Suffolk Ewes Research Articles

296 Ergot Alkaloid Consumption Alters Serotonin Receptor-Induced Vasoactivity in Ovine Umbilical Vasculature

Abstract Consumption of ergot alkaloids during the second half of gestation have been shown to decrease umbilical artery vasoactivity resulting in lighter birth weights. The objective was to evaluate vasoactivity of serotonin (5HT) receptors 5HT2A and 5HT1B/1D in umbilical artery and vein from ewes receiving endophyte-infected (E+ 1.77 mg ergovaline/animal/day) or endophyte-free (E-; 0 mg ergovaline/animal/day) tall fescue seed at d-85 (n=4), 110 (n=4 E+; n=4 E-), 135 (n =4 E+; n=4 E-) of gestation. Suffolk ewes began receiving seed treatments on d-86 of gestation. Umbilical artery and vein were collected from ewes at slaughter. Two-mm blood vessel cross sections were mounted in multi-myographs containing Krebs-Henseleit buffer and exposed to increasing concentrations of rizatriptan benzoate (5HT1B/1D agonist) and TCB-2 (5HT2A agonist). Data were analyzed as a split-plot with mixed models of SAS. The 5HT1B/1D agonist did not stimulate a contractile response in either vessel for any gestation time point evaluated. The 5HT2A agonist caused contractile responses in umbilical artery with the greatest occurring at d-85 and decreasing in magnitude as days of gestation increased (P< 0.05). On d-110 and 133 of gestation, umbilical arteries from ewes receiving E- seed had a greater contractile response than those arteries collected from ewes receiving the E+ treatment (P< 0.05). Umbilical veins also responded to increasing concentrations of 5HT2A agonist and maximal response was not different between d-85 and 110 of gestation, but both were greater than d-133. Unlike the artery, umbilical veins from ewes receiving E+ seed had greater contractile response than E- at d-133 (P< 0.05). These results demonstrate that ovine vascular smooth muscle contractions of the umbilical artery and vein are induced by 5HT2A receptor activity and not 5HT1B/1D. Umbilical artery 5HT2A receptor activity responded to seed treatment differently than umbilical vein and could be the source of ergot alkaloid-induced intra-uterine growth restriction.

278 Awardee Talk: Progesterone Regulates the Abundance of Mrnas and Proteins Involved in Polyamine Metabolism in the Sheep Endometrium

Abstract Agmatine and polyamines (putrescine, spermidine, and spermine), metabolites of L-arginine, are important for development of mammalian conceptuses. The objective of this study was to determine if exogenous progesterone administered during the pre-implantation period, which accelerates conceptus development, alters the expression of mRNAs and proteins involved in the metabolism of agmatine and polyamines in the sheep uterus during pregnancy. Mature Suffolk ewes were bred to fertile rams and assigned randomly to be treated with daily i.m. injections of either 1ml corn oil (CO;n=28) or 25mg progesterone in corn oil (P4;n=20) from Day 1.5 through Day 8 after breeding (Day 0). Ewes were hysterectomized on Day 9, 12, or 125 of gestation and endometrial samples were collected. The expression of candidate mRNAs was quantified using qPCR and proteins localized using immunohistochemistry. On Day 9 of pregnancy, the expression of mRNAs for AMD1 (P < 0.01), ASL (P < 0.01), and SLC12A8 (P < 0.05) was greater in endometria of P4-treated compared to CO-treated ewes. The expression for MAT2B mRNA was greater (P < 0.001) for P4-treated compared to CO-treated ewes on both Days 9 and 12. On Day 125 of gestation, endometria from P4-treated ewes tended to have greater expression of SAT1 (P < 0.10) mRNA compared to CO-treated ewes. In contrast, the expression of AMD1(P < 0.01), MAT2B (P < 0.05), OAT (P < 0.05), SMOX (P < 0.05), and SLC12A8 (P < 0.05) mRNAs was lower in endometria from P4-treated compared to CO-treated ewes on Day 125. Exogenous P4 appeared to increase the relative abundance of SAT1 and SMOX proteins in the uterine luminal and glandular epithelia on Day 125 of pregnancy. These results suggest that early administration of progesterone may alter the biosynthesis of agmatine and polyamines in the sheep uterus during the pre-implantation period of pregnancy with potential ‘programming’ effects manifested in late pregnancy.

