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.

Variability of albumin adducts of 1,4-benzoquinone, a toxic metabolite of benzene, in human volunteers.

A putative haematotoxic and leukaemogenic metabolite of benzene, 1,4-benzoquinone (1,4-BQ), reacts rapidly with macromolecules. The authors previously characterized levels of the albumin (Alb) adduct (1,4-BQ-Alb) of this reactive species in populations of workers exposed to benzene. Since high levels of 1,4-BQ-Alb were also measured in unexposed workers from those investigations, the current study was initiated to determine potential sources of 1,4-BQ in the general population. A single blood sample was collected from 191 healthy subjects from the Research Triangle area, NC, USA, to determine the baseline 1,4-BQ-Alb levels and contributing sources. The median 1,4-BQ-Alb at baseline was 550?pmol?g(-1) Alb (interquartile range 435-814?pmol?g(-1)). A second blood sample was collected approximately 3 months later from a subgroup of 33 subjects to estimate the within- and between-person variation in 1,4-BQ-Alb. Standardized questionnaires were administered to collect information about demographic, dietary and lifestyle factors. Multiple linear regression models identified several significant contributors to 1,4-BQ-Alb levels, including gender, body mass index (BMI), the gender-BMI interaction, automobile refuelling, smoking status, and consumption of fruit and the artificial sweetener, aspartame. The authors predicted that these background levels of 1,4-BQ-Alb were equivalent to occupational exposures between 1 and 3 parts per million of benzene. Mixed effects linear models indicated that the random variation in adduct levels was about equally divided between and within subjects. The observations indicate that levels of 1,4-BQ-Alb cover a wide range in the general population, and they support the hypotheses that demographic, diet and lifestyle factors are contributing sources.

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.

Individual versus group therapy for obesity: effects of matching participants to their treatment preferences.

This study examined the effects of matching participants to treatments on the basis of their preferences for either individual or group therapy for obesity. Seventy-five obese adults who expressed a clear preference for either individual or group therapy were randomly assigned to either their preferred or their nonpreferred treatment modality within a 2 (individual vs. group therapy) x 2 (preferred vs. nonpreferred modality) factorial design. At posttreatment, group therapy produced significantly greater reductions in weight and body mass than individual therapy, and no significant effects were observed for treatment preference or the interaction for treatment preference by type of therapy. All treatment conditions showed equivalent improvements in psychological functioning. These findings suggest that group therapy produces greater weight loss than individual therapy, even among those clients who express a preference for individual treatment.

Relapse prevention training and problem-solving therapy in the long-term management of obesity.

This study compared 2 extended therapy programs for weight management with standard behavioral treatment (BT) without additional therapy contacts. Participants were 80 obese women who completed 20 weekly group sessions of BT and achieved a mean initial weight loss of 8.74 kg. Participants were randomly assigned to a no-further-contact condition (BT only) or to one of two extended interventions consisting of relapse prevention training (RPT) or problem-solving therapy (PST). No significant overall weight-change differences were observed between RPT and BT or between RPT and PST. However, participants who completed the PST intervention had significantly greater long-term weight reductions than BT participants, and a significantly larger percentage of PST participants achieved clinically significant losses of 10% or more in body weight than did BT participants (35% vs. 6%).

Determinants of melanoma in a case-control study of twins (United States).

OBJECTIVES: To estimate the relative contribution of environmental and genetic factors disposing towards the development of melanoma. METHODS: We investigated risk factors for melanoma in a case-control study conducted among 185 North American twin pairs in which one was diagnosed with melanoma and the other (the co-twin of the case) was not. We considered monozygous (MZ) and dyzygous (DZ) twins separately. RESULTS: While greater risk of melanoma was associated with number of large nevi in DZ twins (adjusted OR = 26.6 (4.2-170.8) for three or more large moles), this was not the case for MZ twins (adjusted OR = 1.4 (0.6-3.2) for three or more large moles). Elevated risks of developing melanoma with site-specific sun exposures resulting in sunburn also appeared to be confined to DZ twins. Despite the number of identical twins reporting a difference in mole prevalence, we observed only a modest and inconsistent increase in melanoma risk attributable to that factor, whereas the increase within fraternal twin pairs was larger by an order of magnitude. CONCLUSIONS: If confirmed, this indicates that the significance of mole prevalence as a risk factor for melanoma is largely as a genetic, rather than an environmental, factor. We noticed an increased risk of developing melanoma among DZ twins who drank moderate amounts of beer, but we believe this result may be due to the sun-exposure activities of those people most likely to drink moderate amounts of beer.

