The effect of transferring ovine IVP embryos on Day 2 versus Day 6 postinsemination was investigated. Oocytes were collected from 35 cull ewes and cultured separately for each donor. Embryos were exposed to serum in the maturation and fertilization media, and then cultured in a serum-free SOF system under serum-conditioned silicone oil. Cleaved Day 2 postfertilization embryos were randomly allocated for immediate transfer versus Day 6 transfer. Parturition was induced (with 15 mg dexamethasone) between Days 141 and 143 of pregnancy; those failing to initiate parturition by Day 146 had a Caesarean section. Stillborn lambs and those dying within several days of birth were necropsied. Lambs were weighed at birth; lambs (n=132) derived from the same flock (from natural breeding or AI) were used as a control. An estimate of the 99th percentile of the normal population was used as the point above which lambs from three litter-size categories were classified as abnormally large (analysis was repeated for 95% confidence and median estimates of this value). Thirty-eight lambs were born from early transfer, 45 from late transfer and 12 from unobserved lambings (parentage could not be determined). Lambs from early transfer were heavier at birth (P=6.0×10−11). The early treatment group had fewer lambs produced per embryo transferred (P=0.0002), fewer live lambs per embryo transferred (P=0.00009) and fewer normal lambs per embryo transferred (P=0.0002). There was an effect of donor on the number of dead lambs per embryo transferred (P=0.017). The number of dystocias per transfer was not significantly different for early versus late transfer groups. However, the probabilities of survival for cleaved embryos were 27.3% and 13.1% in the early and late transfer treatment, respectively. There were more lambs produced per cleaved embryo with early transfer (P=0.004) and there was an effect of donor within ram and replicate (P=0.04) on the number of lambs produced. The odds ratio for early/late treatment was 2.449 with a confidence interval of 1.368–4.382. Day 2 transfer of cleaved embryos did not prevent the production of oversized or abnormal offspring; however, there was an increased chance of a cleaved embryo producing a lamb when transferred on Day 2 rather than Day 6 postfertilization.

The differences in the embryo production potential of four rams used in a commercial embryo transfer program were examined in both in vivo and in vitro embryo production systems. Processing frozen-thawed spermatozoa through Percoll density gradients prior to in vitro insemination eliminated differences in the estimates of sperm viability between the four rams, and yet, differences in embryo production persisted throughout the in vitro culture period. However, there was no effect of ejaculate within ram on embryo production rates. In addition, the timing of the onset of the differences between the rams at each stage of in vitro embryo development were revealed. Ram 2 differed from ram 4 in the proportion of fertilized oocytes at 17 h post-insemination (pi) and by 52 h, ram 3 differed from ram 4 in the proportion of cleaved embryos, and the observed differences between ram 1 and ram 2 in their blastocyst production were initiated prior to activation of the embryonic genome. Once differences in embryo development rates were detected among the four rams, they persisted throughout the in vitro culture period. The reduced in vitro fertilization (IVF) rates from ram 2 compared with the other rams was paralleled in vivo by the significantly lower proportion of embryos recovered from ewes mated to ram 2, and this was further exacerbated by a significantly lower embryo survival rate after transfer. However, the subtle differences observed in the timing of the contribution of each sire to embryo development during in vitro culture were not able to be detected in vivo. However, the higher proportions of transferable quality blastocysts obtained from ewes mated to ram 4 did not result in increased embryo survival throughout the remainder of gestation. Therefore, in this study, the blastocyst production potential for a particular sire, either in vitro or in vivo, does not necessarily reflect the potential for the production of live offspring.

