I.S. Robertson

Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors

In the first of two experiments investigating the effect of dietary urea on the survival and metabolism of ovine embryos, 30 Border Leicester x Scottish Blackface ewes received a maintenance diet (milled hay, molasses, minerals, vitamins) with no urea (control, C; n = 10) or with added urea at 15 g (low urea, LU; n = 10) or 30 g (high urea, HU; n = 10) kg-1 feed for a 12 week period. The degraded nitrogen (N) status relative to estimated rumen microbial N requirements was -2, +9 and +20 g per day, respectively. One week after allocation to diets, progesterone priming (12 days) commenced. Ewes received 800 IU of equine chorionic gonadotrophin at progesterone withdrawal, were inseminated 52 h later (Day 0) and embryos were collected from five ewes per group at Day 4 and from five ewes at Day 11. If available, one embryo was returned to each ewe; the rest were cultured in vitro. There was no effect of treatment on progesterone, luteinizing hormone (LH), or time of oestrus onset C, LU and HU plasma urea (P < 0.001) and ammonia levels (C vs. HU, P < 0.01; LU vs. HU, P < 0.05) differed. Day 4 HU embryos were retarded relative to C and LU embryos. After 3 days of culture, 70%, 66% and 0% of C, LU and HU embryos, respectively, were viable. Mid-term pregnancy rates following transfer were 63%, 43% and 33%. Only one HU lamb (male) was born following embryo transfer, its birthweight (10.1 kg) exceeded that of its C (n = 3; 7.0, 7.0, 7.5 kg) and LU (n = 2; 7.3, 8.2 kg) counterparts (P < 0.025). In the second experiment, C2 (2.5 g urea kg-1; n = 5) and HU2 (30 g kg-1; n = 7) diets which provided similar intakes of degraded N relative to microbial requirements as those for C and HU ewes in Experiment 1 were fed to Border Leicester x Scottish Blackface ewes superovulated with 16 mg of porcine follicle-stimulating hormone. Urea and ammonia levels in utero-oviductal samples were elevated in HU2 ewes (P < 0.05). At collection (Day 3), HU2 embryos used more glucose (P < 0.01) and, following culture, some exhibited up to a 2.8-fold increase in metabolism. In conclusion, excess rumen degradable N in ewe diets elevates urea and ammonia in plasma and in utero, with an associated increase in embryo mortality. Nevertheless, metabolism appears to be up-regulated in some embryos and, among those that survive, fetal growth appears to be enhanced.