295 Effects of Prenatal Testosterone Excess on Puberty Attainment of First-Generation ewe Lambs

Abstract Prenatal testosterone excess causes reproductive perturbations in sheep that recapitulate those seen in women with polycystic ovary syndrome (PCOS). Obesity has a significant role in the development and severity of the PCOS phenotype. Our objective was to investigate the effects of testosterone excess from gestational days (GD) 60-90 (unlike the GD 30-90 model, these animals are not virilized) and postnatal body weight (BW) gain on puberty in first-generation ewe lambs. Pregnant Suffolk ewes received testosterone propionate (T; 100 mg) or corn oil (C) i.m. twice weekly from GD 60-90 (term=147d). Ewe lambs were weaned ~3 mo of age. Control and T lambs were assigned to gain either 0.3 kg/d (C and T-maintenance) or 0.4 kg/d (T-overfed). Maintenance diet was designed to promote optimal growth without excess fat deposition. Overfed ration was designed to achieve a BW ~30% above that of maintenance. Progesterone concentrations were measured via radioimmunoassay in blood samples collected twice weekly from ewe lambs (n=15/group) during the first breeding season. Puberty was defined as when progesterone concentrations were < 0.5 ng/mL for four consecutive samples followed by three consecutive samples ≥0.5 ng/mL. Six animals (C, n=1; T-maintenance, n=3; T-overfed, n=2) did not attain puberty during the study and puberty was assigned as last day of sampling. Average daily gain was not different among treatments (C, 0.23±0.02kg; T-maintenance, 0.25±0.01kg; T-overfed, 0.27±0.02kg). Age at puberty did not differ between groups (C, 274.6±9.9d; T-maintenance, 270.0±11.3d; T-overfed, 266.0±10.2d). Similarly, BW at puberty did not differ across treatments (C, 50.44±2.2kg; T-maintenance, 52.5±2.4kg; T-overfed, 57.3±2.5kg). In conclusion, unlike GD 30-90 exposure which advanced puberty, GD 60-90 exposure did not affect age and BW at puberty in ewe lambs. Collectively, these studies suggest that GD 30-60 is a critical developmental window in which prenatal testosterone excess advances puberty in female sheep. Research support: NIH-NICHD (R01HD099096).

Creatine metabolism at the uterine-conceptus interface during early gestation in sheep†.

Ruminant conceptuses that elongate and attach to the uterine luminal epithelium (LE) to establish pregnancy require a large amount of adenosine triphosphate (ATP). The creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system re-generates ATP in dividing and migrating cells such as the conceptus trophectoderm cells. However, little is known about metabolism of Cr within uterine and conceptus tissues in livestock species during early gestation. In this study, Suffolk ewes were ovariohysterectomized on Days 9, 12, 15, 16, 17, 18, 20, or 21 of pregnancy (n = 2-5 animals/per day) to investigate metabolites, mRNAs, and proteins of the Cr-CK-PCr system at uterine-conceptus interface. Amounts of Cr and guanidinoacetate (GA) in uterine flushings increased between Days 12 and 17 of pregnancy. Endometrial expression of mRNAs for GA formation (AGAT), Cr synthesis (GAMT), and Cr/PCr utilization (CKB) was greater on Days 17 and 21 than on Days 9 and 12 of pregnancy. Immunoreactive AGAT was detected in uteri only on Day 21 but not in uteri or conceptuses at earlier days of pregnancy. GAMT, SLC6A8, and CKs were expressed in uterine luminal and glandular epithelia. Immunoreactive CKs (CKB, CKM, and CKMT1) appeared greater on Day 9 than Day 17 of pregnancy. Immunoreactive GAMT and CKs appeared greater in trophectoderm of conceptuses on Day 20 than on Day 15 of pregnancy, whereas the opposite was observed for that of SLC6A8. This study provides insights into cell-, tissue-, and time-specific metabolism of Cr at the uterine-conceptus interface suggesting a role for the Cr-CK-PCr system in ovine conceptus development and implantation.

