Traversing the ovine cervix - a challenge for cryopreserved semen and creative science.

This review brings together research findings on cervical relaxation in the ewe and its pharmacological stimulation for enhancement of the penetration needed for transcervical insemination and embryo transfer. On the basis that the success of artificial insemination is the percentage of ewes lambing, a review is made of recent research aimed at understanding and minimising the sub-lethal effects of freezing and thawing on the viability of spermatozoa, their membrane integrity and their ability to migrate through cervical mucus, as these characteristics have a major influence on fertility, particularly when semen is deposited, artificially, in the os cervix. Milestones of achievement are given for transcervical intrauterine insemination, embryo recovery and transfer and the birth of lambs of pre-determined sex, firstly following intracytoplasmic sperm injection, then laparoscopic intrauterine insemination using highly diluted flow-cytometrically sorted fresh semen and subsequently by os cervix insemination using sexed semen that had been frozen and thawed. Diversity of research endeavour (applied, cellular, molecular), research discipline (anatomy, histology, immunology, endocrinology) and research focus (cell, tissue, organ, whole animal) is embraced within the review as each has significant contributions to make in advancing recent scientific findings from the laboratory into robust on-farm transcervical insemination and embryo transfer techniques.

Lambing rates and litter sizes following intrauterine or cervical insemination of frozen/thawed semen with or without oxytocin administration.

Intrauterine insemination by laparoscopy is required to achieve acceptable lambing rates in ewes when using frozen semen but the procedure has evoked welfare concerns. Oxytocin has been used to dilate the cervix as a means of accessing the uterus during conventional cervical insemination, but its effect on fertility is not well documented. Three hundred crossbred ewes were synchronised in estrus and randomly allocated to one of three insemination procedures using frozen/thawed semen containing 400 x 10(6)/ml progressively motile sperm: single cervical (0.2 ml), multiple cervical (4 x 0.05 ml) or laparoscopic (0.05 ml per uterine horn). The effects of each insemination procedure on lambing rate (percentage of treated ewes lambing) and litter size (lambs per ewe lambing) were tested with and without oxytocin (10 IU given i.m.) prior to fixed-time insemination. Oxytocin did not permit complete cervical penetration in any ewes and neither lambing rate nor litter size was influenced by the number of inseminations. Lambing percentages were 69 and 42 (P < 0.01) for the laparoscopic and cervical insemination methods, respectively, and oxytocin reduced these to 58 (NS) and 10 (P < 0.001) percent, respectively. Corresponding litter sizes for ewes not receiving oxytocin were 1.91 and 1.51 and for those receiving oxytocin, 1.83 and 1.41 (laparoscopic versus cervical, P < 0.02). Thus, in the absence of complete cervical penetration at insemination, 10 IU oxytocin decreased the number of ewes lambing but had no effect on their litter size.

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes.

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.

Seasonal differences in lamb birthweight do not arise from inherent differences in the oocyte and/or early embryo.

The aim of this study was to determine whether previously observed seasonal differences in conceptus development in ewes are attributable to inherent differences in the oocyte and/or early embryo. Day 6 embryos were recovered from 50 ewes subjected to a standard oestrus synchronization, superovulation and laparoscopic artificial insemination protocol during October (peak breeding season) and April (transition to anoestrus). During the following October, 40 grade 1 and 2 embryos from each month, which had been cryopreserved at the late morula or unexpanded blastocyst stage, were thawed and transferred in singleton to synchronous recipients. Resulting pregnancies were monitored to term. For ewes receiving October- and April-produced embryos, overall mean +/- SEM liveweight at the time of embryo transfer was 72 +/- 0.7 kg, body condition score was 3.1 +/- 0.04 units, and the number of corpora lutea on the ovaries was 2.7 +/- 0.11 per ewe. Thirty-one and 27 ewes, respectively, became pregnant and their gestation lengths were 147 +/- 0.2 and 147 +/- 0.3 days. There was no effect of month of embryo production on peripheral ovine pregnancy-associated glycoprotein concentrations during pregnancy or on fetal and placental characteristics at term, but, for each month, male lambs were heavier than females and were associated with larger placentae. Lamb birthweight was positively correlated with placental weight (r2 = 0.474, P<0.001) and the total weight of cotyledonary tissue (r2 = 0.429, P<0.001), but not to the number of cotyledons. Results demonstrate close relationships between fetal and placental weights at term, and that seasonal effects on conceptus development in ewes do not arise from inherent differences in the oocyte and/or early embryo.

Comparative epidemiology of scrapie outbreaks in individual sheep flocks.

Data recording the course of scrapie outbreaks in 4 sheep flocks (2 in Cheviot sheep and 2 in Suffolks) are compared. For each outbreak the data on scrapie incidence and sheep demography and pedigrees cover periods of years or decades. A key finding is that the incidence of clinical cases peaks in sheep 2-3 years old, despite very different forces-of-infection. This is consistent with age-specific susceptibility of sheep to scrapie, as has been reported for cattle to bovine spongiform encephalopathy and for humans to variant Creutzfeldt-Jakob disease. Scrapie incidence was higher in ewes than rams and at certain times of years, though these effects were not consistent between flocks. There was no evidence for high levels of vertical transmission.

Onset and distribution of tissue prp accumulation in scrapie-affected suffolk sheep as demonstrated by sequential necropsies and tonsillar biopsies.