Dietary-induced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova

In the first of three experiments, eight ovariectomised Greyface ewes primed with exogenous progesterone were used to provide quantitative data on the effects of two contrasting feeding levels (0.3 vs. 1.4 × maintenance) on plasma progesterone concentrations. Over the 9 day study period, mean (± SEM) daily progesterone concentrations were 4.3 ± 0.13 and 3.3 ± 0.17 μg l−1 for the low and high feeding regimens, respectively (P = 0.06), indicating that high feed intake suppressed circulating progesterone levels. The second experiment examined the effect in superovulated Finn-Dorset ewes of a diet supplying either 0.6 (Group L, n = 8) or 2.3 (Group H, n = 8) times their daily energy needs for maintenance, from 1 day before introduction of exogenous progesterone to the time of insemination, on plasma progesterone concentrations and the viability of ova recovered 4 days after insemination. Mean (± SEM) plasma progesterone concentrations were 4.5 ± 0.17 μg l−1 and 2.8 ± 0.16 μg l−1 for L and H ewes, respectively, during the 12 day priming period (P 0.10) at ovum collection. Intervals (mean ± SEM) to oestrous onset (14.5 ± 0.38 h) and the luteinising hormone (LH) surge (27.1 ± 0.98 h) were unaffected by feed intake. Mean (± SEM) ovulation rates (8.1 ± 1.57 vs. 7.8 ± 1.10) and numbers of ova recovered (5.0 ± 1.39 vs. 4.8 ± 1.11) were also similar for each group. However, the proportions of ova considered viable (over 32 cells) at recovery were 0.53 and 0.22 for L and H groups, respectively (P < 0.005). Following 72 h culture (Tissue Culture Medium-199 (M199) + 10% foetal calf serum (FCS)), 0.55 and 0.27, respectively, had developed to blastocysts (P < 0.025). Of ova assessed as viable at recovery, similar proportions (0.86 vs. 0.75) from L and H treatments developed to blastocysts, with corresponding nuclei counts (mean ± SEM) of 55 ± 5.2 and 55 ± 13.2. The third experiment used 12 superovulated Greyface ewes, each offered a different feed level within the range 0.6–2.5 × maintenance, to determine the nature of the relationship between feeding level, pre-ovulatory progesterone concentrations and ovum development at Day 2 following insemination and subsequently during 7 day co-culture (M199 + FCS). Increases in feeding level were accompanied by linear decreases in plasma progesterone (r2 = 0.79, P < 0.001), the interval to oestrous onset (r2 = 0.52, P < 0.01) and timing of the LH surge (r2 = 0.32, P < 0.06). Although undetectable at ovum collection, and somewhat equivocal after 4 day culture, high feeding levels prior to ovulation reduced the proportion of ova (0.16 vs. 0.58) developing to or beyond the expanding blastocyst stage after 7 day culture. Quantitative indices of cell division and protein synthesis confirmed this. In conclusion, excessive feeding during follicular recruitment and oocyte maturation in superovulated ewes imparts a legacy of embryonic loss and developmental retardation.

Repeated recoveries of embryos from ewes by laparoscopy

Abstract Eight ewes were synchronised in oestrus and induced to superovulate on three occasions over a period of two months. Eggs a were recovered on Day 7 (Day 0 = oestrus) of each cycle by a laparoscopic technique which avoids the adhesion formation normally associated with the conventional surgical procedure. Egg recovery rates were 35% ( 22 62 ), 76% ( 13 17 ) and 66% ( 21 32 ) for the three consecutive recovery sessions. This technique allows ewes to be used repeatedly as embryo donors and should be valuable to research workers and those involved with commercial embryo transfer in sheep.

LAPAROSCOPY FOR INTRAUTERINE INSEMINATION AND EMBRYO RECOVERY IN SUPEROVULATED EWES AT A COMMERCIAL EMBRYO TRANSFER UNIT

Of 111 variable age, pedigree ewes subjected to a range of superovulatory regimens and then submitted to embryo recovery by laparoscopy, nine had adhesions corresponding to a mid-line laparotomy (presumably from a previous attempt to recover embryos) and could not have their embryos recovered by the laparoscopic technique. Of the remainder, 27 ewes (26.5%) had less than three ovulations or had prematurely regressing corpora lutea at the selected time for embryo recovery (Days 5 to 6 following insemination), and no attempt was made to recover embryos from them. For the 75 ewes subjected to laparoscopic ovum recovery following laparoscopic intrauterine insemination, the average number of ovulations (+/- SEM) was 7.9 +/- 0.6; the average ovum recovery (mean of values for each ewe) was 51.7% +/- 3.5; and the percentage of recovered ova that were fertilized was 87.3%. For a further nine 3-yr-old crossbred ewes the mean values for ovulation and ovum recovery were 7.6 +/- 1.2 and 70.1 +/- 7.7, and were not significantly different for the two insemination methods used (laparoscopic intrauterine vs cervical). In general, ovulation rates for ewes given pregnant mare serum gonadotrophin (PMSG) tended to be lower (5.2 +/- 0.7) than for those given porcine follicle stimulating hormone (pFSH, 7.7 +/- 0.8) or human menopausal gonadotrophin (hMG, 7.7 +/- 2.3). Ova recovery rates were similar on Days 5 and 6 (Day 0 = insemination), and were not affected by method of insemination (laparoscopic intrauterine vs cervical).

