G. E. Moss

Maternal Undernutrition from Early- to Mid-Gestation Leads to Growth Retardation, Cardiac Ventricular Hypertrophy, and Increased Liver Weight in the Fetal Sheep

Abstract 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.

Maternal obesity accelerates fetal pancreatic β-cell but not α-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.

Down-Regulation of Growth Signaling Pathways Linked to a Reduced Cotyledonary Vascularity in Placentomes of Over-Nourished, Obese Pregnant Ewes

Both protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) are down-stream components of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. AMP-activated protein kinase (AMPK) is known to sensitize cells to insulin/IGF-1 signaling. The objective of this study was to assess the activity of AMPK and its role in the observed down-regulation of insulin/IGF-1 signaling in cotyledonary (COT) arteries supplying the placental component of the ewe placentome. Nonpregnant ewes were randomly assigned to a control (C, 100% of NRC recommendations) or obesogenic (OB, 150% of NRC) diet from 60 days before conception until necropsy on day 75 of gestation (n=5/group) or until lambing (n=5/group). At necropsy on day 75 of gestation, the smallest terminal arteries that entered the COT tissues (0.5-1.0 mm in diameter) were collected for analyses. Fetal weights were approximately 20% greater (P<0.05) on OB than C ewes, but birth weights of lambs were similar across dietary groups. Fetal plasma concentrations of glucose, insulin and IGF-1 were higher (P<0.05) in the blood of fetuses from OB than C ewes. Total AMPK and phosphorylated AMPK at Thr 172 (the active form) were reduced (P<0.05) by 19.7+/-8.4% and 25.9+/-7.7%, respectively in the COT arterial tissues of OB ewes. Total acetyl-CoA carboxylase (ACC), a down-stream target of AMPK, and its phosphorylated form were also reduced (P<0.05) by 32.9+/-9.2% and 45.4+/-14.6%, respectively. The phosphorylation of IRS-1 at Ser 789, a site phosphorylated by AMPK, was 24.5+/-9.0% lower (P<0.05) in COT arteries of OB than C ewes. No alteration in total insulin receptor, total IGF-1 receptor or their phosphorylated forms was observed, down-stream insulin signaling was down-regulated in COT arteries of OB ewes, which may have resulted in the observed decrease in COT vascular development in OB ewes.

Active Surveillance for Scrapie by Third Eyelid Biopsy and Genetic Susceptibility Testing of Flocks of Sheep in Wyoming

ABSTRACT 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 PrPSc, the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrPSc 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 PrPSc-positive sheep were identified through the passive surveillance program, and an additional three PrPSc-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrPSc-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.

Estradiol and gonadotropin-releasing hormone (GnRH) interact to increase GnRH receptor expression in ovariectomized ewes after hypothalamic-pituitary disconnection

Gonadotropin-releasing hormone (GnRH) receptor expression is regulated by estradiol and GnRH itself. The objective of this experiment was to determine the extent to which low levels of estradiol, similar to those observed during the transition from the luteal to the follicular phase of the estrous cycle, and GnRH interact to regulate expression of GnRH receptors and GnRH receptor mRNA. Ewes were ovariectomized (OVX) at least 2 wk prior to initiation of the experiment, and the pituitary gland was surgically disconnected from the hypothalamus to remove ovarian and hypothalamic inputs to the pituitary. Within 24 h after hypothalamic-pituitary disconnection, ewes received pulses of GnRH (250 ng/pulse) every 2 h for 6 d. At the end of 6 d, ewes were randomly assigned to treatments in a 2 × 2 factorial arrangement as follows: half of the animals received a single estradiol implant and half received an empty implant (placebo). At the same time, animals also received one of the following treatments: (1) saline or (2) GnRH (100 ng/pulse/2 h). Additionally, one group of ewes was ovariectomized, but not subjected to hypothalamic-pituitary disconnection (OVX controls). Blood samples were collected 15 min prior to each pulse of GnRH or saline and at 15-min intervals for 1 h after each pulse until tissues were collected and concentrations of luteinizing hormone (LH) were determined. Anterior pituitaries were collected 24 h after implant insertion to quantitate steady-state amounts of GnRH receptor mRNA and numbers of GnRH receptors. Mean LH was greatest in ovariectomized control ewes compared to all other treatments (p<0.05). Mean LH and LH pulse amplitude in the placebo and GnRH-treated group most closely mimicked LH secretion in ovariectomized control animals. Mean LH and LH pulse amplitude were similar between both GnRH-treated groups (p<0.05). Mean LH and LH pulse amplitude were significantly lower in all animals treated with saline compared to OVX controls (p<0.05). Treatment with an estradiol implant and pulsatile GnRH increased (p<0.05) relative amounts of GnRH receptor mRNA and the number of GnRH receptors compared to all other treatments. There were no differences in GnRH receptor expression between the remaining treatment groups (p>0.05). Therefore, in OVX ewes after hypothalamic-pituitary disconnection, low levels of estradiol and GnRH are required to increase GnRH receptor mRNA and GnRH receptor numbers. Since we only observed an increase in GnRH receptor expression in the presence of both estradiol and GnRH, we conclude that there is a synergistic interaction between these two hormones in the regulation of GnRH receptor expression.

