Effects of fetal and maternal genotype on placentome morphology in sheep.

Both maternal and fetal genotypes contribute to conceptus development. The objective was to determine how placentome number, size, and type and fetal weight was influenced after reciprocal embryo transfer in Columbia and Romanov sheep. Reciprocal embryo transfer was conducted between Columbia and Romanov ewes where a single embryo was transferred into Romanov and Columbia recipients [Romanov embryo in a Romanov uterus (RinR, n = 9); Romanov embryo in a Columbia uterus (RinC, n = 7); Columbia embryo in a Columbia uterus (CinC, n = 8); Columbia embryo in a Romanov uterus (CinR, n = 4)]. On day 130 of gestation, fetuses were weighed and placentomes were morphologically typed, weighed, and measured. Regardless of maternal genotype, Romanov fetuses were smaller (P < 0.05) compared to Columbia fetuses. Moreover, CinC fetuses were larger (P < 0.05) than CinR fetuses. There was a tendency (P = 0.12) for a fetal by maternal genotype interaction on total placentome weight, but main effects were significant for fetal genotype (P = 0.04) and maternal genotype (P < 0.01). The number of Type A placentomes was greater than any other types. Type A placentomes had a greater (P < 0.05) contribution to total placentome weight within the Romanov uterus, or when associated with a Romanov fetus, than within the Columbia breed, in which placentome type was evenly distributed. The hypothesis that the Romanov uterus would limit the growth of a Columbia conceptus is accepted; however, the Romanov conceptus did not experience augmented growth when transferred into a Columbia uterus as predicted.

Nutritionally induced tanycytic plasticity in the hypothalamus of adult ewes.

The blood-brain barrier regulates the transport of molecules that convey global energetic status to the feeding circuitry within the hypothalamus. Capillaries within the median eminence (ME) and tight junctions between tanycytes lining the third ventricle (3V) are critical components of this barrier. Herein, we tested the hypothesis that altering the plane of nutrition results in the structural reorganization of tanycytes, tight junctions, and capillary structure within the medial basal hypothalamus. Proopiomelanocortin (POMC) neuronal content within the arcuate nucleus of the hypothalamus (ARC) was also assessed to test whether reduced nutritional status improved access of nutrients to the ARC, while decreasing the access of nutrients of overfed animals. Multiparous, nongestating ewes were stratified by weight and randomly assigned to dietary treatments offered for 75 d: 200% of dietary recommendations (overfed), 100% of dietary recommendations (control), or 60% of dietary recommendations (underfed). The number of POMC-expressing neurons within the ARC was increased (P ≤ 0.002) in underfed ewes. Overfeeding increased (P ≤ 0.01) tanycyte cellular process penetration and density compared with control and underfeeding as assessed using vimentin immunostaining. Immunostaining of tight junctions along the wall of the 3V did not differ (P = 0.32) between treatments. No differences were observed in capillary density (P = 0.21) or classification (P ≥ 0.47) within the ME. These results implicate that changes within the satiety center and morphology of tanycytes within the ARC occur as an adaptation to nutrient availability.

Follicle-stimulating hormone receptors expression in ovine corpora lutea during luteal phase: effect of nutritional plane and follicle-stimulating hormone treatment.

Corpus luteum (CL), a transient endocrine gland critical for reproductive cyclicity and pregnancy maintenance, is controlled by numerous regulatory factors. Although LH is widely recognized as the major regulator, other factors may also affect luteal functions. It has been demonstrated that FSH receptors (FSHR) are expressed not only in ovarian follicles but also in other tissues within the reproductive tract, including the CL. To evaluate FSHR expression in nontreated (nonsuperovulated; experiment 1) or FSH-treated (superovulated; experiment 2) sheep fed a control (C; maintenance), excess (O; 2 × C), or restricted (U; 0.6 × C) diet, CL were collected at the early, mid and/or late luteal phases (n = 5-7 per group). Protein and messenger RNA (mRNA) expression of FSHR were detected in the CL from all groups using immunohistochemistry followed by image analysis and quantitative RT-PCR, respectively. Follicle-stimulating hormone receptor was immunolocalized to steroidogenic small and large and nonsteroidogenic luteal cells. In both experiments, FSHR protein expression was not affected by stage of luteal development or diet. In experiment 1, expression of mRNA for all FSHR variants was greater (P <0.02 to 0.0003) at the late phase than mid or early luteal phase, and in experiment 2, it was greater (P < 0.001) at the mid than early luteal phase. Plane of nutrition did not affect FSHR mRNA expression. Comparison of FSH-treated with nontreated ewes demonstrated that FSH increased FSHR protein expression by 1.5- to 2-fold (P < 0.0001) in all groups, and mRNA expression by 7- to 30-fold (P < 0.001) for (1) FSHR-1 in all groups except U at the early luteal phase, (2) FSHR-2 in C, O, and U at the mid-phase, but not early luteal phase, and (3) FSHR-3 in U at the mid-luteal phase. Our data demonstrate that (1) FSHRs are expressed in ovine CL at several stages of luteal development, (2) FSHR protein expression does not change during the luteal phase and is not affected by diet, (3) FSHR mRNA expression not only depends on the stage of the estrous cycle but also not affected by diet in nonsuperovulated or superovulated ewes, and (4) in vivo FSH treatment enhanced FSHR protein and/or mRNA expression in the CL depending on diet and phase of the estrous cycle. Presence of FSHR in the CL indicates a regulatory role of FSH in luteal function in sheep. As very little is known about the possible role of FSH and FSHR in luteal functions, further studies should be undertaken to elucidate the endocrine, molecular, and cellular mechanisms of FSH effects on the CL.