Daily oral administration of melatonin from March onwards advances by 4 months the breeding season of ewes maintained under the ambient photoperiod at 57 °N

Abstract In three experiments, ewes kept under the ambient photoperiod at 57 °N were given daily at 15:00 h from March onwards an oral dose of 3 mg of melatonin in a 4:1 mixture of water and ethanol or, in the case of controls, the vehicle alone. All ewes were challenged daily with a vasectomized ram for the detection of behavioural oestrus. In Experiment 1, the mean interval (± standard error of the mean (s.e.m.)) from the start of melatonin treatment on 5 March to first behavioural oestrus for Border Leicester × Scottish Blackface ewes that had remained barren over the winter period was 87 ± 5.8 days ( n = 8) compared with 222 ± 10.3 ( n = 7) for controls. Corresponding intervals from the start of melatonin treatment on 24 March for the same breed of ewe in Experiment 2 were 80 ± 3.7 ( n = 6) and 211 ± 9.4 ( n = 6) days. In Experiment 3, 22 Scottish Blackface ewes that lambed between 11 January and 22 February were paired by lambing date for weaning at either 6 or 14 weeks. All ewes received melatonin from 22 March until 5 August. Apart from one ewe from the 14 week group that failed to show oestrus, all other ewes responded to melatonin and exhibited first behavioural oestrus at mean intervals from initial dosing of 77 ± 4.6 days and 75 ± 3.2 days for the 6 week and 14 week groups, respectively. Melatonin suppressed prolactin in both dry (Experiments 1 and 2) and lactating ewes (Experiments 1, 2 and 3), but in the only experiment in which it was tested (Experiment 1), its continuous administration throughout pregnancy failed to prevent the late-pregnancy rise in prolactin. It had no effect on gestation length or the subsequent growth rate of lambs; its effect on lamb birth weight was equivocal, causing a significant decrease in twin lambs in Experiment 2 and no effect in Experiment 1. Detailed sequential measures of the nature of the episodic release of luteinizing hormone (LH) (Experiment 2) provided no evidence that melatonin induced a progressive increase in the activity of the gonadotrophin releasing hormone (GnRH) pulse generator. The ovarian response to the daily oral administration of melatonin from March onwards contrasts with literature reports on the failure of continuous-release implants of melatonin to stimulate behavioural oestrus when inserted as early as April or May.

MELATONIN TREATMENT OF EMBRYO DONOR AND RECIPIENT EWES DURING ANESTRUS AFFECTS THEIR ENDOCRINE STATUS, BUT NOT OVULATION RATE, EMBRYO SURVIVAL OR PREGNANCY

Thirty-two Border Leicester x Scottish Blackface ewes that lambed in March were individually penned with their lambs from April 16th and given daily an oral dose of 3 mg melatonin at 1500 h (Group M). A further 32 acted as controls (Group C). Within each group half were used as embryo donors (Group D) following superovulation and half received embryos (Group R) following an induced estrus. Prior to weaning on 21 May ewes received ad libitum a complete diet providing 9 megajoules (MJ) of metabolizable energy and 125 g/kg crude protein. Thereafter each received 1.6 kg of the diet daily. In early June each ewe received an intravaginal device (300 mg progesterone) inserted for 12 d. Donors were superovulated with 4 i.m. injections of porcine FSH 12 h apart, commencing 24 h before progesterone withdrawal. Ovulation in recipients was induced with 800 IU PMSG injected i.m. at progesterone removal. Donor ewes were inseminated 52 h after progesterone withdrawal. Embryos were collected 4 d later and transferred to recipients. Melatonin suppressed plasma prolactin (P < 0.001) and advanced estrus (P < 0.05) and timing of the LH peak (P < 0.05). These events also occurred earlier in donors than in recipients (P < 0.01). Mean (+/- SEM) ovulation rates for melatonin-treated and control donors were 5.5 +/- 0.71 and 4.7 +/- 0.66, respectively (NS). Corresponding recipient values were 3.3 +/- 0.40 and 3.4 +/- 0.39 (NS). Mean (+/- SEM) embryo yields were 2.9 +/- 0.64 and 2.6 +/- 0.73 for melatonin-treated (n = 15) and control (n = 16) donors, respectively, and for the 12 ewes per treatment that supplied embryos, corresponding numbers classified as viable were 2.7 +/- 0.47 and 2.3 +/- 0.61 (NS). Following transfer, 57% of embryos developed to lambs when both donor and recipient received melatonin, 86% when only the donor received melatonin, 91% when only the recipient received melatonin, and 67% when neither received melatonin (NS). Thus, embryo survival following transfer was not improved by treating recipients with melatonin. Gestation length and lamb birthweights were unaffected by melatonin. Unlike nonpregnant control ewes, melatonin-treated recipients that failed to remain pregnant sustained estrous cyclicity following embryo transfer.

