Control Ewes Research Articles

Late-gestation maternal undernutrition induces circulatory redistribution while preserving uteroplacental function independent of fetal glycaemic state.

Programming effects of maternal undernutrition on fetal metabolic and cardiovascular systems are well elucidated, yet a detailed characterization of maternal haemodynamics is not available. This study used comprehensive cardiovascular magnetic resonance (CMR) imaging to quantify maternal haemodynamics after 29days (111-140 days) of late-gestation undernutrition (LGUN) in pregnant sheep. Control ewes received 100% of metabolizable energy requirements (MERs, n=15), whereas LGUN ewes were globally nutrient restricted to 50% MER (n=18), with a subset of fetuses undergoing continuous glucose infusion (LGUN+G, n=6/18). Ewes underwent CMR (138-140 days' gestation), and placental tissue was collected the next day. Ewes in both LGUN groups had reduced body weight and mean blood glucose concentration across gestation. Ventricular dimensions were lower in both LGUN groups. Uterine artery blood flow (QUtA) was elevated in the LGUN group compared with controls, whereas peripheral blood flow was reduced and further diminished in LGUN+G. Maternal weight change correlated with all haemodynamic parameters across all groups. Uteroplacental oxygen and glucose delivery were increased in LGUN compared to control ewes, whereas uteroplacental oxygen consumption was preserved. LGUN did not impact placental or fetal weight, and markers of brain-sparing physiology were absent. Placental expression of insulin-like growth factors (IGF-1 and IGF-2) and their receptors, glucose, fatty acid (FA) or amino acid transporters and markers of angiogenesis was not impacted. FA transporter expression was positively correlated with QUtA, and FA binding protein correlated negatively with maternal weight change. Maternal cardiovascular adaptations in response to LGUN manifest as preservation of placental growth and function, thereby preserving fetal growth. KEY POINTS: Maternal undernutrition during pregnancy alters fetal metabolic and cardiovascular physiology, but little is known about alterations in maternal haemodynamics. Late-gestation undernutrition (LGUN) and LGUN+G redirected maternal blood flow from the periphery to the uteroplacental unit, concomitantly increasing the delivery of glucose and oxygen to the uteroplacental unit. Substrate transporter expression and uteroplacental oxygen consumption were preserved in LGUN and LGUN+G, suggesting prioritization of the placenta. This study is the first to report detailed maternal haemodynamics in the setting of maternal undernutrition, where placental growth and function were maintained, ultimately preserving fetal oxygen metabolism and growth.

Plasma progesterone profiles in ewes using different injectable progesterone formulations

Context The use of progesterone vaginal implants in sheep is frequently associated with vaginitis and adherences; and can hamper fertility in estrus synchronization programs. Aims The aim of this study was to characterize the plasma hormone profile induced by two injectable progesterone (iP4) formulations in ewes. Methods In Experiment 1, ewes received 20 or 40 mg of an iP4, with a third group as a control. In Experiment 2, 75 or 150 mg of a long-acting iP4 were administered, again with a control group. Key results In Experiment 1, progesterone concentrations remained at luteal levels for less than 24 h. The intervals from iP4 treatment to the onset of estrus and ovulation were greater in treated than in control ewes (P < 0.01). The proportion of ewes that ovulated within 8 days after treatment was lower in ewes that received 40 mg iP4 than in control ewes (P < 0.05). In Experiment 2, mean progesterone concentrations remained at luteal levels for 120 h. The interval from iP4 administration to estrus was longer in 150 mg treated ewes than in control ewes (P < 0.01). The interval from treatment to ovulation was longer in ewes treated with 150 mg than 75 mg and control ewes (P < 0.05). The proportion of ewes that ovulated was lower at 150 mg (P < 0.05). Conclusions The iP4 formula used in Experiment 1 could not maintain luteal levels for more than 24 h. The use of long-acting iP4 maintained plasma progesterone concentrations above luteal levels for at least 5 days. The long-acting treatment can therefore be tested for estrous synchronization treatments. Implications The long-acting progesterone may be an alternative to short estrous synchronization protocols.

