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.

Failure to transmit scrapie infection by transferring preimplantation embryos from naturally infected donor sheep.

The objective of the study was to examine whether or not the preimplantation embryo can act as a carrier of classic scrapie infection. The study was carried out on quarantined premises with sheep of highly susceptible scrapie genotypes. Uninfected embryos, collected from New Zealand-derived Suffolk ewes, were surgically transferred into recipient ewes that were also of New Zealand origin. Seventeen negative control lambs were born on the study premises from these embryo transfers. Thirty-nine experimental lambs were from embryos collected from naturally infected donor ewes. The experimental lambs were also born on the study premises after their surgical transfer into recipient ewes of New Zealand origin. These embryos had been collected from donor ewes in a scrapie-infected flock where the ewes were clinically sick with scrapie or developed clinical scrapie after embryo collection. All lambs were confirmed as scrapie susceptible of the ARQ/ARQ genotype. Twenty-eight experimental animals survived to the end point of the study at 5 yr of age with a mean survival of 1579 d. In the negative control group, 12 of 17 sheep survived to 5 yr of age with a mean survival of 1508 d. Postmortem examinations were carried out on all animals derived by embryo transfer, and in none was histologic or immunohistochemical evidence of scrapie found. In contrast, in the originating flock the majority of scrapie cases occurred in ARQ/ARQ genotyped animals where a 56% mortality from scrapie had been recorded in animals of this genotype. Thus, the study provides no evidence for transmission of scrapie and reinforces published evidence that vertical transmission of scrapie may be circumvented by embryo transfer procedures.

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.

Interleukin 8 in the cervix of non-pregnant ewes.

Studies of cervical artificial insemination of ewes at hormone-synchronized oestrus indicate that the cervix remains relatively impenetrable to semen, in contrast to naturally breeding animals. During parturition the inflammatory response plays an important part in cervical dilation and possibly, to a lesser extent, in the non-pregnant cervix at oestrus to facilitate the transcervical transport of semen. The expression of interleukin 8 (IL-8), a pro-inflammatory cytokine in the ovine cervix, has been mapped and quantified, using semi-quantitative in situ hybridization, to ascertain the role played by inflammation in the ovine cervix during natural and artificially induced oestrous cycles. IL-8 gene expression was observed in both the luminal epithelium and fibroblastic cells of the cervix. The presence of IL-8 was confirmed using immunohistochemistry. IL-8 gene expression in the luminal epithelium varied throughout the oestrous cycle and was highest at oestrus and at day 5 of the oestrous cycle. In ewes artificially induced to ovulate, either by the withdrawal of progesterone pessaries after treatment for 12 days, or by two i.m. injections of prostaglandin 9 days apart, IL-8 gene expression at oestrus was significantly lower than it was at natural oestrus. Insemination increased IL-8 gene expression in progesterone-synchronized ewes. These data support the hypothesis that IL-8-induced inflammation is important in normal cervical function and that this process is inhibited during artificial synchronization of the oestrous cycle and is increased by exposure to semen.

Oestrogen and progesterone receptor immunoreactivity and c-fos expression in the ovine cervix.

Immunocytochemistry was used to detect the presence of oestrogen and progesterone receptors in the cervices of prepubertal lambs, seasonally anoestrous ewes, cyclic ewes, and pregnant ewes of known gestational stages, to define the roles of gonadal steroids in cervical function. The presence of the immediate early gene product, c-Fos, a marker for cellular activation, was also investigated using immunocytochemistry and in situ hybridization. Oestrogen receptor immunoreactivity was restricted to the endometrium on days 0-3 of the oestrous cycle (day 0 = oestrus). In immature animals, very few scattered nuclei in the endometrium were immunoreactive. Oestrogen receptor immunoreactivity was not apparent in the endometrium during the remainder of the oestrous cycle or in this region in anoestrous animals. In pregnant ewes, oestrogen receptor immunostaining appeared as relatively few isolated nuclei in the connective tissue stroma. Progesterone receptor immunoreactivity was found in the endometrium at days 0-3 of the oestrous cycle and also in the luminal epithelium, the myometrium and the blood vessels. Progesterone receptor immunoreactivity was also found in these regions, with the exception of the endometrium, at all other stages examined. Immunostaining for c-Fos was present in the endometrium at days 0-3 of the oestrous cycle, and some scattered immunopositive nuclei were present in prepubertal animals. c-Fos immunoreactivity was also found in the myometrium and in blood vessels at all other stages examined. Visualization of c-fos gene expression by in situ hybridization showed that it occurred in the luminal epithelium and blood vessels at oestrus, but was restricted to the blood vessels in all other samples examined.