The effect of two different levels of progesterone priming on the response of ewes to superovulation

The use of either 1 or 3 controlled internal drug release (CIDR) devices for progesterone priming in ewes (n=11) superovulated with 1500 IU pregnant mare serum gonadotrophin (PMSG) at 28 hours prior to CIDR device withdrawal was investigated in relation to the stages of development and viability of the ova produced. Progesterone levels in the ewes (n=6) treated with 3 CIDR devices were significantly higher (P<0.01) during the 11 days of insertion than in those (n=5) treated with 1 CIDR device (7.3 vs 3.3 ng/ml) over the same period. However, following superovulation, the mean (+/-SEM) ovulation rates were similar for both groups (8.2 +/- 1.7 vs 10.2 +/- 1.5). The number of ova (M+/-SEM) recovered by laparoscopy 5 days after insemination was 4.2 +/- 1.0 for ewes treated with 3 CIDR devices and 7.0 +/- 1.1 for those treated with 1 CIDR device (P<0.10). The respective ovum recovery rates (M+/-SEM) were 55+/-9.8 and 74+/-13.2%. There was no effect of progesterone concentration in the priming phase on either the stages of development of the recovered ova or on their ability to develop during in vitro culture. It was concluded, therefore, that progesterone concentrations within the range 3.3 +/- 0.1 to 7.3 +/- 0.3 ng/ml during the priming phase and 2.4 +/- 0.3 to 6.5 +/- 0.2 ng/ml at the time of PMSG administration did not affect the ovulation rate or the viability of ova recovered from superovulated ewes.

A comparison of two dosages of fluorogestone acetate in pessaries on the quality of embryos recovered from superovulated ewes

Abstract The effect of synchronizing estrus using two dosages of progestagen (30- or 40-mg fluorogestone acetate; FGA) pessaries on the quantity and quality of embryos produced following superovulation with pregnant mare serum gonadotropin (PMSG) was investigated using 19 donor ewes. The viability of ova recovered on Day 3 or 6 after insemination was investigated by transfer to synchronized recipient ewes. The ovulation rates and proportions of embryos recovered were not significantly affected by the level of progestagen priming or the day of embryo recovery, although overall the recovery rate was lower at 3 days after insemination than at 6 days (46.5 vs 71.3%). The level of progestagen priming caused no apparent difference in the quality of ova recovered on Day 3, but on Day 6 a significantly smaller proportion of the ova were of transferable quality when recovered from donors treated with the 30-mg FGA pessary compared with the 40-mg pessary (43.3 vs 87.5%). Embryos recovered on Day 6 from the donor ewes treated with 30-mg FGA pessaries exhibited a wider spread in the stages of development than those collected from ewes treated with 40-mg FGA pessaries. Results for the transfer of embryos 3 days after insemination were consistent with the hypothesis that the level of progestagen priming prior to superovulation affects embryo viability, the mean survival rate for the 3-day embryos being 58.3 and 75% for the 30- and 40-mg FGA treatments, respectively.

