Birth weight, growth indices, and seminal parameters in male offspring are resilient features to maternal pre-conceptional dietary manipulation in sheep.

Gestational diet manipulation can lead to inadequate fetal nutrient supply resulting in low birth weight, limited postnatal growth, and consequently, reduced reproductive performance in the progeny. However, effects of short-term maternal pre-conceptional dietary manipulation on postnatal growth and reproductive parameters of male offspring in large animals remains unexplored. To determine these consequences, female crossbred (Polypay x Dorset) sheep were allocated to three groups (n = 33/group) of dietary manipulation for 21 days prior to mating under the following conditions: (1) control at 100 % of maintenance energy requirements (40 Kcal of metabolizable energy/kg body weight [BW]), (2) undernutrition (UN) at 50 % of Control intake, and (3) overnutrition (ON) at 200 % of maintenance energy. Singleton ram lambs (UN:9; C:12; ON:6) were monitored from birth until 8 months of age, including birth weight, weekly weights, weight gain, body mass index (BMI), and circulating testosterone. After weaning, monthly scrotal circumference and subcutaneous fat depth were measured. Semen morphology and motility were evaluated at 7 and 8 months of age. Birth weight, weight gain, and BMI at birth and weaning were not significantly different among nutritional treatments. None of the pre-conceptional diets affected body weight change from weaning until 36 weeks of age, BMI, fat depth, or scrotal circumference across the experiment. A sustained rise in plasma testosterone concentrations was detected when ram lambs were, on average, 82 days old and 37 kg. Both testosterone concentrations and scrotal circumference were positively correlated to body weight regardless of treatment group. In addition, seminal parameters did not differ among treatments, but a transient increase in plasma testosterone at 18 weeks of age was observed in ON ram lambs compared to control rams. In conclusion, birth weight, growth indices, and seminal parameters in singleton rams are resilient features in the progeny upon maternal pre-conceptional dietary manipulation in sheep.

Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model.

Bisphosphonates are commonly used to treat and prevent bone loss, but their effects in active, juvenile populations are unknown. This study examined the effects of intramuscular clodronate disodium (CLO) on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. Forty juvenile sheep (253 ± 6 days of age) were divided into four groups: Control (saline), T0 (0.6 mg/kg CLO on day 0), T84 (0.6 mg/kg CLO on day 84), and T0+84 (0.6 mg/kg CLO on days 0 and 84). Sheep were exercised 4 days/week and underwent physical and lameness examinations every 14 days. Blood samples were collected for SBB every 28 days. Microstructure and BMD were calculated from tuber coxae (TC) biopsies (days 84 and 165) and bone healing was assessed by examining the prior biopsy site. BT and GAG were evaluated postmortem. Data, except lameness data, were analyzed using a mixed-effects model; lameness data were analyzed as ordinal data using a cumulative logistic model. CLO did not have any measurable effects on the skeleton of sheep. SBB showed changes over time (p ≤ 0.03), with increases in bone formation and decreases in some bone resorption markers. TC biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84 (p ≤ 0.04). These changes may be attributed to exercise or growth. The absence of a treatment effect may be explained by the lower CLO dose used in large animals compared to humans. Further research is needed to examine whether low doses of bisphosphonates may be used in active juvenile populations for analgesia without evidence of bone changes.

Utilising male stimulus to improve the reproductive efficiency of 8-month-old nulliparous ewes and adult parous ewes.

