Restricted Ewes Research Articles

PSI-21 The Role of N-Carbamylglutamate Supplementation During Last Third of Gestation in Nutrient Restricted Twin-Bearing Ewes

Abstract Nutrient restriction in sheep during pregnancy normally occurs in Patagonia with negative effects on ewe body condition (BCS) and reproductive performance. N-carbamylglutamate (NCG), an analogue of N-acetylglutamate, is postulated to improve urea recycling, nitrogen utilization and antioxidant capacity in ruminants. The objective of this study was to evaluate the effect of oral NCG supplementation in naturally nutrient restricted grazing twin-bearing ewes, on maternal body weight (BW) and body condition score (BCS), serum biochemical indexes and antioxidant capacity in mid-late gestation. Ten twin-bearing ewes were orally dosed once-daily with 60 mg/kg BW NCG in a water carrier from 100 to 140 days of gestation (dg) and were compared with unsupplemented controls (CON; n = 8). Maternal BW, and BCS were recorded every 10 days, from 100 to 140 dg. Blood samples were collected at the same time points to evaluate plasma metabolites (NEFA, urea, total protein, albumin, globulin, Ca, P, Mg, Cholesterol, ASAT, CK, GGT, GLDH, total bilirubin and triglycerides), antioxidant status [malondialdehyde (MDA)], and total antioxidant capacity (TAC). Ewes were managed as a single mob in a paddock with natural pasture (3.3% CP, 1.9 Mcal/kg ME), supplemented with alfalfa hay (600 g· ewe-1· /day-1; 13% CP and ME 2.2 Mcal/kg ME) from 130 dg, with a stocking rate of 0.9 ewes per hectare and a dry matter availability of 525 kg per hectare, representative of Patagonian prairie conditions. The effect of treatment on ewe BW, BCS and plasma metabolites were analyzed using REML analysis in R using lme4 R package with time (dg) and treatment (and their interaction) as fixed effects and animal as the random effect. Ewe BW increased (P < 0.0001) and BCS decreased (P = 0.005) with advancing gestation, but there was no effect of treatment. There was a tendency for a treatment effect (P = 0.07) on plasma urea with ~10% less concentrations in NCG than CON ewes at 130 dg (P = 0.08) and 140 dg (P = 0.04). There was a treatment by time interaction for total bilirubin (P = 0.005), phosphorus (P = 0.02), and a similar trend for albumin (P = 0.09). Ewes supplemented with NCG had~10% lower concentrations of bilirubin at 100 (P = 0.01) and 120 dg (P = 0.04), less concentrations of phosphorus (P = 0.003) and albumin (P = 0.02) at 140 dg compared with CON ewes. No other effects of treatment on blood metabolites or antioxidant status or capacity were observed. Collectively these results suggest that supplementation of nutritionally restricted ewes with 60 mg/kg BW from 100 to 140 dg did not influence BW or LWT but may have some effects on liver function, protein metabolism, urea recycling, feed intake and/or feed efficiency which warrant further investigation.*Funded by FONDECYT Project N°1221042; AgResearch Strategic Science Investment Fund.

Bisphenol S Alters the Steroidome in the Preovulatory Follicle, Oviduct Fluid and Plasma in Ewes With Contrasted Metabolic Status.

Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.

Effects of restricted nutrition and flushing on reproductive performance and metabolic profiles in sheep

The aim of this study was to assess the impact of dietary restriction during the lactation period on flushing performance and metabolic profiles in sheep. The treatments were performed in a 2 × 2 factorial arrangement with (i) adequate and restricted feeding treatment during lactation and (ii) flushing and control feeding treatment during mating season. Eighty-four Karacabey Merino ewes were divided into two groups for adequate energy (100%) and restricted (70%) at energy requirements for three months of lactation. Each group was also divided into two sub-groups based on flushing diet (AL-F, RL-F; 150%) or control diet (AL-C, RL-C; 100%) during the mating season. Live weight (LW), body condition score (BCS), serum metabolites and hormone concentrations, ovulation rate, estrus performance and lambing performance of ewes were observed. Data from this study showed that average difference (P < 0.001) of LW and BCS between groups were 5.45 kg and 0.54 respectively at the end of lactation. No significant effects of nutritional treatments (P > 0.05) were observed; the average fecundity, litter size and ovulation rates of ewes were 1.30±0.07, 1.44±0.06 and 1.53±0.07 respectively. The onset of estrus was earlier (P = 0.021) in RL-F ewes (7.0 d) than for AL-F, AL-C and RL-C ewes (11.0, 12.2 and 10.9 d respectively). Mean PRG concentration of ewes was lower (P = 0.011) in RL-C group than ewes in AL-C group (1.859±0.266 and 3.143±0.272 ng/ml respectively) at the ram introduction day. Serum biochemical variables blood urea nitrogen (BUN), glucose (GLUC), triglycerides (TGL) and very low density lipoprotein cholesterol (VLDL-CHOL) concentrations- decreased (P < 0.05), while non-esterified fatty acid (NEFA), cholesterol (CHOL), high density lipoprotein cholesterol (HDL-CHOL) and low density lipoprotein cholesterol (LDL-CHOL) concentrations increased (P < 0.05) in the restricted ewes compared with adequate ewes in the different months. In conclusion, in addition to BCS, blood total protein, BUN, GLUC, NEFA and CHOL can be recommended for testing flock metabolic profiles as specific blood metabolites. Flushing did not cause significant differences in reproductive parameters including the ovulation rate. However, the time required for onset of estrus was found to be significantly shorter after the flushing treatment in restricted group of animals.