The effect of time of intrauterine insemination on the development and viability of embryos collected from superovulated ewes

Eighteen Border Leicester x Scottish Blackface ewes, primed with 300 mg progesterone (12 d) and superovulated with decreasing doses (6, 5, 3 and 2 mg) of porcine FSH, were inseminated with fresh semen, using laparoscopic intrauterine procedures at 48 (Group E) or 60 h (Group L) after exogenous progesterone removal. Five days after insemination, embryos were collected and classified on the basis of their morphological development. During the subsequent 3 d of in vitro culture (38.5 degrees C; 5% CO2) the embryos were evaluated at 24-h intervals. After 72 h, the embryos were individually fixed (24 h) and stained with aceto-orcein and the nuclei were then counted to provide an objective index of cell proliferation and development. Mean (+/-SEM) ovulation rates for the 2 groups (9.2+/-1.5 and 7.1+/-1.2, respectively) and the corresponding percentages (53 vs 59) of embryos collected by laparoscopy were unaffected by insemination time. All donors yielded fertilized ova, but whereas all Group-E donors yielded 1 or more viable embryos (i.e., >32 cells), only 5 Group-L ewes yielded viable embryos (P<0.10). At collection, the percentages of embryos at the morula stage of development were 98 (Group E n = 44) and 39 (Group L n = 38; P<0.001). Few of the remaining ova (Group E = 0% Group L = 8%) were at the 1-cell stage of development when collected, indicating that retarded development post fertilization, not fertilization failure, was the principal consequence of delayed insemination. The percentages of embryos that continued to develop during in vitro culture were 91 and 37 for Groups E and L, respectively (P<0.001), and all of these reached the blastocyst stage. Of these blastocysts, 75 and 50% in Groups E and L hatched in vitro (P<0.10), with mean (+/-SEM) nuclei counts of 148+/-22.7 and 76+/-13.8 (P<0.02), respectively. In conclusion, while delayed intrauterine insemination did not affect the efficiency of ovum collection, it caused a major reduction in the yield of embryos that were capable of developing during in vitro culture. However, fertilization failure accounted for only 13% of the loss in viability following late insemination.

The effect of method of oestrous synchronisation on the response of ewes to superovulation with porcine follicle stimulating hormone

Fifty Border Leicester × Scottish Blackface ewes superovulated with 16 mg of porcine follicle stimulating hormone (p-FSH) were used to test the effects on ovulation and ovum quality of five oestrous synchronisation treatments (n=10 per treatment) applied during the breeding season. The treatments were a controlled internal drug release (CIDR) device containing 330 mg progesterone (T1), a 30 mg fluorogestone acetate (FGA) pessary (T2), a 30 mg FGA pessary in which the FGA was redistributed on the sponge matrix (T3), a 30 mg FGA pessary plus 400 mg of progesterone (T4) and an injection of 125 μg of the prostaglandin (PGF2α) analogue, Cloprostenol, on Day 12 following oestrus (T5). Ewes were inseminated (Day 0) by the laparoscopic intrauterine method at 60 h after either ‘progesterone/progestagen’ withdrawal (T1–T4) or PGF2α injection (T5). Mean (±SEM) ovulation rates were 3.5±1.0 (T1), 7.9±1.1 (T2), 6.9±1.3 (T3), 8.8±1.4 (T4) and 5.4±1.7 (T5) (P<0.05 for T1 vs. T2 and T1 vs. T4). Corresponding values for the proportion of ova recovered by laparoscope on Day 6 were 0.46, 0.40, 0.42, 0.68 and 0.44. The greatest number (4.1±0.9) of high quality ova (compact morulae and blastocysts) was obtained with T4. The results indicate that for ewes superovulated with p-FSH, a higher level of steroid priming than that supplied by either a CIDR device or a 30 mg FGA pessary is desirable.