We tested whether utilising the male effect to stimulate ewes before the mating period can reduce the time to conception following the introduction of entire rams, and increase fertility, prolificacy, and reproductive rate (number of fetuses per 100 ewes exposed to fertile rams). A retrospective analysis was used to analyse records from 59,716 ewes collected over 34 years (1986-2020) from seven genotypes: Border Leicester, Composite (crossbred), Dorset, Merino, Dorset x Polypay, Rambouillet, White Suffolk. The dataset also included nulliparous young ewes (mated at age 8 months) and adult parous ewes. Vasectomized rams were used to stimulate 20,632 ewes before a mating period that lasted 2 or 3 estrous cycles, and the outcomes were compared with those from 39,084 ewes that had not been stimulated. Independently of genotype, utilising the male stimulus advanced the average conception date by 8 days for young ewes (P < 0.0001) and by 1 day for adult ewes (P < 0.0001). The male stimulus also increased the proportion of ewes that conceived in their first cycle by 33 % for young ewes and by 6 % for adult ewes (P < 0.0001). For the cycle of conception, there were significant (P < 0.0001) effects of two interactions: male stimulus x age at mating and male stimulus x live weight at mating. The male stimulus improved fertility in both adult ewes (99.8 % vs 89 %; P < 0.001) and young ewes (77.7 % vs 81.3 %; P < 0.001). The male stimulus increased the number of young ewes (41.9 % vs 11.1 %; P < 0.001) and adult ewes (16.6 % vs 2.7 %; P < 0.001) that conceived multiple fetuses in the first 17 days of the mating period. The reproductive rate was improved by the male stimulus in young ewes (129 % vs 135 %; P < 0.001) but not in adult ewes (120 % vs 122 %; P = 0.12). When all animals for all breeds were included in the analyses, there were improvements in fertility, prolificacy, and reproductive rate as age and live weight increased at mating (P < 0.0001). We conclude that, independently of genotype, utilising the male stimulus before the mating period reduces the time to conception and improves reproductive performance in both young and adult ewes.

Effects of the central melanocortin system on feed intake, metabolic hormones and insulin action in the sheep.

Voluntary feed intake is insufficient to meet the nutrient demands associated with late pregnancy in prolific ewes and early lactation in high-yielding dairy cows. Under these conditions, peripheral signals such as growth hormone and ceramides trigger adaptations aimed at preserving metabolic well-being. Recent work in rodents has shown that the central nervous system-melanocortin (CNS-MC) system, consisting of alpha-melanocyte-stimulating hormone (α-MSH) and agouti-related peptide (AGRP) acting respectively as agonist and antagonist on central MC receptors, contributes to the regulation of some of the same adaptations. To assess the effects of the CNC-MC on peripheral adaptations in ruminants, ewes were implanted with an intracerebroventricular cannula in the third ventricle and infused over days with artificial cerebrospinal fluid (aCSF), the α-MSH analog melanotan-I (MTI), or AGRP. Infusion of MTI at 0.03 nmol/h reduced intake, expressed as a fold of maintenance energy requirement (M), from 1.8 to 1.1 M (P < 0.0001), whereas AGRP at 0.3 nmol/h increased intake from 1.8 to 2.0 M (P < 0.01); these doses were used in all subsequent experiments. To assess the effect of MTI on plasma variables, sheep were fed ad libitum and infused with aCSF or MTI or pair-fed to MTI-treated sheep and infused with aCSF (aCSFPF). Feed intake of the MTI and aCSFPF groups was 40% lower than the aCSF group (P < 0.0001). MTI increased plasma triiodothyronine and thyroxine in an intake-independent manner (P < 0.05 or less) but was devoid of effects on plasma glucose, insulin, and cortisol. None of these variables were altered by AGRP infusion in sheep fed at a fixed intake of 1.6 M. To assess the effect of CNS-MC activation on insulin action, ewes were infused with aCSF or MTI over the last 3 d of a 14-d period when energy intake was limited to 0.3 M and studied under basal conditions and during hyperinsulinemic-euglycemic clamps. MTI had no effect on plasma glucose, plasma insulin, or glucose entry rate under basal conditions but blunted the ability of insulin to inhibit endogenous glucose production during hyperinsulinemic-euglycemic clamps (P < 0.0001). Finally, MTI tended to reduce plasma leptin in sheep fed at 0.3 M (P < 0.08), and this effect became significant at 0.6 M (P < 0.05); MTI had no effect on plasma adiponectin irrespective of feeding level. These data suggest a role for the CNC-MC in regulating metabolic efficiency and peripheral insulin action.