Corpora lutea in superovulated ewes fed different planes of nutrition.

The corpus luteum (CL) is an ovarian structure which is critical for the maintenance of reproductive cyclicity and pregnancy support. Diet and/or diet components may affect some luteal functions. FSH is widely used to induce multiple follicle development and superovulation. We hypothesized that FSH would affect luteal function in ewes fed different nutritional planes. Therefore, the aim of this study was to determine if FSH-treatment affects (1) ovulation rate; (2) CL weight; (3) cell proliferation; (4) vascularity; (5) expression of endothelial nitric oxide (eNOS) and soluble guanylate cyclase (sGC) proteins; and (6) luteal and serum progesterone (P4) concentration in control (C), overfed (O), and underfed (U) ewes at the early- and mid-luteal phases. In addition, data generated from this study were compared to data obtained from nonsuperovulated sheep and described by Bass et al. Ewes were categorized by weight and randomly assigned into nutrition groups: C (2.14 Mcal/kg; n = 11), O (2xC; n = 12), and U (0.6xC; n = 11). Nutritional treatment was initiated 60 d prior to day 0 of the estrous cycle. Ewes were injected with FSH on day 13-15 of the first estrous cycle, and blood samples and ovaries were collected at early- and mid-luteal phases of the second estrous cycle. The number of CL/ewe was determined, and CL was dissected and weighed. CL was fixed for evaluation of expression of Ki67 (a proliferating cell marker), CD31 (an endothelial cell marker), and eNOS and sGC proteins using immunohistochemistry and image analysis. From day 0 until tissue collection, C maintained, O gained, and U lost body weight. The CL number was greater (P < 0.03) in C and O than U. Weights of CL, cell proliferation, vascularity, and eNOS but not sGC expression were greater (P < 0.001), and serum, but not luteal tissue, P4 concentrations tended to be greater (P = 0.09) at the early- than mid-luteal phase. Comparisons of CL measurements demonstrated greater (P < 0.01) cell proliferation and serum P4 concentration, but less vascularity at the early and mid-luteal phases, and less CL weight at the mid-luteal phase in superovulated than nonsuperovulated ewes; however, concentration of P4 in luteal tissues was similar in both groups. Thus, in superovulated ewes, luteal cell proliferation and vascularity, expression of eNOS, and serum P4 concentration depend on the stage of luteal development, but not diet. Comparison to control ewes demonstrated several differences and some similarities in luteal functions after FSH-induced superovulation.

Gap junctional connexin messenger RNA expression in the ovine uterus and placenta: effects of estradiol-17ß-treatment, early pregnancy stages, and embryo origin.

Gap junctions play a major role in direct, contact-dependent cell-cell communication, and they have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues. Gap junctional channels, composed of connexin (Cx) proteins, have been detected and shown to be influenced by hormones (eg, estrogen and progesterone) in uterine and placental tissues in several species. We hypothesized that (1) the messenger RNA (mRNA) for Cx26, Cx32, Cx37, and Cx43 is expressed in the uterus of ovariectomized sheep treated with estradiol-17ß (E2) and in ovine placenta during early pregnancy, (2) E2-treatment of ovariectomized ewes would cause time-specific changes in Cx26, Cx32, Cx37, and Cx43 mRNA expression (experiment 1), and (3) expression of these 4 Cx would vary across the days of early pregnancy (experiment 2) and will be affected by embryo origin (ie, after application of assisted reproductive technologies [ARTs]; experiment 3). Thus, we collected uterine tissues at 0 to 24 h after E2 treatments (experiment 1), and placental tissues during days 14 to 30 of early pregnancy after natural (NAT) breeding (experiment 2) and on day 22 of early pregnancy established after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA, parthenotes; experiment 3). In experiment 1, the expression of Cx26, Cx37, and Cx43 mRNA increased (P < 0.05) and Cx32 mRNA decreased (P < 0.06) in both caruncular and intercaruncular tissues after E2 treatment. In experiment 2, during early pregnancy, there were significant changes (P < 0.01) across days in the expression of Cx26, Cx37, and Cx43 mRNA in the maternal placenta, accompanied by changes (P < 0.001) in Cx37 and Cx43 mRNA in the fetal placenta. In experiment 3, in maternal placenta, Cx32 mRNA expression was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared to the NAT group; but in fetal placenta, Cx32 mRNA expression was increased (P < 0.05) in NAT-ET, IVF and IVF groups, and Cx26 mRNA expression was increased (P < 0.05) in IVA compared to NAT group. These data suggest that Cx26, Cx32, Cx37, and Cx43 play specific roles in E2-regulated uterine function and in placental development during early gestation both after natural mating and with application of ART.