The Role of Feed Restriction on DNA Methylation, Feed Efficiency, Metabolome, Biochemical Profile, and Progesterone Patterns in the Female Filial Generation (F1) Obtained From Early Feed Restricted Ewes (F0).

Deficient management of replacement animals in the farm during early developmental windows may promote adverse programming effects on reproductive traits and subsequent transmission to the next generation. In this sense, DNA methylation profiles allow researchers to decode epigenetic regulation mechanisms in mammals and identify novel candidate genes correlated with phenotype differences in both dams and offspring. Therefore, improving knowledge in the field of epigenetics and intergenerational effects caused by prenatal and postnatal early nutritional events (e.g., feed restriction) is crucial for refining strategies dedicated to animal breeding. In this study, we determined differences in the global blood methylation patterns, biochemical profile, and metabolome of ewe lambs (F1) born from either early feed restricted dams (F0-RES) or fed ad libitum (F0-ADL). Our data show that functional categories such as those related to cellular processes, phosphorylation, nervous system, immunity response, or reproductive function were enriched significantly in the F1-RES lambs due to differences in the methylation of genes in these categories. These F1-RES lambs did not show differences in feed efficiency during the replacement period but presented higher levels of insulin and triglycerides and reduced concentration of progesterone, whereas the metabolome profile demonstrated variations in the bile acid composition when compared with the F1-ADL lambs. Taken together, all these results suggest that intergenerational effects caused by early feed restriction of dams (F0) may persist in the F1 female lambs with negative consequences on genes involved in cellular processes and reproductive traits.

Chronic low BPS exposure through diet impairs in vitro embryo production parameters according to metabolic status in the ewe

Bisphenol A (BPA), an endocrine disruptor, has been replaced by structural analogues including bisphenol S (BPS). BPA and BPS exhibited similar effects regarding reproductive functions. Moreover, metabolic status and lipid metabolism are related to female fertility and could worsen BPS effects. The objective was to determine BPS in vivo effects on folliculogenesis and embryo production after chronic exposure through diet, and the influence of metabolic status in adult ewes. Sixty primiparous 2.5 year-old ewes, undergoing a restricted or well fed diet, were exposed to BPS (0, 4 or 50 µg/kg/day) for at least three months. After hormonal oestrus synchronisation and ovarian stimulation, ewes were subjected to ovum pick-up (OPU) procedures to collect immature oocytes, that underwent in vitro maturation, fertilisation and embryo production. Body weight, body condition score and plasma glucose were higher in well-fed compared to restricted ewes, while plasma NEFA was lower during the 4–5 months after the beginning of the diets. Plasma progesterone levels increased on day 5 before OPU session in well-fed compared to restricted ewes. No effect of BPS dose was observed on follicle population, plasma AMH levels and embryo production numbers and rates. However, a significant diet x BPS dose interaction was reported for cleaved embryos, > 4-cell embryos, blastocyst and early blastocyst numbers, and plasma triiodothyronine levels. Our study showed that a contrasted diet did not affect follicle population nor embryo production in adult ewes but could affect the quality and progesterone secretion of the corpus luteum. Chronic low BPS exposure had no effect on follicular population and oocyte competence. Nevertheless, the significant diet x dose interactions observed on embryo production suggest that BPS effect is modulated by metabolic status. Further studies are required to assess the risk of BPS exposure for public reproductive health.