Sudden death due to aortic rupture in New Zealand sheep

ABSTRACT Case history Over a period of 2 months in the spring and early summer of 2021, 13 cases of sudden death in cull ewes due to aortic rupture were diagnosed at a small number of New Zealand abattoirs. Clinical findings In 12/13 (92%) cases, a large blood clot was present in the thorax, and in one case the blood clot was seen in the tissues dorsal to the heart. There were no obvious signs of external trauma. The pluck (heart and lungs) or fixed aorta was submitted for histological examination in seven cases and in all of these, a tear in the aorta was found. Comparing the microscopic appearance of the proximal aorta in these seven cases to three clinically normal ewes from unaffected farms, the aortic wall thickness appeared thinner in the case ewes than the unaffected ewes. Subjectively, there was increased collagen in the tunica media in 3/7 and decreased elastin fibres in 5/7 case ewes compared to the control ewes. Further investigations on the index farm (where the first cases originated), found that the mean liver and serum Cu concentrations in 10 similarly aged, clinically normal ewes were within the normal reference range for New Zealand sheep. Similarly, the liver Cu concentrations of the seven case ewes were within the normal reference range. Diagnosis Aortic rupture due to an unknown aetiology. Clinical relevance Clinicians should be aware of this condition as a differential diagnosis for sudden death in older sheep and to assist the Ministry for Primary Industries in establishing the extent of this problem in New Zealand.

476 Induced hindgut acidosis in sheep affected gut permeability and ruminal fermentation

Abstract Leaky gut may be caused by acidotic conditions in the hindgut and lead to systemic inflammation and negative effects on the gastrointestinal tract. The objective was to determine the effects of induced hindgut acidosis in sheep on cecal pH, ruminal fermentation, and gut permeability. Ruminally and cecally cannulated ewes [n = 11; body weight (BW) = 49 ± 4 kg] were assigned to one of two treatments: control (CON; n = 5) or induced hindgut acidosis (HGA; n = 6). To induce hindgut acidosis, 3 g wheat starch/kg BW per 24 h was continuously infused via the cecal cannula for 4 d. Control ewes received a constant infusion of deionized water. The diet contained 40% grass hay pellets, 35% whole shelled corn, 15% dried distillers grains, and 10% alfalfa cubes. Chromium EDTA was dosed once daily via the cecal cannula as a marker of gut permeability. Rumen, cecal, and fecal samples were collected to determine pH and volatile fatty acids (VFA). Rumen fluid was collected on d 4 for an ex vivo fermentation. Flasks were incubated for 24 h to determine pH, VFA, ammonia and in vitro dry matter digestibility. Tissue samples from the ileum, cecum, and colon were mounted in Ussing chambers to measure transepithelial electrical resistance (TEER). There was a treatment × time effect (P = 0.05) for cecal pH with HGA ewes having lesser cecal pH after d 1. By d 4, cecal pH had declined to 5.07 for HGA ewes compared with CON ewes which remained above 6.40 throughout the experiment. A treatment × time interaction was also observed (P < 0.01) for fecal pH and followed the same trend as cecal pH. Rumen pH was not affected (P = 0.87) by the interaction of treatment and time but was affected (P < 0.01) by treatment as ewes on the HGA treatment had a lesser rumen pH than CON ewes. Control ewes had lesser ruminal VFA and ammonia concentrations than HGA ewes (P < 0.01). Urinary Cr recovery was not affected by the interaction of treatment and time, or treatment (P ≥ 0.13). In vitro dry matter disappearance was not affected by cecal infusion treatment (P = 0.60). There was no effect (P = 0.78) of treatment on ex vivo pH. There were no effects (P ≥ 0.11) of treatment, time, or their interaction on ex vivo ammonia or total VFA concentration. In cecal tissue, TEER tended (P = 0.09) to be greater in CON ewes than HGA ewes. In contrast, TEER was not different (P ≥ 0.83) in ileal or colonic tissues between treatments. Induced hindgut acidosis in sheep altered rumen fermentation and tended to increase ex vivo measures of cecal permeability, but did not affect ex vivo rumen fermentation.