Tonsillar biopsies (single or multiple) or necropsies, or both, were performed on sheep taken from a Suffolk flock in which frequent cases of scrapie had occurred over a period of several years. Clinically affected sheep of the susceptible PrP(AQ/AQ)genotype had widespread disease-specific PrP accumulation in the central nervous system (CNS), lymphoreticular system and peripheral ganglia. In nine healthy PrP(AQ/AQ)Suffolk sheep between 4 and 7 years of age, PrP could not be demonstrated post mortem in any of the lymphoreticular tissues, or in the peripheral ganglia or CNS. Tonsillar biopsies taken from animals of the resistant PrP(AR/AR)and PrP(AR/AQ)genotypes at age 3, 8, 14, 20 or 26 months did not show PrP accumulation. Disease- specific PrP accumulation in tonsillar biopsies from PrP(AQ/AQ)sheep was not seen in 20 animals aged 3 months, but was found in two of 10 animals at age 8 months and in eight of 10 animals at age 20 months. The numbers of PrP-positive tonsillar biopsies obtained from sheep previously biopsied on more than one occasion was greater than the number of positive tonsils obtained from other susceptible sheep of comparable ages. The earliest disease-specific PrP accumulation seen was in tingible body macrophages within germinal centres and only later was it detected in cells resembling follicular dendritic cells. Fourteen PrP(AQ/AQ)sheep examined post mortem at up to 17 months of age and which had not previously been biopsied or were biopsied only once had no CNS or tonsillar PrP accumulations. Two of these sheep subjected to necropsy at 14 months had PrP accumulation in lymphoreticular tissue, where it was confined to the mesenteric lymph nodes. In susceptible sheep, only low levels of immunohistochemically detectable PrP were present in a minority of follicles from tonsillar biopsies of young lambs, but by 14 months of age widespread PrP accumulation, affecting many or even all follicles, was present. Although clinical cases had widespread PrP accumulations in viscera, susceptible survivors had no such accumulations in tissues of the lymphoreticular system, peripheral nervous system or CNS, suggesting that some animals were not exposed to infection or were exposed to a non-infectious dose.

Physiological correlates of maternal-offspring behaviour in sheep: a factor analysis.

The onset of maternal behaviour in sheep is controlled by levels of oestrogen and progesterone in gestation and the release of oxytocin during delivery. Factor analysis was used to investigate the relationships between maternal behaviour, offspring behaviour, and maternal ovarian hormone levels during gestation in sheep. Ewes gave birth to lambs following embryo transfer between two breeds (Suffolk and Scottish Blackface), which differ in their expression of maternal behaviour. Plasma oestradiol-17beta concentration was significantly higher in Blackface ewes in the last 6 weeks of pregnancy, whereas plasma progesterone was higher in Suffolk ewes in early and mid gestation. Factor analysis revealed three factors that accounted for 50% of the total variation between variables. Factor 1 described lamb activity and had positive loadings for lamb behavioural latencies immediately postpartum, and negative loadings for sucking frequency. Factor 2 described some aspects of ewe maternal behaviour, and had positive loadings for ewe-lamb separation and lamb vocalisation, and negative loadings for ewe grooming behaviour and plasma oestradiol concentration. Factor 3 described ewe and lamb-sucking interactions, and had positive loadings for ewe moving as the lamb made sucking attempts, and negative loadings for ewe and lamb vocalisation. Factor 1 scores were significantly affected by lamb breed: Suffolk lambs received positive scores, indicating a longer latency to right and stand, whereas Blackface lambs had negative scores. Maternal progesterone concentration was significantly correlated with Factor 1 scores. Factor 2 and 3 scores were significantly affected by ewe breed. This study has shown that there are two behavioural axes to maternal behaviour in sheep, and that maternal oestradiol concentration is related to affiliative behaviours (e.g., grooming) but only weakly to ewe responsiveness to lamb-sucking attempts.

Association between natural scrapie and PrP genotype in a flock of Suffolk sheep in Scotland.

The incidence of natural scrapie in sheep is associated with polymorphisms of the PrP gene, particularly those at codons 136, 154 and 171. In many breeds, the PrP allele encoding valine at codon 136 confers an extremely high risk of scrapie, but in Suffolk sheep this allele is vanishingly rare. In this study of a single closed flock of Suffolk sheep in Scotland, scrapie occurred primarily in animals which were homozygous for glutamine at codon 171, a genotype which was significantly less frequent in healthy flockmates. However, the apparent linkage between glutamine at codon 171 and scrapie was not completely recessive because two of 64 scrapie cases were heterozygous glutamine/arginine. These results suggest that breeding for increased resistance to scrapie in Suffolks by the selection of animals according to their PrP genotype is a feasible option.

A field study of watery mouth: clinical, epidemiological, biochemical, haematological and bacteriological observations.

More than 500 cases of watery mouth in newborn lambs were recorded in 11 flocks and a detailed examination was performed in more than 200 cases. The condition occurred predominantly in lambs aged 24 to 48 hours. The incidence in a flock varied from 1 per cent to 24 per cent and the mortality in affected lambs varied from 7 per cent to 83 per cent. A bacteraemia was found in 38 per cent of the affected lambs. There were no consistent abnormalities of clinical biochemistry or haematology. Watery mouth was more common in twins and especially in triplets than in single lambs. In twins the highest incidence of watery mouth was recorded in lambs born to ewes in poor body condition. Antibiotic given orally within 15 minutes of birth greatly reduced the incidence of watery mouth.