Impact of supplementing feed restricted ewes’ diet with propylene glycol, monensin sodium and rumen-protected choline chloride during late pregnancy on blood biochemical indices, body condition score and body weight

The purpose of this study was to investigate the effects of supplementing feed restricted diet with propylene glycol, monensin sodium and rumen-protected choline chloride on blood metabolic status, body condition score (BCS) and body weight of ewes in late pregnancy. At the final six weeks of pregnancy, 48 pregnant Ghezel ewes (body weight 65.53 ± 6.90 kg) were randomly allocated into six groups (n = 8) as follows: control (CG, ad libitum); feed restriction (FR, 60 % of the dry matter intake offered to CG group); feed restriction + propylene glycol (67 g/d) (FR-PG); feed restriction + propylene glycol + monensin sodium (30 mg/d) (FR-MS); feed restriction + propylene glycol + rumen-protected choline chloride (6 g/d) (FR-RPC); feed restriction + propylene glycol + monensin sodium + rumen-protected choline chloride (FR-PMC). Blood samples were collected on days 90, 117, 124, 135 and 140 of pregnancy, at lambing and days 7 and 17 after lambing for analysis of glucose, triglyceride, total cholesterol, total protein, albumin, urea, non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and insulin using the automated biochemical analyzer and ELISA microplate reader. Body weight and BCS of ewes were recorded on days 90, 110, 130 and 140 of pregnancy, at lambing and also day 7 after lambing. All statistical analyses were carried out in a completely randomized design using the MIXED procedure of SAS v.9.3. Results of analysis revealed that pre-parturient administration of rumen-protected choline chloride combined with propylene glycol and monensin sodium elevated both pre- and postpartum concentrations of insulin (P < 0.01), glucose (P < 0.001), triglyceride (P < 0.05), total cholesterol (P < 0.05), total protein (P < 0.01) and albumin (P < 0.01) compared with other treatments. The weight and BCS loss of these ewes were also lower during the pre- and postpartum periods (P < 0.05). In this study, except for day 124 of pregnancy, when the liver enzyme levels of the choline chloride supplemented ewes were numerically low, nutritional treatments did not affect the concentrations of these enzymes at other sampling points (P> 0.05). Regarding BHBA, NEFA and urea, FR and FR-MS ewes had a higher level of these parameters than other groups (P < 0.05). It was concluded that although Ghezel ewes showed some ability in tolerating late gestational feed restriction mainly through lipomobilization, supplementing the restricted diet with rumen-protected choline chloride combined with propylene glycol and monensin sodium during the final trimester of pregnancy could result in beneficial effects on metabolic status and finally good health and condition of animals.

Ghrelin antagonist overrides the mRNA expression of NPY in hypothalamus in feed restricted ewes.

A negative energy balance (NEB) is detrimental to reproduction in animals. A suggested link between NEB and reproductive failure is the gastrointestinal hormone ghrelin, because of the association between ghrelin and the hypothalamo-pituitary-gonadal axis. The [D-Lys3]-Growth Hormone Releasing Peptide-6 ([D-Lys3]-GHRP-6) is a ghrelin antagonist that acts on ghrelin receptors (GHS-R1). The objective of this study was to evaluate the effect of [D-Lys3]-GHRP-6 on reproduction variables in feed restricted ewes. Two experiments were conducted. Experiment I was conducted for 30 days; and Experiment II for 13 days. In both experiments the ewes (n = 18) were randomly assigned to: Control (CO): fed to meet maintenance requirements; Feed restriction (FR): 80% of maintenance restriction; or Ghrelin antagonist (GA): feed restricted and daily subcutaneous of 7.5μg/kg of [D-Lys3]-GHRP-6. Plasma was collected to measure hormones and metabolite concentration. In Experiment II, the hypothalamus and ovaries were collected on day 13. In both Experiments, sheep allocated to the FR and GA treatments decreased their body weight compared with sheep in the CO group (P < 0.06); progesterone however, did not differ between treatments (P > 0.10). Experiment I: Plasma ghrelin concentration was greater (P < 0.01) in FR and GA compared with CO ewes. Plasma non-esterified fatty acids concentration was greater (P < 0.01) in GA and FR than CO. Experiment II: Kisspeptin1-Receptor (Kiss1-R) mRNA expression was greater in FR (P < 0.01) and tended to be greater in GA (P = 0.10) compared with CO ewes. The neuro peptide-Y (NPY) mRNA expression was greater (P = 0.03) in FR than CO; and tended to be greater (P = 0.06) compared with GA ewes. Growth hormone releasing hormone (GhRH) mRNA expression was greater in GA (P = 0.04) and tended to be greater in FR (P = 0.07) compared with CO ewes. Feed restriction increased GhRH, NPY, and Kiss-R mRNA expression in hypothalamus without affecting reproductive variables.Ghrelin antagonist may prevent an increase inNPY expression in ewes.

Identification of Pathways Associated with Placental Adaptation to Maternal Nutrient Restriction in Sheep.