The impact of diet and arginine supplementation on pancreatic mass, digestive enzyme activity, and insulin-containing cell cluster morphology during the estrous cycle in sheep.

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-µmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

Placental development during early pregnancy in sheep: estrogen and progesterone receptor messenger RNA expression in pregnancies derived from in vivo-produced and in vitro-produced embryos.

Sex steroids are important regulators of angiogenesis and growth in reproductive tissues, including the placenta. In experiment (exp.) 1, to examine the expression of a suite of sex steroid receptors throughout early pregnancy, maternal (caruncular [CAR]) and fetal (fetal membranes [FM]) placental tissues were collected on days 14 to 30 after mating and on day 10 after estrus (nonpregnant controls). In exp. 2, to examine the hypothesis that assisted reproductive technology would affect the expression of the same suite of sex steroid receptors, pregnancies were achieved through natural mating (NAT) or transfer of embryos from natural mating (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA), and CAR and FM were collected on day 22. In exp. 1, for CAR messenger RNA (mRNA) expression of estrogen receptors (ESR) 1 and 2, nuclear (n) progesterone receptors (PGR) and membrane (m) PGRα, ß, and γ were affected (P < 0.02) by pregnancy stage, as were ESR1, nPGR, and mPGRα, ß, and γ for FM (P < 0.03). In exp. 2, for CAR, mRNA expression of ESR1 and nPGR was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared with NAT. For FM, mRNA expression of ESR1 tended to be greater (P = 0.10) in the IVA group compared with NAT and NAT-ET, and GPER1 was greater (P < 0.05) in NAT-ET and IVF compared with NAT. These data establish the normal pattern of sex steroid receptor mRNA expression in maternal and fetal placenta during early pregnancy in sheep, and in addition, suggest that altered expression of placental sex steroid receptors may be an early event leading to poor placental vascularization and growth after assisted reproductive technology.

Dietary selenium and nutritional plane alter specific aspects of maternal endocrine status during pregnancy and lactation.

Objectives were to examine effects of selenium (Se) supply and maternal nutritional plane during gestation on placental size at term and maternal endocrine profiles throughout gestation and early lactation. Ewe lambs (n = 84) were allocated to treatments that included Se supply of adequate Se (ASe; 11.5 µg/kg BW) or high Se (HSe; 77 µg/kg BW) initiated at breeding and nutritional plane of 60% (RES), 100% (CON), or 140% (EXC) of requirements beginning on day 40 of gestation. At parturition, lambs were removed from their dams, and ewes were transitioned to a common diet that met requirements of lactation. Blood samples were taken from a subset of ewes (n = 42) throughout gestation, during parturition, and throughout lactation to determine hormone concentrations. Cotyledon number was reduced (P = 0.03) in RES and EXC ewes compared with CON ewes. Placental delivery time tended (P = 0.08) to be shorter in HSe ewes than in ASe ewes, whereas placental delivery time was longer (P = 0.02) in RES ewes than in CON and EXC ewes. During gestation, maternal progesterone, estradiol-17ß, and GH were increased (P < 0.05) in RES ewes and decreased (P < 0.05) in EXC ewes compared with CON ewes. In contrast, maternal cortisol, IGF-I, prolactin, triiodothyronine, and thyroxine were decreased in RES ewes and increased in EXC ewes compared with CON ewes during gestation. Selenium supply did not alter maternal hormone profiles during gestation. During parturition and lactation, maternal hormone concentrations were influenced by both Se and maternal nutritional plane. During the parturient process, HSe ewes tended to have greater (P = 0.06) concentrations of estradiol-17ß than ASe ewes. Three hours after parturition a surge of GH was observed in ASe-RES ewes that was muted in HSe-RES ewes and not apparent in other ewes. Growth hormone area under the curve during the parturient process was increased (P < 0.05) in ASe-RES vs HSe-RES ewes. Ewes that were overfed during gestation had reduced (P < 0.05) estradiol-17ß but greater IGF-I, triiodothyronine, and thyroxine (P < 0.05) compared with RES ewes. Even though ewes were transitioned to a common diet after parturition, endocrine status continued to be affected into lactation. Moreover, it appears that gestational diet may partially affect lactational performance through altered endocrine status.

Maternal dietary intake alters organ mass and endocrine and metabolic profiles in pregnant ewe lambs.