Production of corn plus sunflower forage and its preference as silaged by sheep in Mineral de la Reforma, Hidalgo

a) Objective. The objective was to evaluate the forage production of maize, maize + sunflower and its preference for sheep as silage. b) Design/methodology/approach. The design in the field was in completely randomized blocks with three replications and in the laboratory in a completely randomized design (Tukey α = 0.05). Sowing was in spring - summer 2020 under rainfed conditions. The treatments were: 100 % corn, 90 % corn + 10 % sunflower, 80 % corn + 20 % sunflower. Forage production was evaluated at 126 days of sowing, it was ensiled and the quality through bromatological analysis. For silage preference, 10 pregnant Hampshire × Suffolk ewes were used with 44.8 kg of live weight along 20 days. c) Results. The 80 % maize + 20% sunflower combination showed higher green forage yield (P<0.001; 28 t ha-1), higher percentage of soluble protein (P<0.01) and higher percentage of lignin (P<0.001; 4.6 %). The sheep preferred the silage with 100 % corn, since it had a lower percentage of non-fibrous carbohydrates (22.2 %), lower acid detergent fiber (35.3 %) and lower neutral detergent fiber (59.4 %). d) Limitations/implications. Sunflower should be established on soils with low numbers of broadleaf plant seeds, because chemical control cannot be applied. e) Findings/conclusions. A greater amount of forage was produced per surface unit whit 80 % corn + 20 % sunflower. The 100 % maize silage was preferred by sheep due to lower percentage of lignin, and higher in vitro digestibility of neutral detergent fiber.

Cervical immune activation during the luteal phase may compromise subsequent trans-cervical ram sperm transport†.

Worldwide, cervical artificial insemination using frozen–thawed semen yields low pregnancy rates. The only exception to this is in Norway, where vaginal insemination with frozen–thawed semen yields pregnancy rates in excess of 60% and which has been attributed to the specific ewe breed used. Our previous work demonstrated differences in cervical gene expression at the follicular phase of the estrous cycle in ewe breeds with known differences in pregnancy rates. In this study, we characterized the cervical transcriptome of the same ewe breeds [Suffolk, Belclare, Fur, and Norwegian White Sheep (NWS)] during the luteal phase, as an optimal environment at the luteal phase could better prepare the cervix for sperm migration through the cervix at the subsequent follicular phase. High-quality RNA extracted from postmortem cervical tissue was analyzed by RNA sequencing. After stringent filtering, 1051, 1924, and 611 differentially expressed genes (DEGs) were detected in the low-fertility Suffolk breed compared with Belclare, Fur, and NWS, respectively. Gene ontology analysis identified increased humoral adaptive immune response pathways in Suffolk. Increased expression of multiple immune genes supports the presence of an active immune response in the cervix of Suffolk ewes, which differentiates them significantly from the other three ewe breeds. Inflammatory pathways were upregulated in the Suffolk, resulting in higher expression of the potent pro-inflammatory cytokines. Therefore, higher levels of pro-inflammatory cytokines indicate unresolved inflammation in the cervix of the low-fertility Suffolk breed that could contribute to reduced cervical sperm transport in the next follicular phase.

Biochemical and molecular characterization of sialylated cervical mucins in sheep

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen–thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.

Effects of exogenous progesterone on the expression of mineral regulatory molecules by ovine endometrium and placentomes†.

