The fertility of ram sperm held for 24h at 5 degrees C prior to cryopreservation.

Diluted ram sperm can be held for 24h at 5 degrees C prior to cryopreservation without impacting cryosurvival rates, however, the effects this storage has on subsequent fertility are unknown. These studies were conducted to evaluate the fertility of semen held for 24h (to mimic shipping semen to a cryopreservation center), prior to freezing. Semen from Suffolk rams (n=3 in experiment 1 and n=6 in experiment 2) with initial motility of greater than 70%, were diluted to 200 x 10(6)sperm/mL, in one step, with a Tris-egg yolk-glycerol diluent. In experiment 1, diluted samples were cooled to 5 degrees C over 2h, and then divided. Sperm in one fraction were loaded into 0.5mL straws, frozen (T0) and stored in liquid nitrogen until thawing. Sperm in the second fraction were held at 5 degrees C for 24h (T24) before being frozen. In experiment 2 ejaculates were collected and divided into two fractions. Sperm in one fraction were treated with cholesterol-loaded cyclodextrin (CLC) and sperm in the other served as control. Both fractions were diluted, cooled, and cryopreserved as described in experiment 1. Stage of the estrous cycle was synchronized in ewes (n=196) using controlled internal drug releasing devices (CIDR) for 12d and at CIDR removal each ewe was administered PMSG (500IU in experiment 1 and 350IU in experiment 2) immediately before insemination. Ewes were stratified by age and randomly assigned to one of the semen treatments; experiment 1: Fresh (F), T0, or T24; experiment 2: F, T24, or CLC, and inseminated laparoscopically 56h after CIDR removal. Differences in fertility were detected between experiments, but not for treatments within experiments. Differences in fertility were also observed due to ewe age, with the 3-year-old ewes having the greatest fertility (50.7%) and 6-year-old ewes having the least fertility (9.6%; P<0.05). Differences in the prolificacy rates due to semen treatment were also observed but differences due to ewe age were not detected. Therefore, sperm can be held at 5 degrees C for 24h prior to cryopreservation without altering sperm fertility.

Maternal obesity accelerates fetal pancreatic beta-cell but not alpha-cell development in sheep: prenatal consequences.

Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring.

Effect of maternal fatness on fetal steroids and semi-quantitative real-time PCR expression of receptor genes in sheep.

Sexual differentiation of the brain occurs between d 30 and 70 in the fetal lamb. The objective of this experiment was to determine if maternal fatness affects fetal steroid production and expression of their receptors which may ultimately alter endocrine systems postnatally. Fetuses were collected from ewes fed at either 100% (Control; n=5) or 150% (Fat; n=6) of NRC recommendations from 60 d prior to breeding until collection at 75 d of gestation. Hypothalamic and amygdala neural tissues were collected from twin male/female fetuses. Serum concentrations of testosterone were greater (P<0.001) in male fetuses compared to female fetuses. Further, male fetuses from Fat ewes had greater (P<0.05) serum concentrations of testosterone than male fetuses from Control ewes, but differences in testicular steroidogenic enzyme mRNA were not detected (P=0.18). Quantity of hypothalamic mRNA for estrogen receptor (ER) beta tended (P=0.1) to be influenced by a sex by treatment interaction. Messenger RNA for ER-beta was greater in female fetuses than male fetuses from Control ewes (P=0.05). Although amount of ER-beta mRNA did not differ among male fetuses (P=0.7), amounts tended to be less (P=0.07) in female fetuses from Fat ewes compared to those from Control ewes, and did not differ (P> or =0.8) from male fetuses. Hypothalamic ER-alpha mRNA tended (P=0.1) to be less in fetuses from Fat ewes compared to Control fetuses but was not influenced (P=0.3) by fetal sex or their interaction. Amount of mRNA for hypothalamic progesterone receptor tended (P=0.06) to be greater in male fetuses than female fetuses and tended to be less (P=0.06) in fetuses from Fat ewes than in Control fetuses, but did not differ by any sex by treatment interaction (P=0.6). Hypothalamic RNA for the androgen receptor did not differ by sex, dam nutritional treatment, or the interaction. Likewise, amygdala RNA for the estrogen or androgen receptor did not differ (P> or =0.3) by sex, treatment, or their interaction. Dam fatness appears to decrease the expression of progesterone receptor, ER-alpha, and decrease amount of ER-beta in the female fetuses while increasing circulating concentrations of testosterone in male fetuses. Altered expression of hypothalamic receptor genes by the uterine environment may affect adult responses to stress, sexual behavior and/or the pattern of gonadotropin release in response to gonadal steroids.