To determine the impacts of Se supply and maternal dietary intake on ewe organ mass and endocrine and metabolic changes throughout gestation, pregnant first parity ewes (n=77) were allocated to 6 treatments in a 2×3 factorial array. Factors included Se [adequate Se (ASe; 9.5µg/kg BW) vs. high Se (HSe; 81.8µg/kg BW)] initiated at breeding and dietary intake [60% (RES), 100% (CON), or 140% (EXC) of requirements] initiated on d 50 of gestation. Ewes were individually fed and blood samples from the jugular vein were obtained approximately every 14 d from d 50 until parturition. Maternal Se supply did not impact endocrine or metabolic status. There was a nutritional intake by day interaction for NEFA, blood urea nitrogen (BUN), insulin, triiodothyronine (T3), thyroxine (T4), progesterone (P4), and estradiol-17ß (E2). As expected, with increased maternal intake, NEFA concentrations were reduced. During the last weeks of gestation, BUN and insulin were elevated in EXC compared with RES ewes. Although the pattern of T3 and T4 differed throughout gestation within a treatment group, as maternal intake increased, circulating T3 and T4 were increased. For P4 and E2, as maternal dietary intake increased, there was a reduction in the steroid concentrations in jugular blood. There was only a main effect of maternal nutrition (P<0.001) for cortisol concentrations with EXC ewes having greater concentrations than RES and CON ewes, which did not differ. Although Se is known to influence thyroid hormone metabolism, supranutritional levels during pregnancy did not alter circulating T3 and T4 concentrations. Alterations in maternal endocrine status may have influenced placental transport of nutrients to the developing fetus, which we have shown previously is affected by maternal dietary Se and intake. In addition, the alterations in mammary gland weight that we observed may explain the impact of maternal nutrition on mammary gland function and colostrum production, thereby further impairing growth of developing neonates.

Relationships among vasculature, mitotic activity, and endothelial nitric oxide synthase (eNOS) in bovine antral follicles of the first follicular wave.

To determine the relationships among vasculature, mitotic activity, and expression of endothelial nitric oxide synthase (eNOS) of antral follicles in Bos indicus, bovine ovaries were obtained on day 6 of the estrous cycle from 10 crossbred (Brahman to Thai native cows) after a synchronized estrus with prostaglandin F2α analogue. Ovaries were fixed, paraffin-embedded, and used for immunofluorescence detection of factor VIII (a marker of endothelial cells). Immunostaining of eNOS and proliferating cell nuclear antigen (PCNA) were performed with specific monoclonal antibodies. Vasculature and positive staining of eNOS and PCNA were quantitatively evaluated with the image analysis. Follicles were classified by size (small, medium, and large) and by structure as healthy and atretic follicles (n = 82). The expression of factor VIII and eNOS were detected greater in the blood vessels of the theca layers of the healthy follicles than those in atretic follicles. The labeling indices (LIs) in granulosa and theca cells were greater (P < 0.05) in the healthy small and medium follicles than in the healthy large follicles. Vasculature, capillary area density, and capillary number density were positively correlated with eNOS expression and the LIs of granulosa and theca cells but were negatively correlated with the healthy follicle size. During the growing phase of antral follicle in Bos indicus, relationships among vasculature, mitotic activity, and eNOS were observed predominantly in healthy antral follicles. Thus, these data highlight the importance of vasculature, cell proliferation, and eNOS expression of growing and atretic follicles in the first follicular wave.

Impacts of maternal selenium supply and nutritional plane on visceral tissues and intestinal biology in 180-day-old offspring in sheep.

Objectives were to investigate the effects of maternal Se supply and nutritional plane during gestation on offspring visceral tissues and indices of intestinal growth, vascularity, and function at 180 d of age. Rambouillet ewe lambs (n = 82, approximately 240 d of age; 52 ± 0.8 kg BW at breeding) were allocated to a 2 × 3 factorial arrangement of treatments. Treatments included dietary Se [adequate Se (ASe, 9.5 µg/kg BW) or high Se (HSe, 81.8 µg/kg BW)] initiated at breeding and nutritional plane [60% (restricted, RES), 100% (control, CON), and 140% (high, HI) of requirements] initiated at d 50 of gestation. Ewes were fed pelleted diets and housed individually indoors. At parturition, lambs were immediately removed and fed artificial colostrum for the first 20 h followed by ad libitum access to milk replacer. At 180 ± 2 d of age, lambs were euthanized and tissues were harvested. Birth weight was affected by nutritional treatments (P < 0.001), with decreased birth weight in RES and HI compared with CON. Offspring from RES and HI ewes had decreased (P = 0.07) blood volume compared with CON, and those born to HSe ewes had increased (P < 0.04) total visceral adiposity. Within offspring from CON ewes, those from HSe ewes had greater (P < 0.02) intestinal mass compared with ASe ewes. Within offspring from HSe ewes, both RES and HI had reduced (P ≤ 0.05) intestinal mass compared with CON. Jejunal capillary area density was greater (P = 0.08) in offspring from ewes fed HSe compared with ASe. In addition, area per capillary was greater (P ≤ 0.09) in CON compared with RES. Maternal nutritional plane tended (P ≤ 0.11) to alter total small intestinal vascularity, with lambs from CON being greater than RES. Expression of most mRNA for measured angiogenic factors and receptors was not altered (P ≤ 0.13) by maternal treatments; however, expression of glucagon-like peptide-2 (GLP-2) was decreased (P = 0.07) in offspring from RES compared with CON ewes. Offspring from ewes fed HI diets had increased (P = 0.08) jejunal mucosal maltase activity. In conclusion, maternal Se supply and nutritional plane during gestation resulted in measurable changes in offspring visceral tissues and intestinal biology, including perirenal fat, blood volume, intestinal mass, total jejunal crypt cell proliferation, area per capillary in jejunal villi, GLP-2 mRNA expression, and maltase activity at 180 d. Additional work is needed to determine impacts on intestinal function and nutrient uptake.