Maternal nutrient restriction impairs placental growth and development, but available evidence suggests that adaptive mechanisms exist, in a subset of nutrient restricted (NR) ewes, that support normal fetal growth and do not result in intrauterine growth restriction (IUGR). This study utilized Affymetrix GeneChip Bovine and Ovine Genome 1.0 ST Arrays to identify novel placental genes associated with differential fetal growth rates within NR ewes. Singleton pregnancies were generated by embryo transfer and, beginning on Day 35 of pregnancy, ewes received either a 100% National Research Council (NRC) (control-fed group; n = 7) or 50% NRC (NR group; n = 24) diet until necropsy on Day 125. Fetuses from NR ewes were separated into NR non-IUGR (n = 6) and NR IUGR (n = 6) groups based on Day 125 fetal weight for microarray analysis. Of the 103 differentially expressed genes identified, 15 were upregulated and 88 were downregulated in NR non-IUGR compared to IUGR placentomes. Bioinformatics analysis revealed that upregulated gene clusters in NR non-IUGR placentomes associated with cell membranes, receptors, and signaling. Downregulated gene clusters associated with immune response, nutrient transport, and metabolism. Results illustrate that placentomal gene expression in late gestation is indicative of an altered placental immune response, which is associated with enhanced fetal growth, in a subpopulation of NR ewes.

Early compensatory Placentomal differentiation in nutritional restricted ewes at 70 days of gestation

Early compensatory Placentomal differentiation in nutritional restricted ewes at 70 days of gestation

Effects of nutrient restriction and arginine treatment on oxidative stress in the ovarian tissue of ewes during the luteal phase

The aim of this study was to determine whether nutrient restriction and arginine treatment affect energy metabolism changes and oxidative stress through the mitochondrial pathway in the ovarian tissue of ewes during the luteal phase. On days 6–15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ± 1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9), and l-arginine group (AG; n = 9) administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. In this study, the expression levels of antioxidase enzymes (SOD2, CAT and GPX1) and mitochondrial biogenesis-related genes (ESRRA and TFAM), as well as antioxidase activity and mitochondrial function were examined in ovarian tissue. Nutrient restriction resulted in activation of ESRRA and TFAM and an increase in relative mtDNA copy number, whereas arginine treatment led to a pronounced recovery of ovarian tissue. In addition, we observed increased AMPK phosphorylation at Thr172 and SIRT3 levels in nutrient restricted ewes, and these effects decreased with arginine treatment. In conclusion, the present results indicated that short-term nutritional restriction led to changes in energy metabolism and oxidative stress. These changes disrupted the redox balance, thus leading to apoptosis through the mitochondria-dependent apoptosis pathway. Arginine treatment altered gene expression in ovarian tissue and increased the resistance to oxidative stress and the anti-apoptosis capacity. The results presented here suggest a potential method to increase agricultural productivity and economic benefits in the sheep industry by using dietary supplementation with arginine to decrease temporary undernutrition of ewes.

Growth, meat and feed efficiency traits of lambs born to ewes submitted to energy restriction during mid-gestation

The objective of this study was to evaluate the effects of the energy restriction of gestation of adult ewes from day 45 to day 115 on lamb live performance parameters, carcass and meat traits. In experiment I, dietary energy was restricted at 70% of the metabolizable energy (ME) requirements, after which ewes were re-fed ad libitum until lambing. In experiment II, dietary energy was restricted at 60% of the ME requirements, and ewes were re-fed to ME requirements until lambing. All ewes grazed together from the end of the restriction periods to weaning. Lambs were weaned and lot fed until slaughter. Feed intake, weight gain and feed efficiency were recorded, and body fat thickness and ribeye area (REA) were measured in the longissimus thoracis muscle. After slaughter, carcass weight and yield, fat depth, carcass and leg length, and frenched rack and leg weights and yields were determined. Muscle fiber type composition, Warner-Bratzler shear force, pH and color were determined in the longissimus lumborum muscle. In experiment I, energy restriction followed by ad libitum feeding affected lamb birth weight (P<0.05); however, no effects (P>0.05) were observed on later BW, REA, BF or carcass traits. Lambs born to non-restricted-fed ewes had higher (P<0.05) weight and yield of the frenched rack cut and their meat tended (P=0.11) to be tender compared with that of lambs from restricted ewes. The percentage of oxidative muscle fibers was lower for lambs born to non-restricted ewes (P<0.05); however, no effects of ewe treatment were observed on other muscle fiber types. For experiment II, energy restriction followed by ME requirements feeding, affected (P<0.01) pre-weaning live weight gain, weaning and final weights. Lambs from restricted ewes had higher (P<0.05) feed intake as % of leg weight and a trend to be less efficient (P=0.16) than lambs from unrestricted dams. Ribeye area and BF were not influenced by treatment. Treatment significantly affected slaughter weight, but had no effects on carcass yield and traits or on meat traits. The results obtained in both experiments indicate submitting ewes to energy restriction during gestation affects the performance of their progeny but the final outcome would depend on the ewe’s re-feeding level during late gestation and the capacity of the offspring to compensate the in utero restriction after birth.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Alterations in uteroplacental hemodynamics during melatonin supplementation in sheep and cattle.