This study aimed to determine whether the acceleration of conceptus development induced by the administration of exogenous progesterone (P4) during the preimplantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal-conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil (CO) vehicle or 25mg of progesterone in CO (P4) for the first 8days of pregnancy and hysterectomized on either Day 9 (CO, n = 5; P4, n = 6), 12 (CO, n = 9; P4, n = 4) or 125 (CO, n = 14; P4, n = 10) of gestation. The expression of S100A12 (P < 0.05) and fibroblast growth factor receptor (FGFR2) (P < 0.01) messenger RNAs (mRNAs) was lower in endometria from P4-treated ewes on Day 12. The expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. The expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), solute carrier (SLC)20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, the expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, vitamin D receptor (VDR), S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered the expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal-conceptus interface.

Utilization of Wool Integral Lipids to Determine Milk Fat Content in Suffolk Down Ewes

The identification of higher fat content in ewe milk during lactation can help to improve the nutritional value and quality of the derived dairy products. In this study, we characterized fatty acids from the wool of Suffolk ewes at two time points during lactation and assessed whether they were related to milk fat content through discriminant analysis and, thus, could be potentially used to identify ewes with a high fat content. Eighty single-bearing Suffolk ewes of similar body weight, body score, and age were selected for this study. The overall fat contents of milk and wool were determined, as well as the fatty acids in the wool. The wool fat content was 1.14% on average. The proportions of wool fatty acids were 65.82% saturated, 21.70% monounsaturated, and 12.48% polyunsaturated fatty acids. The wool fatty acid concentrations of C18:1n9c, C18:2n6c, and C22:2 were higher in ewes whose milk had a high fat content at both time points. Moreover, the levels of these fatty acids were positively correlated with milk fat content. Discriminant analyses using C18:1n9c and C18:2n6c were the best candidates for the prediction of high milk fat content, with an accuracy of 87.50%. The wool fatty acids C18:1n9c and C18:2n6c could potentially be used to determine the milk fat content of ewes.

Breed and diet influence the ruminal bacterial community of sheep

Context Ruminal microbial communities are important in production ruminants, as they can affect health and production efficiency. Differences between meat- and wool-producing sheep breeds have not yet been fully explored. Aims In this study, we aimed to determine the impact of diet on ruminal microbial populations of sheep selected for different production traits, namely, meat production (White Suffolk) and wool production, and quality (Poll Merino). Methods The study utilised 24 White Suffolk and 24 Poll Merino ewes, assigned to either a roughage (20% pellet and 80% chaff) or high-grain diet (80% pellet and 20% chaff). Following acclimatisation for 21 days, they each had a ruminal sample taken and analysed for bacterial communities, using 16S rRNA sequencing. Entry and exit weight of the ewes and their feed intake were measured. Key results There was a preference for the high-grain diet (P &lt; 0.0001), with greater amounts consumed, although there was no significant difference in ewe weights between the two dietary treatments. However, White Suffolk ewes lost weight on the roughage diet, whereas all other groups gained weight (White Suffolk roughage −5.9 ± 2.6 kg, White Suffolk high-grain 7.8 ± 1.7 kg, Poll Merino roughage 9.2 ± 2.7 kg, Poll Merino high-grain 5.0 ± 1.8 kg). There were significant ruminal bacterial differences associated with both diet and breed. The average dissimilarity in ruminal bacterial phyla associated with diet was 14.13%, with the top 50% of phyla contributing to the dissimilarity being Verrucomicrobia, Lentisphaerae, Elusimicrobia, SR1 and Fibrobacteres, which were significantly more abundant in the roughage dietary group, and Proteobacteria, which were significantly more abundant in the high-grain dietary group. Conclusions We have demonstrated that although diet strongly influences the ruminal microbiota, there is a significant interaction between diet and breed in effects on ruminal microbiota and also animal performance. Implications The differences in microbial composition between breeds were related to some of the animal productivity differences of the two breeds, indicating that at least some of the genetic differences in animal productivity are generated by differences in the responsiveness of the ruminal microbiota to diet.