The efficacy of oocyte selection for in vitro embryo production depends on the abundance and diameter of follicles, cumulus layers around the oocytes and subsequent fertilization. Application of 'ovum pick-up' technique allows us to utilize partially matured oocytes for embryo production even from juvenile subjects. To compare their developmental competence, oocytes derived from lambs and ewes and cultured in maturation medium for up to 26 h were assessed at 2 h intervals by confocal microscopy after chromatin and microtubulin-specific fluorochrome labelling. Lamb oocytes reached second meiotic metaphase (MII) at lower numbers at 24 h (60.0%) and 26 h (28.6%) whereas 85.7% of adult-derived oocytes attained MII status by 24 h of maturation. Radiolabelling of oocyte proteins revealed higher incorporation of [(35)S-]-methionine and [(35)S]-cysteine in adult-derived oocytes compared to lamb oocytes. Although the cleavage rate of lamb oocytes was similar to that of ewe oocytes, the proportion reaching blastocyst stage was significantly lower (p < 0.05) in the lamb-derived oocytes. However, blastocysts from both types of oocytes displayed similar cell lineage allocations to inner cell mass and trophectoderm.

To select rams suitable for ovine in vitro embryo production (IVP), the predictive values of the screening tests used to identify unsuitable rams need to be established. The present study examined some characteristics of frozen-thawed ram spermatozoa that might be evaluated routinely in a commercial breeding programme. These included sperm motility, plasma membrane integrity, morphology, and acrosome and capacitation status of the sperm population. Cryopreserved spermatozoa from four Dorset rams, which had previously satisfied the selection criteria for inclusion in a commercial breeding programme, were used for IVP. The overall contribution of the four rams and the ejaculates within each ram to the variability (R2) in the production of blastocysts was very small (2.1% and 2.5% respectively). The analysis of the sperm characteristics by logistic regression revealed a significant and positive association between total post-thaw sperm motility, viability and longevity with in vitro blastocyst production. However, there was no association between the other surface characteristics of the spermatozoa measured in this study with embryo production. Despite the absence of differences between the rams in the low incidence of polyspermic fertilization, the significant and detrimental effects of polyspermic fertilization on in vitro blastocyst production rates were quantified by logistic regression analysis. A large proportion of the variability within the IVP system was unaccounted for by the analysis of sperm and oocyte characteristics evaluated in this study. Thus, the identification of other factors contributing to the variability in the production of embryos in vitro warrants further investigation. No single sperm characteristic was sufficient to predict the ultimate outcome of blastocyst production. Rather, assessments of multiple characteristics within the IVP system are required to make accurate predictions.

Embryos were generated by in vitro fertilization of in vitro-matured oocytes, cultured to the blastocyst stage, biopsied for sex determination by a PCR-based procedure, and transferred to synchronized recipients. Three out of 5 sheep (60%) were diagnosed pregnant, and 4 lambs of predicted sex were born.

The efficacy of using human interleukin 8 (huIL-8) as an agent for inducing cervical relaxation in estrous and diestrous sheep was assessed in a small pilot study. Multiparous, estrus-synchronized ewes were treated for either 2 or 5 consecutive days with vaginal suppositories with or without 5 micrograms cytokine. Cervical penetration with an insemination instrument was then assessed in vivo. After euthanasia, physical, histological and enzymological properties of the cervix were examined. Treatment of diestrous sheep with huIL-8 did not result in recruitment of neutrophils into the cervix. Treatment of estrous sheep with huIL-8 usually led to neutrophil recruitment to the cervix and to either full or partial penetration of the cervix. However, some animals receiving placebo treatment had neutrophil infiltration of both the vagina and cervix and, in one of these, partial penetration of the cervix was also achieved. Thus, treatment with IL-8 as the sole agent in the vaginal suppository was not sufficient to relax the cervix of the nonpregnant ewe in this study.

The objectives of this study were to evaluate ovarian changes in cows with follicular ovarian cysts following treatment with either GnRH or saline. The parameters determined were the intervals from treatment to observation of a CL and from treatment to disappearance of the cyst, and the association between serum concentrations of LK, FSK and the LH/FSH ratio, before and after treatment, with the test intervals. Thirty-nine cows were identified as having follicular cysts. The GnRH treatment induced a significant increase in LH and the LH/FSH ratio. The gonadotropin response was not associated with the intervals from treatment to CL detection or to disappearance of the cyst. Survival curves for the intervals from treatment to CL detection and cyst disappearance indicate that treatment with GnRH or saline did not yield significantly different results for either parameter. The results question the efficacy of treating cystic ovarian disease with GnRH. (C) 1999 by Elsevier Science inc.