Observations on the transmission of scrapie in experiments using embryo transfer.

This investigation studied the maternal transmission of scrapie in sheep by using embryo transfer to examine the viability of highly susceptible offspring derived from scrapie-affected and uninfected donors. The study also examined the effect of washing the embryos. Scrapie occurred in both washed and unwashed embryo-derived Sip sAsA progeny from both groups of donor ewes. As a result, the earlier observation that scrapie might pass via the unwashed embryo to develop as disease in adult sheep has to be reassessed. Several other implications of the work are considered, including the possibility that natural scrapie is not purely a genetic disease.

Prevalence of Actinobacillus seminis in the semen of rams in the United Kingdom.

A survey was conducted to establish the prevalence of Actinobacillus seminis in ovine semen and the possible importance of the organism as a cause of ram infertility. A seminis was isolated from three of 16 infertile rams and the infected animals had lesions of the genital tract and produced poor quality semen. A seminis was also isolated from two of 96 fertile rams used as donors of semen for artificial insemination programmes. The strains of A seminis could be identified on the basis of their API ZYM profiles. The results suggest that in the United Kingdom A seminis should be considered as a common cause of infertility in rams.

Johne's disease in a herd of farmed red deer.

An outbreak of Johne's disease in a herd of farmed red deer was studied for four years. Serological, histopathological and cultural techniques were used to monitor the progress of the disease, and delayed type hypersensitivity skin tests were also applied. The results of the serological tests showed that they were poor predictors of future clinical cases and did not consistently identify animals harbouring mycobacteria. The histopathological methods provided a sensitive and specific means of confirming the infection. The skin tests had a low sensitivity and the results were poorly correlated with the serological results in seropositive animals. A vaccination policy was instituted which was accompanied by a change in the pattern of disease. Although the histopathological evidence suggested that the infection was still occurring, there was a marked reduction in the incidence of clinical disease. Vaccinated animals showed a good response to the skin test.

The effects of subnutrition on the components of the gravid uterus in the doe.

The effects of subnutrition on the caprine fetus and the other products of pregnancy were investigated in does. Two groups of does were fed on rations calculated to provide 100 and 25% of their energy and protein requirements for maintenance from 19 days before mating until 60 days after mating. Estrus was synchronized in does using PGF(2x). Approximately 60 days after natural mating, pregnant does were slaughtered, and the products of pregnancy were measured. Fetuses from the feed-restricted group were significantly lighter (P<0.05), had shorter crown-rump length (P<0.05), and the uterus contained a smaller volume of fetal fluids (P<0.02). Curved crown-rump length tended to be shorter and fetal placental membranes and cotyledons tended to be lighter (P<0.1) in the feed-restricted group. No significant difference was found between the 2 groups in the head length, number of placentomes, and weight of empty uterus. The number of fetuses affected the number of placentomes (P<0.001), weight of empty uterus (P<0.001), mass of total fetal fluids (P<0.001) and weight of ovaries (P<0.05), but not fetal measurements. Gestation length was found to significantly (P<0.001) affect all the fetal measurements but none of the placental measurements except for the total weight of cotyledons (p<0.001). The results of the study demonstrated detrimental effects of underfeeding on the caprine fetus and placenta.

The effects of low level of feeding on response to synchronization of estrus, ovulation rate and embryo loss in goats.