A second look at the relation between colorectal adenomas and consumption of foods containing partially hydrogenated oils.

The trans fatty acids in partially hydrogenated vegetable oil may cause colorectal neoplasia by interfering with cell membrane function or eicosanoid metabolism. This possibility provided a rationale for looking at the relation between colorectal adenomas and consumption of foods containing partially hydrogenated vegetable oils in 234 cases and 407 controls recruited from referrals for colonoscopy at University of North Carolina Hospitals in Chapel Hill, between 1988 and 1990. Foods containing partially hydrogenated vegetable oils were divided into four groups: sweetened baked goods, chocolate candy, oils and condiments, and french fries and chips. We observed no evidence of increased adenoma prevalence associated with consumption of fries and chips (200+ vs 0 kcals/day: odds ratio (OR) = 0.70; 95% confidence limits (CL) = 0.27, 1.8) or chocolate candy (50+ vs 0 kcals/day: OR = 0.49; 95% CL = 0.23, 1.1). We did, however, find evidence of increased adenoma prevalence associated with consumption of sweetened baked goods (400+ vs < 100 kcals/day: OR = 1.9; 95% CL = 0.95, 3.8) and oils and condiments (200+ vs < 100 kcals/day: OR = 2.4; 95% CL = 1.3, 4.2).

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.

A case-control study of colorectal adenomatous polyps and consumption of foods containing partially hydrogenated oils.

The trans fatty acids produced by partially hydrogenating vegetable oils may cause colorectal neoplasia by interfering with cell membrane function or eicosanoid synthesis. This possibility provides a rationale for looking at the relation between colorectal adenomatous polyps and consumption of foods containing partially hydrogenated vegetable oils (PHVOs). A total of 516 cases and 551 controls who underwent screening sigmoidoscopy from 1991-1993 were recruited from a prepaid Los Angeles health plan. Subjects were interviewed and given a self-administered food frequency questionnaire. Food items containing PHVOs were divided into four groups characterized by principal ingredients and preparation methods: sweetened baked goods, candy bars, oils and condiments, and french fries and chips. After adjusting for age, sex, physical activity, body mass index, smoking, total energy, and red meat and vegetable intake, there was a positive association between polyps and sweetened baked goods [350+ versus <50 kcal/day (odds ratio, 2.1; 95% confidence interval, 1.3-3.5)]. No association was found with the other food groups after adjustment for dietary and nondietary covariates. Neither was total dietary trans fatty acid associated with adenomas after adjustment for sweetened baked goods and other covariates. These results do not support the hypothesis that eating foods containing PHVOs increases the risk of colorectal adenomas, but they are consistent with the hypothesis that foods high in fat and sugar and low in fiber and correlated micronutrients increase the risk of adenomas.

Experimentally induced bovine spongiform encephalopathy did not transmit via goat embryos.

Goats are susceptible to experimental challenge with bovine spongiform encephalopathy (BSE). This study set out to investigate whether the transmission of BSE could occur in goats following the transfer of embryos from experimentally infected donor females into uninfected recipient females. The results showed no evidence of transmissible spongiform encephalopathy disease in any of the offspring which developed from embryos from infected donors, nor indeed in any of the recipient females used as surrogate dams. In addition, there was no indication of experimental BSE spreading as either a venereal infection to males used in mating or by maternal transmission to offspring born naturally to experimentally infected donors, although numbers were small.

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.