α-Trifluoromethylstyrene, gem-difluoroalkenes, organolithium reagentsThe treatment of α-trifluoromethyl styrene with organolithium reagents results in the selective formation of gem-difluoroalkenes in good to excellent yield. This reaction has been applied to the synthesis of a range functionalised gem-difluoroalkenes.

Objective: To review the available evidence concerning the fitting, management, and efficacy of pessaries used to treat stress incontinence.To describe a method of fitting and managing incontinence pessaries.Methods: An electronic search of the English language literature discussing pessaries and stress urinary incontinence was conducted using the following databases: PubMed (1968–2000), Cochrane Database of Systematic Reviews (CHID), HSTAT, Old Medline, Research Index, and Serline.Results: Twelve journal articles, two book chapters, and a pamphlet were found that discussed the use of pessaries to treat stress incontinence.None of the articles provided detailed discussions of fitting and management of incontinence pessaries and none had any results of this treatment option for stress urinary incontinence.Conclusion: The current literature contains few details concerning pessary care and no scientific evidence for the effectiveness of pessary treatment of stress urinary incontinence.Objectif : Passer en revue les données publiées sur la mise en place, la gestion et l’efficacité des pessaires utilisés pour le traitement de l’incontinence à l’effort et décrire une méthode de mise en place et de gestion de ces dispositifs.Méthode : Une recherche des publications en langue anglaise portant sur les pessaires et l’incontinence urinaire à l’effort a été faite à partir des bases de données suivantes : PubMed (1968–2000), Cochrane Database of Systematic Reviews (CHID), HSTAT, MEDLINE (ancien), Research Index, et SERLINE.Résultats : Cette recherche a produit douze articles de revues, deux chapitres de livres et un feuillet portant sur l’utilisation des pessaires pour le traitement de l’incontinence à l’effort. Aucun des articles ne comportait une discussion détaillée de la mise en place et de la gestion des pessaires pour le traitement de l’incontinence et aucun ne présentait de résultats sur ce traitement pour l’incontinence urinaire à l’effort.Conclusion: Les publications actuelles contiennent peu de détails sur les soins au pessaire et il n’existe aucune donnée scientifique sur son efficacité pour le traitement de l’incontinence urinaire à l’effort.

The effect of a number of procedures involved in the preparation of ram semen for freezing or artificial insemination on sperm mitochondrial function were examined. Ram sperm mitochondrial function was assessed using the membrane potential probe rhodamine 123 (R123).Sperm R123 uptake was increased rather than reduced by incubation of sperm at a high rate of dilution (1: 30), and was unaffected by the addition of 1% serum albumin (BSA). Washing sperm also had no effect on R123 uptake.R123 uptake was reduced in ram sperm exposed to either hypertonic (450 mosmol l−1) or hypotonic (150 mosmol l−1) media (P < 0.05) but was unaffected by varying pH from 5.9 to 8.9. There was no interaction between the effects of osmolality and pH.Mitochondrial injury due to cold shock became apparent when sperm were rapidly cooled below 10°C (P < 0.05). Further damage occurred between 10 and 5°C (P < 0.01). The effects of cold shock were not ameliorated by the addition of 1% BSA, 2 mM butylated hydroxytoluene (BHT) or 50 mM erythritol.The effect of freezing on ram sperm R123 uptake was greater than that of cold shock (P < 0.01). R123 uptake after freezing and thawing in sperm treated with 250 mM sorbitol or 250 mM dimethylsulphoxide (DMSO) was greater than that of unprotected sperm, or sperm exposed to 4% glycerol (P < 0.05).Accumulated R123 was released rapidly from frozen ram sperm (P < 0.01) but released more slowly from cold shocked sperm (P < 0.01). This suggests that the two stressors induce different types of mitochondrial injury.