Mature nonlactating British Saanen and Toggenburg does with a body score 2 were fed 25% (n=24) and 100% (n=16) maintenance rations from about 19 days before mating until slaughter at approximately 60 days after mating. Estrus was synchronized using PGF2alpha, and the ovulation rate was determined by laparoscopic examination of the ovaries once between Days 6 and 10 after mating. Pregnancy rate, potential kidding rate and embryo loss were determined by counts of viable fetuses at slaughter. The proportion of does in estrus within 96 hours of PGF2alpha administration was not different (P<0.5) between the feed-restricted and the maintenance groups (71.0% and 87.5%, respectively); however, the time of onset of estrus after PGF2alpha tended to be longer (P=0.12) in the feed-restricted group. Ovulation rate, incidence of multiple ovulations and proportion of does pregnant at 60 days were significantly lower (P=0.0004, P=0.025, P=0.05, respectively) in the restricted group. More embryos from single than multiple ovulations were lost in the restricted group (P=0.01). There was no difference in the overall ovulatory activity between right and left ovaries in the 2 groups. Transuterine migrations were observed in all does that had unilateral multiple ovulations. No migration was observed in does which had single ovulations. These data indicate that restricted feed intake in goats tended to delay the onset of estrus and lowered the ovulation rate, incidence of multiple ovulations, and pregnancy rate.

Studies on maternal transmission of scrapie in sheep by embryo transfer.

The technique of embryo transfer was used to investigate the maternal transmission of scrapie in sheep. Embryo donor ewes were experimentally infected with scrapie (all eventually developing the disease) and artificially inseminated six months later with semen from an uninfected scrapie-susceptible ram. Embryos were harvested five and six days after insemination and transferred by laparoscopy, unwashed, into recipient ewes which had been genetically selected for very low susceptibility to scrapie. Six of the 26 lambs born to these recipients developed scrapie.

Diagnosis of pregnancy and prediction of fetal age in red deer by real-time ultrasonic scanning.

Sixteen pregnant red deer hinds were scanned using real-time ultra sound at regular intervals during the first 150 days of pregnancy. In the early stages of pregnancy, an intra-rectal linear array transducer was used but later in pregnancy the hinds were scanned externally with a sector scanner. Pregnancy was determined from 30 days of gestation by intra-rectal scanning, and from 50 days by external scanning. With advancing age the size of the fetus increased, and linear regressions of size on age gave residual standard deviations of 0.508 days for head diameter and 0.506 days for trunk diameter. It is concluded that this technique can be used to determine pregnancy with a high degree of accuracy from 30 days of gestation and to predict calving date from fetal measurements within the range 1.5 cm to 7 cm for head diameter or 0.5 cm to 8 cm for trunk diameter, corresponding to a fetal age range of 35 to 150 days.

Effect of the body condition of ewes on the secretion of LH and FSH and the pituitary response to gonadotrophin-releasing hormone.

The effects of body fat content (body condition) of ewes on hypothalamic activity and gonadotrophin-releasing hormone (GnRH) secretion and on pituitary sensitivity to GnRH were investigated using Scottish Blackface ewes. Two groups of 12 ewes were fed so that they achieved either a high body condition score (2.98, S.E.M. = 0.046; approximately 27% of empty body weight as fat) or a low body condition score (1.94, S.E.M. = 0.031; approximately 19% of empty body weight as fat) by 4 weeks before the period of study. Thereafter, they were differentially fed so that the difference in mean condition score was maintained. Oestrus was synchronized, and on day 11 of the subsequent cycle half of the ewes of each group were ovariectomized. On day 12, the remaining ewes were injected (i.m.) with 100 micrograms prostaglandin F2 alpha analogue and ovariectomized 30 h later. Numbers of large ovarian follicles and corpora lutea present at ovariectomy were recorded. Blood samples were collected at 15-min intervals for 12 h on day 10 of the cycle (luteal phase) and at 10-min intervals from 24 to 30 h after prostaglandin injection (follicular phase). At days 2 and 7 after ovariectomy, samples were collected at 15-min intervals for 8 h and ewes were then injected with 10 micrograms GnRH and samples were collected for a further 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood biochemical reference values for farmed red deer.

The activities and concentrations of 30 whole blood and plasma constituents including enzymes, substrates, electrolytes and vitamins were determined in samples from 91 clinically healthy farmed red deer (Cervus elaphus) hinds ranging in age from one to 15 years. The hinds were randomly selected from a herd maintained on a mixture of reseeded and permanent hill pasture at an experimental research station in north east Scotland. The results were analysed statistically and the calculated means, standard errors and tolerance intervals provide a basis for diagnostic clinical chemistry in red deer. The data are compared with data from earlier reports on blood biochemistry in deer and other livestock species.