Maternal nutritional plane and selenium supply during gestation impact visceral organ mass and intestinal growth and vascularity of neonatal lamb offspring.

To investigate effects of nutritional plane and Se supply during gestation on neonatal offspring visceral organ mass and intestinal growth and vascularity, 84 nulliparous Rambouillet ewes (age = 240 ± 17 d, BW = 52.1 ± 6.2 kg) were allocated to a 2 × 3 factorial design. Ewes were fed 1 of 2 Se diets [adequate Se (ASe, 11.5 µg/kg BW) or high Se (HSe, 77.0 µg/kg BW)], initiated at breeding, and 1 of 3 nutritional planes [60% (restricted; RES), 100% (control; CON), or 140% (high; HIH) of NRC requirements], initiated at d 40 of gestation. Ewes were fed individually and remained on treatments through parturition. All lambs were removed from their dams at birth and fed milk replacer. At 20.6 ± 0.9 d of age, lambs were necropsied, visceral organs dissected, and jejunal samples collected. Lambs born to ewes fed CON and HIH had greater (P < 0.05) BW, gastrointestinal tract, stomach complex, and liver masses at necropsy than RES. Large intestinal and pancreatic masses, as well as stomach complex, large intestinal, and liver proportional masses, demonstrated (P ≤ 0.08) a nutritional plane × Se supply interaction. Proportional pancreatic mass was greater (P = 0.03) for lambs born to RES ewes than HIH. Although small intestinal mass was not affected (P ≥ 0.18) by gestational treatments, lambs born to HIH-fed ewes had greater (P ≤ 0.09) jejunal DNA concentration than RES and CON, and greater (P = 0.01) total DNA than RES. Nutritional plane and Se supply interacted to affect (P ≤ 0.003) jejunal percent proliferation and total proliferating small intestinal cells, although jejunal crypt depth and villus length were not affected by gestational treatment (P ≥ 0.17). Jejunal glucagon-like peptide-2 mRNA expression was greater (P ≤ 0.07) in lambs born to ewes fed RES compared with CON and HIH. Jejunal capillary size was affected (P = 0.09) by the interaction of nutritional plane × Se supply. Lambs from CON ewes had greater (P ≤ 0.04) jejunal capillary surface density than RES. Nutritional plane and Se supply interacted to affect (P = 0.07) jejunal soluble guanylate cyclase mRNA expression in a manner opposite of capillary size. In conclusion, neonatal lamb visceral organ mass was affected by gestational nutrition, even when lambs had ad libitum intake and similar management postnatally. Despite similar small intestinal mass at 20 d of age, jejunal growth, vascularity, and gene expression were altered by maternal nutrition during gestation.

Overfeeding and underfeeding have detrimental effects on oocyte quality measured by in vitro fertilization and early embryonic development in sheep.

To determine effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 48) were divided into control, overfed (ad libitum feeding), and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Follicular development was induced by twice-daily injections of FSH on days 13 and 14 of the estrous cycle, and ovaries and blood samples were collected on day 15 of the estrous cycle. During the 8-wk experiment, for control ewes BW and BCS did not change, but for overfed ewes mean (± SEM) BW and BCS increased (11.8 ± 1.1 kg and 2.0 ± 0.1, respectively) and for underfed ewes decreased (14.2 ± 0.9 kg and 0.7 ± 0.1, respectively). The number of follicles was determined; oocytes were collected and subjected to in vitro maturation and fertilization. After IVF, developing embryos were evaluated throughout the 8-d culture period. The proportion of cleaved oocytes after IVF and developing morula and blastocyst were less (P < 0.0001) in overfed and underfed ewes than in control ewes. However, number of visible follicles, total number of oocytes, number of healthy oocytes, and percentage of healthy oocytes were similar for control, overfed, and underfed ewes. Serum insulin concentration was greater (P < 0.05) in overfed ewes than in underfed ewes, estradiol 17-ß (E(2)) concentration was greater (P < 0.05) in underfed ewes than in overfed ewes, but triiodothyronine (T(3)) and thyroxine (T(4)) concentrations were similar in all treatment groups. These data show that inadequate feeding has a negative effect on oocyte quality which results in lower oocyte cleavage after IVF and morula and blastocyst formation; overfeeding increased serum insulin and underfeeding increased serum E(2) but not T(3) or T(4). These data emphasize the importance of diet for reproductive and metabolic functions. Furthermore, the mechanisms through which enhanced or decreased energy in diet affect oocyte quality and serum insulin and E(2) concentrations remain to be elucidated.