Follicular dynamics and ovulation were compared in 3 groups of anestrous ewes: those treated with medroxyprogesterone acetate (MAP) sponges for 12 d, then with 750 IU PMSG at the time of sponge removal (P4 + PMSG, n = 6), or PMSG alone (n = 6) and untreated controls (n = 6). Waves of follicular activity were observed in all the animals. In the P4 + PMSG treatment group, MAP priming permitted more ovulatory follicles (P < 0.001) to be recruited without changing follicle growth rate; MAP priming also delayed the time of ovulation (P < 0.001) and the time of the LH surge (P < 0.01), which allowed for an increase in the size of ovulatory follicles (P < 0.05). Ovulation also resulted in normal luteal function after P4 + PMSG (P < 0.01) but not after PMSG alone, since premature luteal regression occurred in 80% of the cases and was related to the presence of follicles > 4 mm when P4 levels were < 1 ng/mL on the day following ovulation. The results showed that MAP priming increased the ovulation rate by increasing the number of follicles that responded to PMSG.

To determine the outcome of interbreeding sheep and goats, ewes and does were bred to rams and bucks, and their embryos recovered. Pregnancy was monitored in 2 does bred to a ram. Fertilization rates in ram X does, buck X does, ram X ewes, and buck X ewes were 72%, 96%, 90%, and 0%, respectively. Ram X doe fetuses died at 5 to 10 weeks.

A total of 104 oocytes were aspirated from 11 mature ewes and 4 prepubertal lambs. After in vitro maturation, fertilization and culture, 11 of the 104 oocytes developed into blastocysts. These were cryopreserved in 1.5 M ethylene glycol. After being thawed, all were transferred into four recipients, two of which became pregnant and delivered a normal lamb. Key words: Oocyte, cryopreservation, in vitro maturation and fertilization, sheep, embryo

This study evaluated the influences of seasons and genotype on the superovulatory response to a standardised oFSH regimen in red deer (Cervus elaphus scoticus) and its hybrids with either wapiti (C.e. nelsoni) or Père David's (PD) deer (Elaphurus davidianus). Adult red deer (n=9), F1 hybrid wapiti×red deer (n=6), and maternal backcross hybrid PD×red deer (i.e., 14 PD hybrid; n=9) were kept together in the presence of a vasectomised stag for 13 months. At 6 weekly intervals, all hinds received a standardised treatment regimen used routinely to induce a superovulatory response in red deer hinds, with 10 consecutive treatments spanning an entire year. This involved synchronisation with intravaginal progesterone devices and delivery of multiple injections of oFSH (equivalent to 72 units NIH-FSH-S1). Laparoscopy to assess ovarian response was performed 6–7 days after the removal of the devices. Both season and genotype had significant effects on ovulation rate (OR) and total follicular stimulation (TFS) (P<0.05). For all the three genotypes, ovarian responses were highest from March to November (breeding season) and lowest in the period from December to January, inclusive. Mean OR for red deer hinds ranged from 3.7 to 1.8 during the breeding season, with no observable trend. All red deer hinds were anovulatory during December and January. A similar pattern occurred for 14 PD hybrids, although mean OR during the breeding seasons were twofold lower than for the red deer. For F1 wapiti hybrids, the first two treatments in March and April resulted in the highest mean OR observed (15.6 and 11.7, respectively). Thereafter, mean values ranged between 6.3 and 4.7 for the remainder of the breeding season. Furthermore, mean OR of 3.0 and 0.5 were recorded in December and January, respectively. For the red deer and F1 wapiti hybrids, between-hind variation in OR was not randomly distributed across the treatment dates, indicating that the individuals varied significantly in their ability to respond to oFSH, at least within a given season.In conclusion, the study has shown that relative to red deer, F1 wapiti hybrid hinds exhibit a higher sensitivity to oFSH, whereas 14 PD hybrid hinds have a lower sensitivity. However, individual variation within genotype was very marked. A seasonal effect was apparent for all genotypes, although some F1 wapiti hybrid hinds exhibited ovulatory responses throughout the year.

