lambed outdoors in March and were weaned at the end of April and individually penned indoors under the natural photoperiod at 57°N were used to determine whether the ovine ovary that was deprived of gonadotrophic support was capable of early activation by melatonin. From 5 May (day 0), 16 of the ewes received an oral dose of 3 mg melatonin in a 4:1 (v:v) mixture of water and ethanol daily at 15:00 h. The remaining 16 ewes received the vehicle alone. Within each of these groups, eight were implanted s.c. on day 0 with an osmotic minipump which infused 50 μg of the gonadotrophin releasing hormone agonist (GnRHa), buserelin day−1. On day 25, a second minipump was inserted to ensure continued infusion of the agonist and on day 50 (24 June) both minipumps were removed. Ovarian activity was assessed by laparoscopy at intervals of 3 weeks from day 29 until the experiment was terminated on day 200 (21 November). Blood samples taken by jugular venepuncture three times a week for the first 50 days, daily from days 51–78 and thereafter three times a week were analysed for progesterone, prolactin and LH. Samples taken at intervals of 15 min for 10 h on days −1, 14, 28, 49, 56, 70 and 91 were assayed for LH. Treatment with GnRHa reduced LH concentrations and abolished pulsatile LH secretion. The onset of ovarian activity (progesterone >3.8 nmol l−1) was not affected by the 50-day GnRHa treatment and occurred for the melatonin-treated ewes at mean (± sem) intervals from 5 May of 66 ± 2.9 (range 51–75) and 71 ± 0.9 (range 68–75) days for non-GnRHa and GnRHa ewes, respectively. For the ewes not receiving melatonin the corresponding intervals were 113 ± 11.6 and 125 ± 9.2 days, respectively. The mean numbers of corpora lutea at first oestrus were not affected by GnRHa treatment and were 1.4 ± 0.13 and 1.5 ± 0.13 for control and melatonin-treated ewes, respectively. First ovulation following GnRHa treatment resulted in luteal concentrations of progesterone of normal duration and magnitude but, within the melatonin-treated ewes, those that received GnRHa returned to anoestrus 21 days earlier (P < 0.03) than did their non-GnRHa counterparts. After their initial suppression by melatonin, prolactin concentrations began to increase after 80 days of melatonin treatment in both non-GnRHa and GnRHa ewes and approximately three months before the ewes returned to anoestrus. The results demonstrate that a period of isolation of the ovaries of the ewe from the pituitary gonadotrophins does not alter their ability to respond to the melatonin-induced activation of the GnRH pulse generator.
J. J. Robinson, J. M. Wallace, R. P. Aitken and S. Wigzell
Summary. Forty-two Scottish Blackface ewes that lambed outdoors in March were removed from their lambs at the end of April and housed under natural daylength at 57°N. Treatments (n = 7 ewes per treatment) commenced on 1 May and comprised daily oral dosing at 15:00 h with 3 mg melatonin dissolved in water and ethanol (4:1, v/v) for 30, 60, 90, 120 or 150 days. Control ewes received the vehicle alone. Ovarian activity was assessed by laparoscopy at monthly intervals with an additional interim observation in mid-July. Blood was sampled three times a week by jugular venepuncture and assayed for progesterone, prolactin and follicle-stimulating hormone (FSH). Luteinizing hormone (LH) was determined in blood samples collected at 15 min intervals for 10 h on days 28, 60, 91, 119 and 150.
Thirty days of melatonin treatment delayed (P < 0·01) first ovulation by about 1 month (mean interval ± sem from 1 May to progesterone > 1 ng ml−1, 165 ± 4·5 days versus 132 ± 9·2 days for controls). None of the ewes that received melatonin for 60 days ovulated before the end of melatonin treatment, but subsequently six of them did; the mean interval from 1 May to increased progesterone concentration was 75 ± 1·2 days. All ewes receiving melatonin for 90, 120 and 150 days ovulated with corresponding mean intervals of 83 ± 2·7, 85 ± 1·3 and 87 ± 2·2 days, respectively (P < 0·001 compared with controls). Ewes treated for 60, 90 and 120 days returned to anoestrus 4–6 weeks after the end of melatonin treatment; for the 120 day treatment this coincided with the onset of oestrous cyclicity in control ewes.
Melatonin had no effect on the concentrations of FSH. Thirty days of melatonin treatment in May delayed the autumn decline in LH pulse amplitude and increase in pulse frequency, whereas treatment for 120 and 150 days advanced these changes to late July–early August. Prolactin concentrations declined to minimum values after 30 days of melatonin treatment, but began to increase again after about 100 days. After the treatments for 90, 120 and 150 days, prolactin increased to higher concentrations (P < 0·001) than in controls and failed to return to control values before sampling ended in late October, i.e. 95, 65 and 35 days later, respectively.
Keywords: melatonin; ovulation; luteinizing hormone; prolactin; ewe