Highly productive ruminants face short-term nutritional deficits during demanding phases of their life cycle. They remain productive and healthy during these periods through a series of metabolic adaptations. Current models in ruminant biology attribute the coordination of these adaptations to circulating hormones and bioactive metabolites but have not considered the possibility that the central nervous system (CNS) is also involved. The latter appears likely given recent work in rodents implicating the CNS-melanocortin system in the regulation of some of these adaptations. To test this possibility, mature ewes were surgically implanted with a cannula accessing the brain allowing chronic infusion of melanocortins, and used in experiments assessing peripheral effects. These experiments showed that the CNS-melanocortin system regulates the circulating concentrations of some metabolic hormones as well as the ability of insulin to regulate glucose production. Overall, these studies suggest a role for the CNS-melanocortin system in regulating metabolic adaptations in ruminants.

Effect of pre-conceptional nutrition and season on fetal growth during early pregnancy in sheep.

Gestational age in sheep can be closely predicted through ultrasonographic measurement of fetal bones when correlated to standardized fetal growth curves. However, these standardized curves do not account for factors that are known modulators of fetal growth, such as maternal nutrition or health status. Despite being seasonal breeders, and studies reporting an effect of season on birth weight, the influence of season on fetal growth has not been well characterized. In this study, we hypothesized that season of conception will affect fetal growth curves during mid-gestation and that pre-conceptional nutrition would have no effect. We investigated this by provisioning treatments of low, control, and high planes of nutrition during the lactation and flushing pre-conceptional periods to multiparous Dorset x Polypay and Dorset ewes over two seasons (the optimal breeding season [n = 97] and the suboptimal breeding season [n = 104]). Females were mated naturally with mating dates recorded, fetal biparietal diameter measured via ultrasound between gestational days 35-71, and newborn weights recorded at lambing. Pre-conceptional nutritional treatments did not affect fetal biparietal diameter. However, low vs. high nutrition in the pre-conceptional lactation (but not flushing) period resulted in reduced lamb birth weights (P < 0.001). Early fetal growth tended to be faster in the suboptimal breeding season than in the optimal breeding season (P < 0.061) with lambs being heavier at birth in the optimal breeding season (P < 0.001). There was no effect of fetal sex or litter size on fetal biparietal diameter during the first half of pregnancy, however both sex and litter size influenced lamb birth weight (P < 0.001) with males being heavier than females and singletons being heavier than twins and triplets. Mating date within the flushing period had a significant effect on lamb birth weight regardless of season and independent of treatment, with ewes that conceived later in the flushing period having heavier lambs at birth (P = 0.007). These findings suggest that pre-conceptional under- or overnutrition resulting in substantial changes in body condition does not affect fetal growth during the first half of pregnancy. However, the reduction in lamb birth weight indicates that pre-conceptional maternal nutrition during the previous lactation period may affect fetal growth later in pregnancy.

Toxicokinetics of bisphenol A, bisphenol S, and bisphenol F in a pregnancy sheep model.

Bisphenol A (BPA), S (BPS), and F (BPF) are among the most abundant bisphenols detected in humans, yet pregnancy toxicokinetics for BPS or BPF remain unknown. Because gestational BPS can disrupt placental function and result in reproductive and metabolic disorders in the progeny, the aim of the study was to investigate BPS and BPF toxicokinetics during pregnancy using an in vivo approach. Fetal catheterizations were conducted in pregnant sheep (n = 6) at mid-pregnancy and injected with either a single dose of BPS (n = 3, 0.5 mg/kg, s.c.), or a combination of BPS, BPF, and BPA (n = 3, 0.5 mg/kg for each chemical, s.c.). Maternal and fetal blood and urine and amniotic fluid were collected over 72 h and analyzed for bisphenols by HPLC-MS/MS. We observed significant differences in half-life, maximum concentration, and total body clearance in maternal circulation among bisphenols. Longer half-lives were observed in fetal vs. maternal circulation for all bisphenols. Fetal toxicokinetics differed among bisphenols with BPS having the longest fetal half-life. All bisphenols reached basal levels at 48 h in maternal plasma, but were still detectable in amniotic fluid, fetal urine, and fetal plasma at 72 h. In this first pregnancy toxicokinetic study of BPS and BPF we have demonstrated maternal and fetal toxicokinetic differences among all three bisphenols. Higher BPS persistence in the fetal compartment warrants studies into progeny adverse outcomes following gestational exposure. Additionally, toxicokinetic differences among bisphenols call for a more careful approach when extrapolating kinetic information from one bisphenol chemical to another.