The fertility of ram sperm held for 24 h at 5°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.

Fos-like immunoreactivity in brain regions of domestic rams following exposure to rams or ewes

Limbic and basal forebrain-hypothalamic regions from male sheep differing in sexual performance were quantified for fos-like immunoreactivity. Rams classified as high-sexually performing (HP), low-sexually performing (LP), and male-oriented (MO) received noncontact sensory stimulation from either ewes in estrus (HP, n=5; LP, n=4; MO, n=4) or other males (HP, n=5; LP, n=4; MO, n=5) for a 4-h period on each of 3 consecutive days. Following exposure to stimulus animals on the third day, rams were euthanized and their brains were perfused with a 1% paraformaldehyde/1.5% glutaraldehyde solution and sections were analyzed for fos-like immunoreactivity. Brain regions analyzed were the medial amygdala (meAMY), medial preoptic area (mPOA), bed nucleus of the stria terminalis (BNST), and ventromedial hypothalamic nucleus (VMH). Fos-like immunoreactivity differed between groups in the mPOA and BNST but not in the meAMY or VMH. LP rams exposed to estrous ewes had more (P<.05) neurons staining positive for fos and fos-related antigens (FRA) in the mPOA and BNST than LP rams exposed to other rams or MO rams exposed to either estrous ewes or other rams. Numbers of neurons staining positive for FRA in the mPOA and BNST of LP rams exposed to estrous ewes, however, were not different (P>.05) from HP rams exposed to either estrous ewes or other rams. The similar fos-like immunoreactivity in areas important for the display of sexual behavior in HP and LP rams may reflect similar sensory input in these two groups of rams; however, LP rams, in contrast to HP rams, do not appear to respond similarly to the same sensory stimulus.

Influence of Prepartum Fat Supplementation on Subsequent Beef Cow Reproduction and Calf Performance

Abstract High fat range supplement ( HFRS ) and HFRS with lipid from soybean soapstock (HFRS-SPH; Consolidated Nutrition, Omaha, NE) were compared with a corn-soybean meal supplement (control). In Exp. 1, primiparous cows were individually fed the control supplement (n = 12), HFRS (n = 12), or HFRS-SPH (n = 10) for 62 ± 2 d prepartum. Heifer body condition score pre- and postpartum did not differ (P=0.78) among groups. Milk production was not influenced (P=0.15) by source of supplement. Somatic cell counts, however, tended to be less (P=0.07) in HFRS-supplemented heifers than in heifers fed the control supplement. At birth, calf body temperature (P=0.8), vigor (P=0.7), and BW (P=0.6), as well as BW gain through 90 d postpartum (P=0.6), did not differ among prepartum supplementation treatments. Plasma concentrations of linoleic acid were greater (P=0.02) in fat-supplemented heifers at 30 d prepartum and at calving compared with heifers on the control treatment; however, concentrations of plasma linoleic acid returned to levels comparable with those in control heifers by 30 d postpartum. Neither number of cows cycling by 90 d postpartum (P=0.15) nor length of the postpartum interval (P=0.25) differed among treatment groups. In Exp. 2, multiparous cows were pen-fed the control supplement (n = 49), HFRS (n = 47), or HFRS-SPH (n = 49) for 59 ± 2 d prepartum. Prior to parturition, cows fed the control supplement had better body condition scores (5.8 ± 0.1; P=0.004) than cows fed either commercial supplement (5.4 ± 0.1). Calf performance (P=0.7) and conception rates (P= 0.5) did not differ among treatments. Productivity of cows and calves was not improved with provision of supplemental fat prepartum.