PSXII-13 Blood flow parameters in ovine fetuses from different sires

Abstract Placental and fetal development are influenced by paternal genes. Doppler ultrasonography was performed on pregnant ewes to examine the effect of sire on fetal and placental blood flow parameters. Twenty Suffolk ewes were randomly assigned to either a Suffolk ram or a Texel ram. Only ewes carrying twins were used for the study. Ultrasound exams were performed at 60, 90, and 120 (± 3) days post-breeding. Umbilical artery and fetal aorta blood-flow velocities, and aorta vessel diameter were measured. Blood flow velocities were used to calculate the resistance index (RI), which was used for statistical analysis (Glimmix, SAS). Gestational age and weight at birth were recorded. There were 5 Suffolk x Suffolk (SxS) and 6 Suffolk x Texel (SxT) ewes carrying twins to term. For umbilical artery RI, there was no significant (P &amp;lt; 0.05) effect of breed; there was significant effect of time (0.89 ± 0.03a; 0.63 ± 0.03b; 0.59 ± 0.03b LSM ± SE for days 60, 90, and 120 respectively); and there was no interaction. For aorta RI, there was no significant effect of breed; there was significant effect of time (0.73 ± 0.03a; 0.67 ± 0.03ab; 0.63 ± 0.03b for days 60, 90, and 120 respectively); and there was no interaction. For aorta diameter, the effect of breed approached significance (P = 0.054), SxS had a larger average diameter (0.50 ± 0.02 cm) than SxT (0.45 ± 0.01 cm); there was significant effect of time (0.24 ± 0.03a; 0.46 ± 0.02b; 0.72 ± 0.02c cm for days 60; 90; and 120 respectively); and there was no interaction. There were no significant differences between sires for weight or gestational age at birth. The results correspond with the changes in fetal and placental vascular resistance over time; however, further research is needed to expand our knowledge on the effect of sire.

PSVIII-23 Mycotoxin exposure on fetal skeletal muscle fiber hypertrophy and miRNA transcriptome

Abstract The objective of this study was to evaluate the miRNA transcriptome and muscle fiber characteristics of lambs from ewes consuming endophyte-infected tall fescue (E+) seed during two stages of gestation at two animal ages. Pregnant Suffolk ewes (yr 1 n = 36; yr 2 n = 60) were randomly assigned to one of two gestational treatments: E+ seed (1.77 mg/hd/d of ergovaline/ergovalinine) during mid- (gd 35 - 85; MID) or late-gestation (gd 86 – 133/parturition; LATE). Longissimus muscle samples (n = 3/E+ treatment/time) were collected from fetuses on gd 133 (FETAL; expt. 1) or from wethers after finishing to market weight (MKT; expt. 2) from ewes from MID and LATE E+ treatments. Data were analyzed as a 2 x 2 factorial with E+ treatment (MID or LATE), time (FETAL or MKT), and the two-way interaction in the model. Exposure to E+ fescue seed during LATE gestation reduced (P = 0.03) cross-sectional area of Type II muscle fibers at MKT but not at FETAL. Cross-sectional area of Type II muscle fibers were larger (P &amp;lt; 0.05) at MKT than FETAL. Animal age influenced miRNA expression with 120 miRNA differentially expressed. miRNA-22-3p and -29a were down regulated (P &amp;lt; 0.001; &amp;gt; -5 log fold change) with animal age; whereas miR-3958-3p, -410-3p, -299-5p and -487b-3p (P &amp;lt; 0.0001; &amp;gt; 3 log fold change) were up-regulated. Exposure to E+ during MID or LATE gestation did not alter miRNA expression. Muscle fiber hypertrophy increased from FETAL to MKT age altered the expression of miRNA in longissimus muscle.