Scrapie resistance and production traits in Rambouillet rams: ram performance test 2002-2006.

Sheep possessing alleles for the prion protein with glutamine (Q) or histidine, both reported as Q, at codon 171 are highly susceptible to scrapie. Incidence of scrapie infection is rare when animals possess at least one allele for arginine (R) at codon 171. The current USDA APHIS scrapie eradication program utilizes genotyping for alleles that confer resistance to scrapie. Although it has not been a criterion of registration, genotyping has been utilized in the University of Wyoming Ram Performance test since 2002. Performance test records from 2002 to 2006 (n=518) were analyzed to determine if any production loss or benefit was associated with scrapie resistance in rams. Performance of rams with scrapie resistant genotypes was equal to or better than scrapie susceptible rams. Based on test performance as an acceptable indicator of sire superiority, selection for rams with scrapie resistant genotypes should not negatively impact flock performance.

Dietary supplementation with safflower seeds differing in fatty acid composition differentially influences serum concentrations of prostaglandin F metabolite in postpartum beef cows.

Synthesis and secretion of prostaglandin F2alpha (PGF2alpha) is elevated following parturition and exerts divergent effects on the re-establishment of fertile estrous cycles in cows. The objective of these experiments was to determine if oil seed supplements differing in fatty acid composition differentially influence serum concentrations of the specific PGF2alpha metabolite, PGFM. Safflower seed supplements were formulated to provide 5% of dry-matter intake as fat. In Trial 1, 24 multiparous beef cows were individually fed control (beet pulp-soybean meal) or cracked high-linoleate safflower seed (78% 18:2n-6) supplements for 80 d postpartum. Linoleate supplemented cows had greater (P < 0.001) serum concentrations of PGFM than control cows. In Trial 2, primiparous beef cows (n = 36) were individually fed control (cracked corn-soybean meal), cracked high-linoleate (76% 18:2n-6) or -oleate (72% 18:1n-9) safflower seed supplements for 92 d postpartum. As in Trial 1, serum concentrations of PGFM were greater (P < or = 0.04) in linoleate than control or oleate supplemented cows. Serum concentrations of PGFM, however, did not differ (P = 0.40) among oleate and control supplemented cows. Although potential impacts on reproductive performance remain to be proven, dietary oil supplements high in linoleate, but not oleate, increased serum concentrations of PGFM compared to control supplements.

Maternal undernutrition from early- to mid-gestation leads to growth retardation, cardiac ventricular hypertrophy, and increased liver weight in the fetal sheep.

Early gestation is critical for placentomal growth, differentiation, and vascularization, as well as fetal organogenesis. The fetal origins of adult disease hypothesis proposes that alterations in fetal nutrition and endocrine status result in developmental adaptations that permanently change structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine disease in adult life. Multiparous ewes were fed to 50% (nutrient restricted) or 100% (control fed) of total digestible nutrients from Days 28 to 78 of gestation. All ewes were weighed weekly and diets adjusted for individual weight loss or gain. Ewes were killed on Day 78 of gestation and gravid uteri recovered. Fetal body and organ weights were determined, and numbers, morphologies, diameters, and weights of all placentomes were obtained. From Day 28 to Day 78, restricted ewes lost 7.4% of body weight, while control ewes gained 7.5%. Maternal and fetal blood glucose concentrations were reduced in restricted versus control pregnancies. Fetuses were markedly smaller in the restricted group than in the control group. Further, restricted fetuses exhibited greater right- and left-ventricular and liver weights per unit fetal weight than control fetuses. No treatment differences were observed in any gross placentomal measurement. However, caruncular vascularity was enhanced in conceptuses from nutrient-restricted ewes but only in twin pregnancies. While these alterations in fetal/placental development may be beneficial to early fetal survival in the face of a nutrient restriction, their effects later in gestation as well as in postnatal life need further investigation.

Active surveillance for scrapie by third eyelid biopsy and genetic susceptibility testing of flocks of sheep in Wyoming.

Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrP(Sc), the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrP(Sc) assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrP(Sc)-positive sheep were identified through the passive surveillance program, and an additional three PrP(Sc)-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrP(Sc)-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program.