Gestational bisphenol S impairs placental endocrine function and the fusogenic trophoblast signaling pathway.

Exposure to bisphenolic chemicals during pregnancy occurs in > 90% of pregnancies. Bisphenolic compounds can cross the placental barrier reaching fetal circulation. However, the effects of emerging bisphenolic compounds, such as bisphenol S (BPS), on placental function remain untested. The aim was to determine if bisphenol A (BPA) or BPS, at an environmentally relevant dose, impairs placental function. Pregnant sheep were randomly distributed into three treatment groups (n = 7-8/group): control, BPA, and BPS. All animals received daily injections of corn oil (control), BPA, or BPS (0.5 mg/kg; s.c.; internal fetal doses were ~ 2.6 ng/mL unconjugated BPA and ~ 7.7 ng/mL of BPS) from gestational day 30-100. After a 20-day washout period, placentas were weighed and placentomes collected. Placental endocrine function was assessed on biweekly maternal blood samples. Gestational exposure to BPS, but not BPA, reduced maternal circulating pregnancy-associated glycoproteins without change in placental weight or placental stereology. BPS-exposed placentas had 50% lower e-cadherin protein expression, ~ 20% fewer binucleate cells, and ~ threefold higher glial cell missing-1 protein expression. BPA placentas were not affected highlighting the intrinsic differences among bisphenolic chemicals. This is the first study to demonstrate that gestational BPS can result in placental endocrine dysfunction and points to a dysregulation in the fusogenic trophoblast signaling pathway.

Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.

Mammals meet the increased nutritional demands of lactation through a combination of increased feed intake and a collection of adaptations known as adaptive metabolism (e.g., glucose sparing via insulin resistance, mobilization of endogenous reserves, and increased metabolic efficiency via reduced thyroid hormones). In the modern dairy cow, adaptive metabolism predominates over increased feed intake at the onset of lactation and develops concurrently with a reduction in plasma leptin. To address the role of leptin in the adaptive metabolism of early lactation, we asked which adaptations could be countered by a constant 96-h intravenous infusion of human leptin (hLeptin) starting on day 8 of lactation. Compared to saline infusion (Control), hLeptin did not alter energy intake or milk energy output but caused a modest increase in body weight loss. hLeptin reduced plasma glucose by 9% and hepatic glycogen content by 73%, and these effects were associated with a 17% increase in glucose disposal during an insulin tolerance test. hLeptin attenuated the accumulation of triglyceride in the liver by 28% in the absence of effects on plasma levels of the anti-lipolytic hormone insulin or plasma levels of free fatty acids, a marker of lipid mobilization from adipose tissue. Finally, hLeptin increased the plasma concentrations of T4 and T3 by nearly 50% without affecting other neurally regulated hormones (i.e., cortisol and luteinizing hormone (LH)). Overall these data implicate the periparturient reduction in plasma leptin as one of the signals promoting conservation of glucose and energy at the onset of lactation in the energy-deficient dairy cow.

Insulin regulates hepatic leptin receptor expression in early lactating dairy cows.