Low-sexually performing rams but not male-oriented rams can be discriminated by cell size in the amygdala and preoptic area: a morphometric study.

Brain regions of male sheep behaviorally classified as high-sexually performing (n=10), low-sexually performing (n=8) or male-oriented (n=9) were examined to determine if differences in reproductive behavior were associated with differences in density or sizes of neurons. High-sexually performing rams actively mounted estrous ewes, low-sexually performing rams failed to mount or had long latencies to mounting estrous ewes, and male-oriented rams mounted other rams in preference to ewes in estrus. Cell densities and sizes were quantified in Nissl stained sections through the medial amygdala (meAMY), preoptic area (POA), bed nucleus of the stria terminalis (BNST), ventromedial hypothalamic nucleus (VMH), lateral geniculate nucleus (LG) and medial geniculate nucleus (MG). Multivariate discriminant analysis based on soma sizes within nuclei of known importance for reproductive behavior and/or gonadotropin release (meAMY, POA, BNST and VMH) discriminated (Wilks Lambda P<0.05) low-performing rams from high-performing and male-oriented rams, but did not discriminate (Wilks Lambda P=0.14) between high-performing and male-oriented rams. Cell size in the parvocellular and magnocellular layers of the LG along with cells of the MG, structures without a specific role in reproduction, did not discriminate any of the three behaviorally defined groups of rams (Wilks Lambda P=0.57). Density of cells present in structures important for the display of reproductive behavior (POA, meAMY, BNST) and/or gonadotropin release (POA, VMH) had no discriminating power nor did density of cells in structures important for the processing of visual (LG) or auditory (MG) stimuli. In conclusion, significant differences in sizes of cells located within nuclei that are specifically important for the display of male reproductive behavior were found in low-sexually performing rams compared to high-sexually performing and male-oriented rams. These differences may result from neuron development in utero or occur later as a consequence of endocrine factors or behavioral experience. Neuronal cell size is a critical variable that determines excitability to synaptic inputs because cell surface area varies exponentially with cell diameter. Relatively small differences in neuron diameter could relate to functionally important differences in neuronal excitability.

Insulin-like growth factor binding proteins in the bovine anterior pituitary.

Insulin-like growth factor binding proteins (IGFBPs) were characterized in bovine anterior pituitary tissue, pituitary conditioned media, and serum collected during the preovulatory and early luteal phases of the estrous cycle. Effects of in vitro treatments of pituitaries with luteinizing hormone-releasing hormone (LHRH), estradiol, and progesterone on IGFBP secretion were also evaluated. Predominant IGFBPs detected in anterior pituitary tissue by immunoprecipitation, ligand blotting, and Northern blotting were IGFBP-5 (29 kDa), IGFBP-2 (32 kDa), and IGFBP-3 (36 and 39 kDa doublet). Conditioned culture media contained IGFBP-5, a slightly larger form of IGFBP-3 (33 kDa), the 36- and 39-kDa forms of IGFBP-3, and a more extensively glycosylated form of IGFBP-3 (44 kDa). In serum, IGFBP-5 was not readily detected, and IGFBP-3 (40- and 44-kDa doublet) and IGFBP-2 (34 kDa) were larger than in pituitary tissue. Levels of IGFBP-2,-3, and-5 in pituitary tissue decreased during the preovulatory period and were lowest in the early luteal phase. Treatment with LHRH increased IGFBP-2 levels in media twofold. Estradiol or progesterone did not alter IGFBP secretion in vitro. Predominant IGFBPs produced and released by anterior pituitary tissue were IGFBP-2,-3 and-5. The activity of IGFBPs fluctuates in the pituitary in association with changes in stage of estrous cycle, implicating IGFBPs as potential regulators of gonadotrope function.