Effects of nutritional plane and selenium supply during gestation on visceral organ mass and indices of intestinal growth and vascularity in primiparous ewes at parturition and during early lactation.

Objectives were to investigate effects of nutritional plane and Se supply during gestation on visceral organ mass and intestinal growth and vascularization in ewes at parturition and during early lactation. Primiparous Rambouillet ewes (n = 84) were allocated to 2 × 3 × 2 factorial arrangement of treatments. Factors included dietary Se [adequate Se (ASe, 11.5 µg/kg BW) or high Se (HSe, 77.0 µg/kg BW)], nutritional plane [60% (restricted; RES), 100% (control; CON), or 140% (high; HIH)], and physiological stage at necropsy (parturition or d 20 of lactation). At parturition, lambs were removed and 42 ewes (7 per treatment) were necropsied. Remaining ewes were transitioned to a common diet which met lactation requirements and mechanically milked for 20 d. In the absence of interactions (P > 0.10), main effects are reported. At parturition, stomach complex and liver masses were greatest for HIH, intermediate for CON, and least for RES (P < 0.02). Small intestinal mass was greater (P ≤ 0.002) for HIH than RES and CON, and greater (P < 0.01) for ASe than HSe. During early lactation, RES and CON gastrointestinal masses increased disproportionally to BW (P < 0.05). At parturition, jejunal mucosal density was less (P ≤ 0.01) for RES than CON and HIH, whereas CON had greater (P < 0.003) jejunal mucosal RNA concentration and RNA:DNA than RES and HIH. Although there were no differences (P > 0.17) at parturition, jejunal cell percent proliferation was greatest in RES, intermediate in CON, and least in HIH (P ≤ 0.09) at d 20 lactation. At both stages, RES had less (P = 0.01) jejunal capillary area density than HIH and less (P ≤ 0.03) capillary surface density than CON and HIH. During lactation, jejunal capillary size was greater (P = 0.04) for ewes previously fed HSe compared with ASe. At parturition, ASe-HIH had greater (P < 0.02) jejunal mucosal endothelial nitric oxide synthase 3 mRNA than all other treatments and greater (P = 0.10) vascular endothelial growth factor (VEGF) than all treatments, except ASe-RES. In addition, CON had less (P ≤ 0.08) jejunal VEGF receptor-1 (FLT1) mRNA compared with RES and HIH, and ASe had greater (P = 0.003) FLT1 than HSe at parturition. Ewes fed HIH had greater (P = 0.04) jejunal VEGF receptor-2 mRNA compared with RES. Results indicate that maternal intestinal growth and vascularization are responsive to nutritional plane and dietary Se during gestation and undergo changes postpartum when under similar lactational management.

Decreasing maternal nutrient intake during the final third of pregnancy in previously overnourished adolescent sheep: effects on maternal nutrient partitioning and feto-placental development.

When pregnant adolescent sheep are overnourished during pregnancy normal nutrient partitioning priorities to the gravid uterus are altered, leading to impaired placental development and fetal growth restriction. We hypothesized that decreasing dietary intake in overnourished dams during the final third of gestation may reverse this inappropriate nutrient partitioning in favor of the fetus. Adolescent ewes were offered control (C; n = 12) or high (H; n = 20) dietary intakes to induce normal vs. compromised placental development. Ten ewes receiving the H intake were switched to a low intake at d90 of gestation (HL). Between d90 to 130, HL dams lost weight and adiposity, and metabolic hormones and glucose at d130 were less than H and similar to C. In spite of these maternal changes, at d130 fetal bodyweight was equivalent in HL and H groups and ∼20% less than in C. A greater degree of brain sparing was evident in HL fetuses and glucose and insulin concentrations were more perturbed than in H fetuses. Relative to C, placentome weight was reduced by 46 and 32% in H and HL and the fetal:placentome weight ratio was H > HL > C. Placental vascular morphology was largely unaffected by maternal diet during late gestation but mRNA expression of five angiogenic genes was up-regulated in the fetal cotyledon of HL pregnancies, commensurate with blood vessel remodeling. Nevertheless, overfeeding to promote maternal anabolic growth during adolescent pregnancy impairs feto-placental development that cannot be rescued by reducing maternal intake during the final third of gestation.

Expression of gap junctional connexin proteins in ovine fetal ovaries: effects of maternal diet.

Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been shown that several environmental factors, including maternal diet, may affect fetal organ growth and function. To determine whether maternal diet affects expression of Cx26, Cx32, Cx37, and Cx43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 d before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from the ASe and HSe groups was fed a restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of Cxs followed by image analysis. All four Cxs were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary, and antral follicles; in granulosa and theca layers of secondary and antral follicles; stroma; and blood vessels. Cx32 was in oocytes, granulosa, and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression, Cx26 in granulosa layer of antral follicles was decreased (P < 0.01) by HSe in the M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P < 0.05) by the M diet with HSe. Thus, Cxs may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance of maternal diet in fetal growth and development.

Effects of maternal selenium supply and plane of nutrition during gestation on passive transfer of immunity and health in neonatal lambs.

To investigate the influence of maternal Se supply and plane of nutrition on lamb morbidity, mortality, and passive transfer of IgG, pregnant ewe lambs were used in 2 experiments with 2 × 3 factorial treatment arrangements. Supplementation of Se began at breeding and was either adequate Se (ASe, 9.5 µg/kg of BW) or high Se (HSe, 81.8 µg/kg of BW) in Exp. 1 or ASe (11.5 µg/kg of BW) or HSe (77.0 µg/kg of BW) in Exp. 2. On d 50 or 40 of gestation for Exp. 1 or 2, respectively, ewes were assigned randomly to 1 of 3 nutritional planes: 60% (RES), 100% (control, CON), or 140% (HI) of NRC requirements. This resulted in the following treatments: ASe-RES, ASe-CON, ASe-HI, HSe-RES, HSe-CON, and HSe-HI. Upon parturition, lambs were separated from their dams and serum samples obtained. Lambs were fed artificial colostrum for the first 20 h and then placed on milk replacer and grain pellets until completion of the study (Exp. 1, 57 d; Exp. 2, 21 d). Twenty-four hours after parturition, lamb serum samples were collected for IgG analysis. All lambs were reared similarly and morbidity and mortality assessed. Main effects were considered significant when P ≤ 0.05. In Exp. 1, there was a Se × plane of nutrition interaction (P ≤ 0.01) for lamb morbidity from birth to weaning and for 24-h IgG concentration. Lambs from ASe-RES and HSe-HI ewes were treated more frequently (P < 0.01) for respiratory and gastrointestinal disease, and lambs from HSe-HI ewes had the smallest (P < 0.01) 24-h serum IgG concentration. In Exp. 1, lambs from HI ewes also had the greatest (P < 0.01) mortality rates from birth to weaning compared with lambs from CON and RES ewes. In Exp. 2, there was an effect (P < 0.01) of maternal plane of nutrition with lambs from RES ewes having increased 24-h IgG compared with lambs from CON and HI ewes. There was no effect of maternal Se supplementation on lamb 24-h IgG in Exp. 2; however, there was a Se × plane of nutrition interaction (P < 0.01) for morbidity. From birth to 21 d of age, lambs from ASe-CON ewes had fewer (P < 0.01) treatment days compared with lambs from any of the other treatment groups. There also tended (P = 0.08) to be an effect of maternal Se supplementation on lamb mortality with increased mortality observed in lambs from HSe ewes. Results from the studies show a restricted maternal plane of nutrition can increase lamb serum IgG concentration. Selenium results were not consistent between the 2 experiments and may be due to differences in maternal Se.

Maternal nutrition during pregnancy influences offspring wool production and wool follicle development.

The effects of maternal nutrition on offspring wool production (quality and quantity) were evaluated. Primiparous Rambouillet ewes (n = 84) were randomly allocated to 1 of 6 treatments in a 2 × 3 factorial design. Selenium treatment [adequate Se (ASe, 9.5 µg/kg of BW) vs. high Se (HSe, 81.8 µg/kg of BW)] was initiated at breeding, and maternal nutritional intake [control (CON, 100% of requirements) vs. restricted (60% of CON) vs. overfed (140% of CON)] was initiated at d 50 of gestation. Lamb birth weight was recorded at delivery, and all lambs were placed on the same diet immediately after birth to determine the effects of prenatal nutrition on postnatal wool production and follicle development. At 180 ± 2.2 d of age, lambs were necropsied and pelt weights were recorded. Wool samples were collected from the side and britch areas, whereas skin samples were collected from the side of each lamb only. Although Se status did not influence side staple length in males, female lambs born from ewes on the ASe treatment had a shorter staple length (P < 0.05) when compared with females from ewes on the HSe treatment. Maternal nutritional intake and Se status did not influence (P ≥ 0.23) wool characteristics on the britch. However, at the britch, wool from female lambs had a reduced comfort factor (P = 0.01) and a greater (P = 0.02) fiber diameter compared with wool from male lambs. Maternal Se supplementation, maternal nutritional plane, sex of the offspring, or their interactions had no effect (P > 0.13) on primary (29.10 ± 1.40/100 µm(2)) and secondary (529.84 ± 21.57/100 µm(2)) wool follicle numbers. Lambs from ASe ewes had a greater (P = 0.03) secondary:primary wool follicle ratio compared with lambs from HSe ewes (20.93 vs. 18.01 ± 1.00). Despite similar postnatal diets, wool quality was affected by maternal Se status and the maternal nutritional plane.