Energy balance controls the expression of the leptin receptor (Lepr) in the ruminant hypothalamus but whether similar regulation occurs in peripheral tissues is unknown. To address this issue, we measured Lepr expression in the liver and adipose tissue of dairy cows during the transition from late pregnancy (LP) to early lactation (EL). This period is characterized by the development of a profound state of energy insufficiency and is associated with reduced plasma insulin and leptin and with increased plasma growth hormone. Hepatic expression of the short (Lepr-a) and long (Lepr-b) isoforms was 40% higher during EL (8 days postpartum) than LP (30 days prepartum). A similar effect was observed when negative energy balance was induced in nonpregnant, late-lactation dairy cows by food restriction, implicating energy insufficiency as a specific cause in EL. The stimulation of hepatic Lepr expression was reversed after a 48-h period of hyperinsulinemic euglycemia in EL. Changes in hepatic Lepr expression during chronic elevation of plasma leptin in EL or plasma growth hormone in nonpregnant, late-lactation cows did not support a role for these hormones in mediating the effects of energy insufficiency on hepatic Lepr expression. In adipose tissue, Lepr expression was increased 10-fold during the transition from LP to EL. Overall, these data indicate that hypoinsulinemia is partly responsible for the induction of Lepr expression in the liver, and perhaps adipose tissue, of energy-deficient dairy cows.

Propionate is not an important regulator of plasma leptin concentration in dairy cattle.

Propionate was recently shown to increase leptin synthesis in rodents. To determine if a similar effect occurs in ruminants, propionate was administered to lactating dairy cows. In experiment 1, 31 cows were given an intrajugular Na propionate bolus (1,040 micromol/kg body weight), increasing plasma propionate from 160 to 5,680 microM and plasma insulin from 6.8 to 77.8 microIU/mL. Plasma leptin concentration decreased from 2.11 ng/mL before bolus to 1.99 ng/mL after dosing (P<0.05) with no differences in leptin concentrations at 20, 50, and 100 min post-bolus (P>0.10). In experiment 2, 12 cows were used in a duplicated 6 x 6 Latin square experiment to assess the dose-response effect of ruminal propionate infusion on plasma leptin concentration. Sodium propionate was infused at rates of 0, 260, 520, 780, 1040, or 1,300 mmol/h, while total short-chain fatty acid infusion rate was held constant at 1,300 mmol/h by addition of Na acetate to the infusate. Coccygeal blood was sampled following 18 h of infusion. Increasing the rate of propionate infusion linearly increased plasma propionate concentration from 180 to 330 microM (P<0.001) and plasma insulin concentration from 6.7 to 9.1 microIU/mL (P<0.05). There was a quadratic response in plasma leptin concentration (P=0.04) with a maximum at 780 mmol/h propionate, but leptin concentrations increased by no more than 8% relative to the 0 mmol/h propionate infusion. Leptin concentrations were correlated with insulin concentrations but not with propionate concentrations in plasma. Propionate is not a physiological regulator of leptin secretion in lactating dairy cows.

Plasma leptin is regulated predominantly by nutrition in preruminant lambs.

In juvenile and mature animals, the plasma concentration of leptin is regulated by adiposity and nutrition. However, the timing of these influences on plasma leptin, and their relative importance in early postnatal life, are unknown. We investigated these plasma leptin influences in sheep, a species characterized during fetal life by leanness and insensitivity of leptin to variation in maternal nutrition. Small and large neonatal lambs were randomly assigned to either a diet sustaining an average daily weight gain (ADG) of 148 g/d (Low plane) or ate ad libitum a diet sustaining an ADG of 337 g/d (High plane). A subset of animals were slaughtered at 7.5, 10, 15 and 20 kg of body weight. Birth size had no effect on plasma leptin concentrations and adiposity at birth or at later times. Plasma leptin concentrations increased within 6 d of birth in the High plane lambs (P < 0.01) and continued to rise over time. In contrast, plasma leptin concentrations never changed in the Low plane lambs despite increasing adiposity. The positive association between plasma leptin concentration and adiposity was greater in the High plane than in the Low plane lambs, suggesting an independent effect of nutrition. Consistent with this finding, lipid accretion rates, a variable that is mostly independent of adiposity, was a strong predictor of plasma leptin concentrations only in the High plane lambs (R(2) = 0.77, P < 0.01). A positive association between plasma insulin and leptin developed over time in the High plane lambs (R(2) = 0.75, P < 0.01 on d 40), but was not seen in the Low plane lambs. These data indicate that both nutrition and adiposity regulate plasma leptin synthesis in early postnatal life, but in contrast to adulthood, the effects of nutrition appear to be predominant.

Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.

After parturition, dairy cows suffer from an intense energy deficit caused by the onset of copious milk secretion and an inadequate increase in voluntary food intake. We previously showed that this energy deficit contributes to a decline in plasma leptin. This decline mirrors that of plasma insulin but is reciprocal to the profile of plasma growth hormone (GH), suggesting that both hormones may regulate plasma leptin in periparturient dairy cows. To study the role of insulin, hyperinsulinemic-euglycemic clamps were performed on six dairy cows in late pregnancy (LP, 31 days prepartum) and early lactation (EL, 7 days postpartum). Infusion of insulin (1 microg.kg body wt-1.h-1) caused a progressive rise in the plasma concentration of leptin that reached maximum levels at 24 h during both physiological states. At steady states, the absolute increase in plasma leptin was greater in LP than in EL cows (2.4 vs. 0.4 ng/ml). Insulin infusion increased leptin mRNA in adipose tissue during LP but not during EL. During lactation, mammary epithelial cells expressed leptin mRNA but insulin did not increase milk leptin output. In contrast, a 3-day period of GH administration had no effect on plasma leptin during LP or EL. Therefore, insulin increases plasma leptin in LP by stimulating adipose tissue synthesis but has only marginal effects in EL, when cows are in negative energy balance. Other factors, such as increased response of adipose tissue to beta-adrenergic signals, probably contribute to the reduction of plasma leptin in early lactating dairy cows.

Spatial and developmental regulation of leptin in fetal sheep.

To better understand the biology of leptin during prenatal life, the developmental and spatial regulation of leptin was studied in ovine fetuses. Fetal plasma leptin increased steadily between days 40 and 143 postcoitus (PC), but it was unrelated to fetal weight or placental weight at day 135 PC. Leptin gene expression was detected in fetal brain and liver during most of gestation and in fetal adipose tissue after day 100 PC. At day 130 PC, expression in fetal perirenal adipose tissue was approximately 10% of maternal expression. In contrast, leptin gene expression was never detected in the placenta and other uteroplacental tissues. When ewes were fed 55% of requirements between days 122 and 135 PC, fetal plasma leptin remained constant despite acute reduction in maternal concentration. We conclude that fetal plasma leptin originates mostly from nonadipose tissue in early pregnancy and, in addition, from fetal adipose tissue near term. The role of fetal plasma leptin remains uncertain given the lack of nutritional regulation and association with fetal growth.

Regulation of placental nutrient transport and implications for fetal growth.

Fetal macronutrient requirements for oxidative metabolism and growth are met by placental transport of glucose, amino acids, and, to a lesser extent that varies with species, fatty acids. It is becoming possible to relate the maternal-fetal transport kinetics of these molecules in vivo to the expression and distribution of specific transporters among placental cell types and subcellular membrane fractions. This is most true for glucose transport, although apparent inconsistencies among data on the roles and relative importance of the predominant placenta glucose transporters, GLUT-1 and GLUT-3, remain to be resolved. The quantity of macronutrients transferred to the fetus from the maternal bloodstream is greatly influenced by placental metabolism, which results in net consumption of large amounts of glucose and, to a lesser extent, amino acids. The pattern of fetal nutrient supply is also altered considerably by placental conversion of glucose to lactate and, in some species, fructose, and extensive transamination of amino acids. Placental capacity for transport of glucose and amino acids increases with fetal demand as gestation advances through expansion of the exchange surface area and increased expression of specific transport molecules. In late pregnancy, transport capacity is closely related to placental size and can be modified by maternal nutrition. Preliminary evidence suggests that placental expression and function of specific transport proteins are influenced by extracellular concentrations of nutrients and endocrine factors, but, in general, the humoral regulation of placental capacity for nutrient transport is poorly understood. Consequences of normal and abnormal development of placental transport functions for fetal growth, especially during late gestation, and, possibly, for fetal programming of postnatal disorders, are discussed.