Serum biochemical reference ranges for pregnant sheep

Late pregnancy is accompanied by dramatic changes in the metabolism of sheep, in which the nutrient requirements increase and the metabolic capacity of ewes is under severe stress due to the rapid foetal growth and development of the mammary gland. The blood metabolic profile can be used to monitor these alterations, which can lead to metabolic disorders such as pregnancy toxaemia. However, data available on serum parameters in sheep do not consider physiological state. Therefore, the present study aimed to determine the biochemical reference ranges for pregnant ewes, including serum energy, protein and enzyme-related metabolites. Data from a variety of metabolites were obtained from experiments performed in several institutions and commercial farms using Santa Inês, Dorper, Lacaune, Morada Nova, Bergamacia and Suffolk ewes reared under different conditions (grazing, feedlot, semi-feedlot, collective and/or individual pens, and metabolic cages) from 2006 to 2017. All animals were healthy and without feed restriction. Data from ewes with any clinical manifestations were removed. The metabolic energy profile included data of glucose, cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein and very-low-density lipoprotein levels; the metabolic protein profile included the metabolites total protein, uric acid, urea, albumin, and creatinine; and the metabolic enzymatic profile included the enzymes aspartate aminotransferase, gamma-glutamyl transferase and alkaline phosphatase. The reference ranges were estimated using the software RefVal 4.11. Dixon’s test was used to identify and remove outliers. The confidence intervals and percentiles were estimated using the nonparametric method of bootstrap when data were not normally distributed. A 95% confidence level was used. The serum biochemical reference ranges for pregnant sheep determined in our study were strongly divergent from those established by one of the most cited books on the topic, especially considering the high serum urea and cholesterol concentrations and low levels of blood glucose observed. Therefore, it is essential to consider physiological status when evaluating the blood metabolic profile of pregnant ewes in order to maintain an adequate nutritional management and to prevent health disorders that may lead to productive and reproductive losses.

Decrease in dysbaric osteonecrosis severity as a result of 45-minute oxygen pre-breathe

Sudden decompression can result in bubble formation as the result of nitrogen gas (N2) dissolved in tissue during disabled submarine escape (DISSUB). This may cause dysbaric osteonecrosis (DON), a condition in long bones where bubbles in fatty marrow result in ischemia and necrosis. Previous research has shown that oxygen (O2) pre-breathe of two hours resulted in a reduction of DON; however, effects of shorter O2 pre-breathe remain uncertain. This study’s aim was to understand the effect of shorter lengths of O2 pre-breathe. Eight adult Suffolk ewes (89.5± 11.5 kg) were exposed to 33 feet of seawater (fsw) for 24 hours. They were placed randomly into four groups and exposed to either 45, 30 or 15 minutes of O2 (91-88%) pre-breathe; the controls received none. They were then rapidly decompressed. Alizarin complexone was later injected intravenously to visualize the extent of DON in the right and left long bones (radii, tibiae, femur and humeri). The 30- and 15-minute pre-breathe groups saw the greatest deposition. There was significant decrease of variance in the 45-minute group when compared with all other treatments, suggesting that 45 minutes of O2 pre-breathe is required to effectively increase confidence in the reduction of DON. Similar confidence was not reflected in the 30-minute and 15-minute groups: 45 minutes of pre-breathe was the minimum amount needed to effectively prevent against DON in DISSUB escape at 33 fsw. However, future research is needed to determine how to calculate effective dosages of O2 pre-breathe to prevent DON in any given scenario.

Pre-implantation exogenous progesterone and pregnancy in sheep. II. Effects on fetal-placental development and nutrient transporters in late pregnancy