Supranutritional selenium increases mammary gland vascularity in postpartum ewe lambs.

Objectives were to determine the effects of maternal dietary supranutritional Se and nutritional plane during gestation on capillary surface density, capillary area density, and angiogenic factor expression in the developing mammary gland of primiparous ewes. Selenium treatments were initiated at breeding [adequate Se (ASe; 9.5 µg/kg of body weight) vs. high Se (HSe; 81.8 µg/kg of body weight)] and nutritional planes at d 50 of gestation [Low, 60%; moderate (Mod), 100%; and High, 140% of requirements). Mammary glands were collected within 24h postpartum. Vascular development was assessed in the glandular portion of the mammary gland. Vascularity was determined for mammary tissue with the following measurements taken: the cross-sectional capillary area density (total capillary area as a proportion of tissue area) and capillary surface density (CSD; total capillary circumference per unit of tissue area). High-Se ewes had greater capillary surface and area densities compared with ASe ewes. A tendency existed for an Se × plane of nutrition interaction for CSD with maternal diet not affecting CSD in HSe ewes, but Low ewes had a decreased CSD compared with Mod ewes, with High being intermediate in ASe ewes. Moreover, HSe-Low and HSe-High ewes had increased CSD compared with ASe-Low and ASe-High, respectively. Although Se status did not influence angiogenic factor mRNA expression, mammary glands from Low ewes tended to have increased VEGF and FLT1 mRNA expression compared with High ewes, with Mod being intermediate. Maternal plane of nutrition did not affect mammary gland glutathione peroxidase activity, but it was increased in HSe compared with ASe ewes. Increased mammary capillary nutrient exchange area may contribute to previously observed changes in colostrum quality.

Nutritional plane and selenium supply during gestation affect yield and nutrient composition of colostrum and milk in primiparous ewes.

The objectives were to investigate effects of nutritional plane and Se supply during gestation on yield and nutrient composition of colostrum and milk in first parity ewes. Rambouillet ewe lambs (n = 84, age = 240 ± 17 d, BW = 52.1 ± 6.2 kg) were allocated to 6 treatments in a 2 × 3 factorial array. Factors included Se [adequate Se (ASe, 11.5 µg/kg of BW) or high Se (HSe, 77.0 µg/kg of BW)] initiated at breeding, and nutritional plane [60 (RES), 100 (CON), or 140% (HIH) of requirements] initiated at d 40 of gestation. Ewes were fed individually from d 40, and lambs were removed at parturition. Colostrum was milked from all ewes at 3 h postpartum, and one-half of the ewes (n = 42) were transitioned to a common diet meeting lactation requirements and mechanically milked for 20 d. Colostrum yield was greater (P = 0.02) for HSe ewes than ASe, whereas CON had greater (P < 0.05) colostrum yield than RES and HIH. Colostrum Se (%) was greater (P < 0.01) for HSe than ASe. Colostrum from ewes fed HSe had less (P = 0.03) butterfat (%), but greater (P ≤ 0.05) total butterfat, solids-not-fat, lactose, protein, milk urea N, and Se than ASe. Colostrum from HIH ewes had greater (P ≤ 0.02) solids-not-fat (%) than RES, whereas RES had greater (P ≤ 0.04) butterfat (%) than CON and HIH. Colostrum from ewes fed the CON diet had greater (P = 0.01) total butterfat than HIH. Total solids-not-fat, lactose, and protein were greater (P < 0.05) in colostrum from CON than RES and HIH. Ewes fed HSe had greater (P < 0.01) milk yield (g/d and mL/d) than ASe, and CON and HIH had greater (P < 0.01) yield than RES. Milk protein (%) was greater (P ≤ 0.01) in RES compared with CON or HIH. Ewes fed HSe had greater (P < 0.01) milk Se (µg/g and mg/d) than ASe on each sampling day. Milk from CON and HIH ewes had greater (P < 0.01) total solids-not-fat, lactose, protein, and milk urea N than RES. Total Se was greater (P = 0.02) in milk from ewes fed the CON diet compared with RES. Somatic cell count and total somatic cells were greater (P ≤ 0.05) in milk from CON than RES. A cubic effect of day (P ≥ 0.01) was observed for milk yield (g and mL). Butterfat, solids-not-fat, lactose, milk urea N, and Se concentration responded quadratically (P ≤ 0.01) to day. Protein (%), total butterfat, and total Se, and somatic cells (cells/mL and cells/d) decreased linearly (P < 0.01) with day. Results indicate that gestational nutrition affects colostrum and milk yield and nutrient content, even when lactational nutrient requirements are met.