Compromised placental function can result in fetal growth restriction which is associated with greater risk of neonatal morbidity and mortality. Large increases in transplacental nutrient and waste exchange, which support the exponential increase in fetal growth during the last half of gestation, are dependent primarily on the rapid growth and vascularization of the uteroplacenta. The amplitude of melatonin secretion has been associated with improved oxidative status and altered cardiovascular function in several mammalian species; however, melatonin mediated alterations of uteroplacental capacity in sheep and cattle are lacking. Therefore, our laboratories are examining uteroplacental blood flow and fetal development during maternal melatonin supplementation. Using a mid- to late-gestation ovine model of intrauterine growth restriction, we examined uteroplacental blood flow and fetal growth during supplementation with 5 mg/d of dietary melatonin. Maternal nutrient restriction decreased uterine arterial blood flow, while melatonin supplementation increased umbilical arterial blood flow compared with non-supplemented controls. Although melatonin treatment did not rescue fetal weight in nutrient restricted ewes; we observed disproportionate fetal size and fetal organ development. Elevated fetal concentrations of melatonin may result in altered blood flow distribution during important time points of development. These melatonin specific responses on umbilical arterial hemodynamics and fetal development may be partially mediated through vascular melatonin receptors. Recently, we examined the effects of supplementing Holstein heifers with 20 mg/d of dietary melatonin during the last third of gestation. Uterine arterial blood flow was increased by 25% and total serum antioxidant capacity was increased by 43% in melatonin supplemented heifers vs. non-supplemented controls. In addition, peripheral concentrations of progesterone were decreased in melatonin supplemented heifers vs. non-supplemented controls. Using an in vitro model, melatonin treatment increased the activity of cytochrome P450 2C, a progesterone inactivating enzyme, which was blocked by treatment with the melatonin receptor antagonist, luzindole. Elucidating the consequences of specific hormonal supplements on the continual plasticity of placental function will allow us to determine important endogenous mediators of offspring growth and development.

Placental restriction in multi-fetal pregnancies and between-twin differences in size at birth alter neonatal feeding behaviour in the sheep.

Most individuals whose growth was restricted before birth undergo accelerated or catch-up neonatal growth. This is an independent risk factor for later metabolic disease, but the underlying mechanisms are poorly understood. This study aimed to test the hypothesis that natural and experimentally induced in utero growth restriction increase neonatal appetite and milk intake. Control (CON) and placentally restricted (PR) ewes carrying multiple fetuses delivered naturally at term. Outcomes were compared between CON (n=14) and PR (n=12) progeny and within twin lamb pairs. Lamb milk intake and feeding behaviour and ewe milk composition were determined using a modified weigh-suckle-weigh procedure on days 15 and 23. PR lambs tended to have lower birth weights than CON (-15%, P=0.052). Neonatal growth rates were similar in CON and PR, whilst heavier twins grew faster in absolute but not fractional terms than their co-twins. At day 23, milk protein content was higher in PR than CON ewes (P=0.038). At day 15, PR lambs had fewer suckling bouts than CON lambs and in females light twins had more suckling attempts than their heavier co-twins. Birth weight differences between twins positively predicted differences in milk intakes. Lactational constraint and natural prenatal growth restriction in twins may explain the similar milk intakes in CON and PR. Within twin comparisons support the hypothesis that prenatal constraint increases lamb appetite, although this did not increase milk intake. We suggest that future mechanistic studies of catch-up growth be performed in singletons and be powered to assess effects in each sex.

Fetal and organ development at gestational days 45, 90, 135 and at birth of lambs exposed to under- or over-nutrition during gestation,.