BackgroundAdministration of progesterone (P4) to ewes during the first 9 to 12 days of pregnancy accelerates blastocyst development by day 12 of pregnancy, likely due to P4-induced up-regulation of key genes in uterine epithelia responsible for secretion and transport of components of histotroph into the uterine lumen. This study determined if acceleration of blastocyst development induced by exogenous P4 during the pre-implantation period affects fetal-placental development on day 125 of pregnancy. Suffolk ewes (n = 35) were mated to fertile rams and assigned randomly to receive daily intramuscular injections of either corn oil vehicle (CO, n = 18) or 25 mg progesterone in CO (P4, n = 17) for the first 8 days of pregnancy. All ewes were hysterectomized on day 125 of pregnancy and: 1) fetal and placental weights and measurements were recorded; 2) endometrial and placental tissues were analyzed for the expression of candidate mRNAs involved in nutrient transport and arginine metabolism; and 3) maternal plasma, fetal plasma, allantoic fluid, and amniotic fluid were analyzed for amino acids, agmatine, polyamines, glucose, and fructose.ResultsTreatment of ewes with exogenous P4 did not alter fetal or placental growth, but increased amounts of aspartate and arginine in allantoic fluid and amniotic fluid, respectively. Ewes that received exogenous P4 had greater expression of mRNAs for SLC7A1, SLC7A2, SLC2A1, AGMAT, and ODC1 in endometria, as well as SLC1A4, SLC2A5, SLC2A8 and ODC1 in placentomes. In addition, AZIN2 protein was immunolocalized to uterine luminal and glandular epithelia in P4-treated ewes, whereas AZIN2 localized only to uterine luminal epithelia in CO-treated ewes.ConclusionsThis study revealed that exogenous P4 administered in early pregnancy influenced expression of selected genes for nutrient transporters and the expression of a protein involved in polyamine synthesis on day 125 of pregnancy, suggesting a ‘programming’ effect of P4 on gene expression that affected the composition of nutrients in fetal-placental fluids.

Novel mineral regulatory pathways in ovine pregnancy: II. Calcium-binding proteins, calcium transporters, and vitamin D signaling.

Mineralization of the fetal mammalian skeleton requires a hypercalcemic gradient across the placenta from mother to fetus. However, the mechanisms responsible for maintaining the placental transport of calcium remain poorly understood. This study aimed to identify calcium and vitamin D regulatory pathway components in ovine endometria and placentae across gestation. Suffolk ewes were bred with fertile rams upon detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n=3-14/Day), ewes were euthanized and hysterectomized. Calcium abundance was influenced by gestational day in uterine flushings and allantoic fluid (P<0.05). The expression of S100G, S100A9, S100A12, ATP2B3, ATP2B4, TRPV5, TRPV6, CYP11A1, CYP2R1, CYP24, and VDR mRNAs known to be involved in calcium binding, calcium transport, and vitamin D metabolism were quantified by qPCR. Mediators of calcium and vitamin D signaling were expressed by Day 17 conceptus tissue, and endometria and placentae across gestation. Gestational day influenced the expression of S100G, S100A9, S100A12, TRPV6, VDR, and CYP24 mRNAs in endometria and placentae (P<0.05). Gestational day influenced endometrial expression of ATP2B3, and placental expression of TRPV5, ATP2B4, and CYP11A1 (P<0.05). VDR protein localized to the endoderm and trophectoderm (Day 17 conceptus) and was expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile suggests a potential role of calcium and vitamin D in the establishment of pregnancy and regulation of fetal and placental growth, providing a platform for further mechanistic investigation.

Novel mineral regulatory pathways in ovine pregnancy: I. phosphate, klotho signaling, and sodium-dependent phosphate transporters.

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n=3-14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P<0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1-4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P<0.05). Gestational day influenced endometrial expression of FGF7 (P<0.001), and placental expression of FGF23 (P<0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.

Transplacental transmission of Neospora caninum to lambs in successive pregnancies of naturally infected sheep in Southern Brazil.

This study aims to report the detection of N. caninum DNA in a newborn lamb (1) with neurological signs and congenital neosporosis and in a stillborn lamb (2), both born from the same ewe in a herd of Southern Brazil. The lambs were born during different pregnancies of a Suffolk ewe seropositive to N. caninum and seronegative to T. gondii. Histopathological lesions were observed only in the central nervous system of the lambs. The newborn lamb (1) showed mild and focal gliosis in the frontal lobe. In the hippocampal region of the stillborn lamb (2), lymphoplasmacytic perivascular cuffs and N. caninum cysts were observed in the cytoplasm of neurons and confirmed by IHC. PCR was performed using brain samples to detect the protozoa N. caninum and Toxoplasma gondii. The infection with N. caninum was confirmed in the newborn lamb (1) by PCR and in the stillborn lamb (2) by histopathology, immunohistochemistry, and PCR tests.