Experiments were conducted to devise an efficient method to cryopreserve ovine embryos for field application. Embryos were surgically collected from superovulated ewes on Day 6 after natural breeding; oocytes were collected from ovaries obtained at the abattoir. Osmotic behavior of oocytes and embryos was determined by measuring their responses to hypertonic solutions of CsCl or sucrose. Embryos and oocytes contracted osmotically by decreasing their volumes proportionally to the reciprocal of the solution's osmolality. The respective nonosmotic volumes of embryos in CsCl and sucrose were 13.8 and 13.5% of their isotonic volume. and those of oocytes were 18.5 and 19.6%. Tests of the permeability of morulae to commonly used cryoprotectants, ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), and glycerol (Glyc), showed that the order of permeability was EG > PG > DMSO ≈ Glyc. Comparison of the efficacy of cryoprotective agents indicated that the respective survivals of embryos frozen in EG, PG, and DMSO were 76.9, 62.5, and 55.6%, based on their development into hatched blastocysts in vitro. Therefore, EG appeared to be superior to the other two cryoprotectants for freezing sheep embryos. To determine the functional survival of embryos in vivo. 67 embryos frozen in EG were thawed and directly diluted with phosphate-buffered saline: 47 of these (70%) appeared morphologically normal and were transfected into 14 recipients. Five of these recipients, which had received a total of 16 embryos, became pregnant. Ten lambs were born, showing that the method employed in this study for cryopreservation of sheep embryos followed by their direct dilution out of EG has potential application for practical field use.

The objectives of this study were to describe the serological prevalence of maedi-visna in a sample of Ontario sheep flocks, and to identify management and demographic variables that were associated with seroprevalence for maedi-visna. A sample of 103 sheep flocks in Ontario was randomly selected from those flocks participating in the Red Meat Plan. The owners of these flocks were surveyed regarding management procedures on their farms, and blood samples were taken from a random sample of ewes in each flock. At least one ewe tested serologically positive, based on the agar gel immuno-diffusion test, in 69.9% of the farms. Positive serological reactions occurred in 20.9% of the 3880 sheep tested. Flock demographics and farm management variables were considered in a multiple regression model, and several factors were positively associated with higher maedi-visna seroprevalence rates. These included the average age of the flock, the number of years the owner had been sheep farming, the practice of using foster ewes, the practice of allowing lambs to have contact with other ewes that are lambing, and the average pasture acreage per ewe.

We compared conventional methods for laparoscopic and cervical artificial insemination (AI) to a transcervical AI procedure (Guelph System for Transcervical AI; GST-AI) for use with frozen semen in Merino ewes. The GST-AI procedure was performed by an experienced operator in Experiment 1 (771 ewes) and by 2 inexperienced operators in Experiment 2 (555 ewes). In Experiment 1, intrauterine insemination by GST-AI was achieved in 76% of the ewes. The pregnancy rate at Day 70 for ewes inseminated by laparoscopy (48%, 120 251 ) was higher (P<0.01) than for ewes inseminated by either intrauterine GST-AI (32%, 64 201 ) or cervical AI (9%, 24 256 ). The overall (intrauterine and intracervical) pregnancy rate for GST-AI was 26% (68 264 ) and was unaffected by depth of insemination within the cervix. Pregnancy rates were unaffected by ram or day of insemination. In Experiment 2, the operators achieved intrauterine inseminations by GST-AI in 43% (78 182 ) of the ewes, with a significant operator effect (P<0.01) on depth of cervical penetration. The pregnancy rate to intrauterine GST-AI (40%, 31 78 ) did not differ from that to laparoscopic insemination. The total pregnancy rate for GST-AI in Experiment 2 (19%, 34 182 ) was lower (P<0.05) than that for laparoscopic AI (39%, 72 187 ) but superior (P<0.05) to that for cervical AI (1%, 1 186 ). The GST-AI pregnancy rates were affected by depth of AI (P<0.01) and by operator (P<0.05). It is concluded that GST-AI is superior to cervical AI, and may have application in Merinos if cervical penetration rates can be improved.