To determine the effects of poor maternal nutrition on offspring body and organ growth during gestation, pregnant Western White-faced ewes (n = 82) were randomly assigned into a 3 × 4 factorial treatment structure at d 30.2 ± 0.2 of gestation (n = 5 to 7 ewes per treatment). Ewes were individually fed 100% (control), 60% (restricted) or 140% (over) of NRC requirements for TDN. Ewes were euthanized at d 45, 90 or 135 of gestation or underwent parturition (birth) and tissues were collected from the offspring (n = 10 to 15 offspring per treatment). Offspring from control, restricted and overfed ewes are referred to as CON, RES and OVER, respectively. Ewe data were analyzed as a completely randomized design and offspring data were analyzed as a split-plot design using PROC MIXED. Ewe BW did not differ at d 30 (P ≥ 0.43), however restricted ewes weighed less than overfed and overfed were heavier than controls at d 45, and restricted weighed less and overfed were heavier than controls at d 90 and 135 and birth (P ≤ 0.05). Ewe BCS was similar at d 30, 45 and 90 (P ≤ 0.07), however restricted ewes scored lower than control at d 135 and birth (P ≤ 0.05) and over ewes scored higher than control at d 135 (P ≤ 0.05) but not at birth (P = 0.06). A maternal diet by day of gestation interaction indicated that at birth the body weight (BW) of RES offspring was less than CON and OVER (P ≤ 0.04) and heart girth of RES was smaller than CON and OVER (P ≤ 0.004). There was no interaction of maternal diet and day of gestation on crown-rump, fetal, or nose occipital length, or orbit or umbilical diam. (P ≥ 0.31). A main effect of maternal diet indicated that the RES crown-rump length was shorter than CON and OVER (P ≤ 0.05). An interaction was observed for liver, kidney and renal fat (P ≤ 0.02). At d 45 the liver of RES offspring was larger than CON and OVER (P ≤ 0.002), but no differences observed at d 90, 135 or birth (P ≥ 0.07). At d 45, the kidneys of OVER offspring were larger than CON and RES (P ≤ 0.04), but no differences observed at d 90, 135 or birth (P ≥ 0.60). At d 135, OVER had more perirenal fat than CON and RES (P ≤ 0.03), and at birth RES had more perirenal fat than CON and OVER (P ≤ 0.04). There was no interaction observed for offspring heart weight, length or width, kidney length, adrenal gland weight, loin eye area or rib width (P ≥ 0.09). In conclusion, poor maternal nutrition differentially alters offspring body size and organ growth depending on the stage of gestation.

Effects of vitamin E supply during late gestation and early lactation upon colostrum composition, milk production and quality in nutritional restricted ewes

The aim of this study was to test if supplemental vitamin E during late gestation and early lactation to ewes facing a moderate nutritional restriction and suckling lambs from different litter size and sex would affect colostrum composition and milk yield and composition. Mature Rambouillet ewes (n=37, 22°N) receiving 70% of energy and 80% of protein requirements were randomly assigned to either of two treatments: intramuscular injections of vitamin E (VitE; n=20, 4 IU vitamin Ekg−1 of live weight) in weekly intervals from 50 days before partition until 60 days of lactation, and control (CON; n=17, without VitE treatment). While colostrum protein and fat concentrations did not differ between treatments (P>0.05), lactose content favoured to the VitE-ewes (1.9% vs. 1.2%, P<0.001). Colostrum protein concentration was affected by litter size favouring to twin lambs (14.3% vs. 17.3%, P<0.05). In addition, litter size tended to affect lactose content, favouring to those ewes suckling twins (1.4% vs. 1.8%, P=0.08). The average milk yield was 2039gday−1, without differences (P>0.05) between treatments and litter size. The average milk concentrations of protein, lactose and solids-non-fat did not differ (P>0.05) between treatments. Yet, milk fat content favour to the VitE-ewes (5.7% vs. 5.2%, P<0.05). Milk concentrations of protein, fat, lactose and solids-non-fat were not affected (P>0.05) by litter size. Non-significant treatment×litter size interactions (P>0.05) were observed for milk yield, milk compositions of protein, fat and lactose and solids-non-fat. When the nutritional requirements are not met, treatment of vitamin E to ewes during late gestation and early lactation might be an strategy to improve the quality of both colostrum (>lactose) and milk (>fat).

Changes in the metabolic profile of pregnant ewes to an acute feed restriction in late gestation