Feeding Tall Fescue Seed Reduces Ewe Milk Production, Lamb Birth Weight and Pre-Weaning Growth Rate.

Simple SummaryThis study was conducted to examine how ergovaline/ergovalinine ingestion during pregnancy in ewes with different DRD2 genotypes altered ewe and lamb performance. Feeding ergovaline/ergovalinine in last trimester reduced lamb birth weight; however, milk production was lower for all ewes fed ergovaline/ergovalinine. Lambs born to dams fed ergovaline/ergovalinine had slower growth rates and lower weaning weights. These results demonstrate that ingestion of ergot alkaloids during pregnancy can negatively influence lactation, growth of offspring and birth and weaning weights. Recommendations would include strategies to mitigate mycotoxin exposure in pregnant ewes to improve production.Endophyte-infected tall fescue (E+) produces ergovaline and ergovalinine, which are mycotoxins that act as dopamine agonists to suppress prolactin and induce vasoconstriction. The experiment was designed as a 3 × 2 × 2 factorial with DRD2 genotype (AA, AG, GG), fescue seed (endophyte-free, E− or endophyte-infected, E+), stage of gestation (MID, d (day) 35–85; LATE, d 86–parturition) and all interactions in the model. Pregnant Suffolk ewes (n = 60) were stratified by genotype and fed E+ or E− seed in a total mixed ration according to treatment assignment. Serum prolactin concentrations were lower (p < 0.05) in ewes fed E+ seed but did not differ by maternal DRD2 genotype or two-way interaction. Lamb birth weight was lower (p < 0.05) in ewes fed E+ seed in last trimester. Pre-weaning growth rate, milk production and total weaning weight was reduced (p < 0.05) in ewes fed E+ fescue seed during MID and LATE gestation. Ingestion of ergovaline/ergovalinine in last trimester reduces lamb birth weight; however, lamb growth rate, milk production and total weaning weight are reduced in all ewes fed E+ during mid and last trimester.

Ewe breed differences in cervical anatomy and cervicovaginal mucus properties: An international study

In sheep, cervical artificial insemination (AI) involves depositing semen at the cervical opening, as it is not possible to traverse the cervix due to its complex anatomy. However, internationally this method yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway, in which vaginal deposition of frozen-thawed semen to a natural estrus yields pregnancy rates around 70%. As the cervix and its secretions are the principal factors influencing sperm transport to the site of fertilization the aim of this study was to characterise the differences in the cervical anatomy as well as the cervicovaginal mucus properties of six European ewe breeds across three countries known to have differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk and Belclare in Ireland, Fur and Norwegian White Sheep (NWS) in Norway and Ile de France and Romanov in France (n = 28–30 ewes/breed). Cervicovaginal mucus was collected at the follicular and luteal phases of both a synchronized and natural cycle and assessed for mucus weight, viscosity and colour. The anatomical characteristics of the cervix (length of the cervix, number of cervical rings and the appearance of the external os) were assessed post-mortem. There was a type of the cycle by ewe breed interaction represented by no differences in mucus production between ewe breeds at the natural cycle for both the follicular and luteal phases of the cycle. However, there were differences between ewe breeds at the synchronized cycle (P < 0.05). Belclare had the lowest mucus production at the follicular phase while NWS had the lowest amount of mucus at the luteal phase of the synchronized cycle. Overall, across all ewe breeds, mucus production was higher at the follicular than at the luteal phase (P < 0.05). Despite reports of Suffolk and NWS having the most divergent pregnancy rates following cervical AI with frozen-thawed semen, both breeds had the lowest overall mucus viscosity at the follicular phase of both types of cycle with no differences between both ewe breeds (P > 0.05). The length of the cervix, number of cervical rings and the external os type were affected by ewe breed (P < 0.05). Suffolk ewes had longer cervices but lower number of cervical rings than NWS and Fur ewes (both with higher pregnancy rates). In conclusion, while mucus production and mucus viscosity was affected by breed, these changes are not consistent with the known differences between ewe breeds in their pregnancy rates following cervical AI with frozen-thawed semen.