AIM: To detect early changes in the metabolic profile of pregnant ewes subject to acute feed restriction at 130 days of gestation, and to establish indicators of risk for ovine pregnancy toxaemia (OPT) for diagnostic purposes.METHODS: Twenty Corriedale ewes with known mating dates, carrying a single fetus, were used. Ewes were maintained on meadow grasslands and at 130 days of gestation were randomly divided in two groups of 10 ewes. The control group had ad libitum access to pasture. Ewes in the restricted group were subjected to an acute feed restriction for a maximum of 144 hours (6 days), with free access to water. From the start (0 hours) until the end of feed restriction, blood samples were collected from all ewes to monitor concentrations of cortisol, non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BOHB) daily, and glucose in plasma every 6 hours; urinary pH was also measured. Every 6 hours the food restricted ewes were observed to detect clinical signs of OPT e.g. apathy, grinding teeth, empty chewing movements, head leaning against the wall, tachypnea and not drinking water.RESULTS: In food-restricted ewes, concentrations of glucose decreased and differed from control ewes from 54 to 90 hours (p<0.001), and 96 to 102 hours (p<0.05). Concentrations of BOHB, cortisol and NEFA increased following feed restriction and differed from control ewes after 48 to 144 hours (p<0.01). Eight of the 10 restricted ewes showed clinical signs of OPT after 102–132 hours. Mean concentrations of glucose, BOHB and cortisol differed between control and restricted ewes prior to the onset of clinical signs of OPT, after 48–96 hours of feed restriction (p<0.01). Mean gestational length, and time from birth to placental expulsion was not affected by the feed restriction.CONCLUSIONS: Our results suggest that concentrations of glucose, BOHB and cortisol in plasma may provide a precocious diagnosis of subclinical OPT, using values of 1.59 (SD 0.24) mmol/L, 2.26 (SD 1.03) mmol/L and 15.09 (SD 7.75) nmol/L, respectively. The identification of a potentially harmful metabolic imbalance could lead to the improvement of treatment success.

Heat stress, divergent nutrition level, and late pregnancy in hair sheep: effects upon cotyledon development and litter weight at birth.

The effect of two divergent nutritional levels during late pregnancy upon some physiological variables and the number (NC) and diameter (DC) of placental cotyledons along with litter weight at birth (LWB) on heat-stressed (42-45°C) hair ewes was evaluated. Multiparous Katahdin x Pelibuey ewes (n = 24) at the onset of the 3/3 of pregnancy were randomly assigned to two treatments (n = 12): (1) non-nutritionally restricted (NNR) ewes, with free access to wheat straw plus 500g/day of concentrate, and (2) nutritionally restricted (NR) ewes, receiving only wheat straw ad libitum. On days 100, 115, 130, and 145 of gestation, the body weight (BW), body condition score (BCS), rectal temperature (RT), respiration rate (RR) were registered in the afternoon (15:00) while the temperature-humidity index (THI) was calculated. At lambing, NC, DC, and LWB were also registered. Analyses considered a completely random design (CRD)-ANOVA with repeated measures across time, considering to litter size (LS) as covariable to reduce any possible influence of LS upon the response variables along experimental diets. BW and BCS were higher in NNR ewes at days 115, 130, and 145. Despite RT similarities (P < 0.05) between treatments, RR was greater (P < 0.01) in the NNR ewes, particularly towards the end of the experimental period. The observed THI averages confirmed severe heat stress conditions on ewes all day round across the experimental period, yet, NC, DC, and LWB favored (P < 0.05) to the NNR ewes. Despite that NNR ewes faced a significant heat stress based on the observed THI values, they consumed a diet with an increased energy-protein density, suggesting that the increased RR in the NNR group was exerted as a compensatory thermoregulation mechanism. Nutritional supplementation in hair ewes besides to an increase the energy body reserves (BW and BCS) also improved both the number and size of cotyledons, while generated an increased litter weight at birth.

Effects of nutrient restriction and melatonin supplementation on maternal and foetal hepatic and small intestinal energy utilization.

To determine how nutrient restriction and melatonin supplementation influence ewe and foetal hepatic and small intestinal energy use, 32 primiparous ewes on d 50 of gestation were fed 60% (RES) or 100% (ADQ) of NRC recommendations with 0 (CON) or 5 mg/d (MEL) of dietary melatonin. On d 130 of gestation, small intestine and liver were weighed and collected. Data were analysed as a completely randomized design with a 2 × 2 factorial arrangement of treatments. Liver weight (g/kg EBW) decreased (p = 0.02) in RES ewes. Jejunum weight (g/kg BW) increased (interaction p = 0.04) in ADQ-MEL ewes compared with all other treatments. Total in vitro O2 consumption (mol/min/tissue) and total citrate synthase activity (mol/min/tissue and mol/min/kg EBW) in liver decreased (p ≤ 0.03) in RES ewes. Oxygen consumption (mol/min/kg EBW) increased (interaction p = 0.02) in jejunum of ADQ-CON versus RES-MEL and ADQ-CON. Citrate synthase activity (mol/min/kg of EBW) increased (interaction p = 0.03) in jejunum of ADQ-MEL compared with RES-MEL and ADQ-CON. Foetal liver weight (g/kg BW) decreased (p = 0.02) in RES versus ADQ. Foetal small intestine weight (g/kg BW) decreased (interaction p = 0.05) in RES-MEL versus ADQ-MEL. Total O2 consumption (mol/min/tissue) and total citrate synthase activity (mol/min/kg of BW) in foetal liver decreased (p ≤ 0.05) in RES versus ADQ. Foetal small intestinal O2 consumption (mol/min/kg of BW) was greater (interaction p = 0.03) in RES-CON and ADQ-MEL than RES-MEL and ADQ-CON. Maternal nutrient restriction had a greater effect than melatonin supplementation on liver and jejunum mass and energy utilization in dams and foetuses. Because intestinal mass and energy utilization were more responsive to melatonin supplementation in ewes fed adequate nutrition compared with restricted ewes, melatonin may have limited use as a therapeutic supplement to help overcome potential negative effects of nutrient restriction.

Thermoregulation of nutrient-restricted hair ewes subjected to heat stress during late pregnancy

In summers with severe hot environmental temperatures, thermoregulation is expected to be critical during the last third of gestation of hair ewes, and this effect can be overstated by suboptimal feeding conditions. Twenty-four multiparous hair sheep ewes with approximately 100d of pregnancy were randomly assigned to two treatments to evaluate the effects of nutritional restriction (NR) during late gestation on some serum metabolites, physiological variables, and thyroid hormone concentrations under intense heat load (maximum temperature 42–45°C). Treatments were as follows: (1) control, ewes fed free choice wheat straw plus a concentrate at a rate of 500g/d, and (2) nutritionally-restricted (NR) ewes, which were offered wheat straw only ad libitum. Body weight of NR ewes dropped 5.1kg (P<0.05), while control ewes gained 4.7kg during the study period. Rectal temperature was not affected (P>0.05) by feeding regime either in the morning or afternoon hours. Respiration rate was greater (P<0.05) in control ewes (20 and 40breaths/min higher in the day and night, respectively) on d 130 and 145 of pregnancy compared to NR ewes. Skin temperatures during the morning and afternoon were affected (P<0.05) by feeding regime at d 130 and 145 of pregnancy. In the morning, NR ewes presented greater (P<0.05) head and rump temperature at day 145 of gestation, and lower (P<0.05) udder temperatures at d 130 and 145 than control ewes. In the afternoon, skin temperatures of NR ewes were higher (P<0.05) in head and right flank on d 130 and 145 of pregnancy, and in udder at day 145 compared to control ewes. Serum glucose was higher (P<0.05) in NR ewes than control animals at day 145 of pregnancy. Serum cholesterol, triglyceride and thyroid hormones were not affected by nutritional restriction. Overall, it was found that nutritionally restricted ewes were less affected by intense heat loads than well-fed ewes, apparently due to the lower metabolic heat produced by this underfed animals. Also, it was apparent that the lower respiration rate of NR ewes was compensated by a greater body surface temperature.

Influence of Maternal Nutrient Restriction during the Period of Embryonic and Maximal Placental Growth on Organ Development in the Adult Sheep

Objective: It is apparent that maternal under nutrition at specific period during pregnancy has differential effects on placental and fetal development, such that the resulting offspring is programmed to be at risk of disease in later life. The study investigated the influence of restricted maternal nutrition during the period of embryonic and maximal placental growth (i.e. 0 &ndash; 95 days gestation; term &asymp;147 days) on the developmental and programming of mitochondrial voltage dependent anion channel (VDAC) and cytochrome c in the lung of the resulting ovine adult. Materials and Methods: Singleton bearing ewes were nutrient restricted between 0 and 95 days of pregnancy and lung weight, the abundance of VDAC and cytochrome c were determined in the resulting adult at 3 years of age, using immunoblotting. Nutrient restricted ewes were fed 50% of their metabolisable energy (ME) requirements (4.0 MJ/day), and diet consisted of 250g hay/day and 210g/day of dried grass pellets (2.2 MJ/day). All diets contained adequate amount of vitamins and minerals. Results: Maternal nutrient restriction had no effect on lung weight, protein and total mitochondrial protein; but caused a significant reduction (P&lt;0.05) in VDAC and cytochrome c abundance. Kidney weight was not affected but cytochrome c abundance was significantly (P&lt;0.05) down regulated. Liver weight was significantly (P&lt;0.05) reduced by nutrient restriction, but no effect on VDAC and cytochrome c abundance. Conclusion: Although maternal nutrient restriction over the period of embryonic and maximal placental growth has no effect on lung weight, it reduces the production of mitochondrial VDAC and cytochrome c at 3 years of age and this may affect the adaptation of the adult sheep.Key words: Maternal, nutrient restriction, pregnancy, placenta, organ, development, Sheep