Milk transcriptome biomarker identification to enhance feed efficiency and reduce nutritional costs in dairy ewes

In recent years, rising prices for high-quality protein-based feeds have significantly increased nutrition costs. Consequently, investigating strategies to reduce these expenses and improve feed efficiency (FE) have become increasingly important for the dairy sheep industry. This research investigates the impact of nutritional protein restriction (NPR) during prepuberty and FE on the milk transcriptome of dairy Assaf ewes (sampled during the first lactation). To this end, we first compared transcriptomic differences between NPR and control ewes. Subsequently, we evaluated gene expression differences between ewes with divergent FE, using feed conversion ratio (FCR), residual feed intake (RFI), and consensus classifications of high− and low-FE animals for both indices. Lastly, we assess milk gene expression as a predictor of FE phenotype using random forest. No effect was found for the prepubertal NPR on milk performance or FE. Moreover, at the milk transcriptome level, only one gene, HBB, was differentially expressed between the NPR (n = 14) and the control group (n = 14). Further, the transcriptomic analysis between divergent FE sheep revealed 114 differentially expressed genes (DEGs) for RFI index (high-FERFI = 10 vs low-FERFI = 10), 244 for FCR (high-FEFCR = 10 vs low-FEFCR = 10), and 1 016 DEGs between divergent consensus ewes for both indices (high-FEconsensus = 8 vs low-FEconsensus = 8). These results underscore the critical role of selected FE indices for RNA-Seq analyses, revealing that consensus divergent animals for both indices maximise differences in transcriptomic responses. Genes overexpressed in high-FEconsensus ewes were associated with milk production and mammary gland development, while low-FEconsensus genes were linked to higher metabolic expenditure for tissue organisation and repair. The best prediction accuracy for FE phenotype using random forest was obtained for a set of 44 genes consistently differentially expressed across lactations, with Spearman correlations of 0.37 and 0.22 for FCR and RFI, respectively. These findings provide insights into potential sustainability strategies for dairy sheep, highlighting the utility of transcriptomic markers as FE proxies.

Accumulation Rate, Depuration Kinetics, and Tissue Distribution of Polychlorinated Dibenzo-p-Dioxins and Dibenzofurans (PCDD/Fs) in Suckler Ewes (Ovis aries).

Understanding the transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in farm animals is essential for ensuring food safety, but such information for suckler ewes (Ovis aries) has been lacking. This work quantifies the accumulation, tissue distribution, and depuration kinetics of PCDD/Fs in these animals. Six suckler ewes (EXP group) were exposed to PCDD/Fs through contaminated hay (2.3-12.7 ng toxic-equivalent kg-1 dry matter) and then allowed to depurate by switching to noncontaminated hay from 29 days of lactation. Four control ewes were fed continuously with noncontaminated hay. At different time points covering depuration, weaning and slaughter, PCDD/F analysis of milk (three time points), blood and sternal adipose tissue (five time points), Longissimus thoracis muscle, liver, and empty body homogenate at slaughter (188 days of depuration) was performed. A relevant PCDD/F bioaccumulation was observed from oral intake in milk and adipose tissue (biotransfer factors of 1.24 and 1.06 day kg-1 lipids for the sum toxic-equivalent, respectively) in the EXP ewes, especially for penta- and hexa-chlorinated congeners. The EXP ewes' adipose tissue started at 10-fold the EU maximum level (ML) and showed depuration below the ML after 130 days. Specific PCDD/F accumulation in the ewe liver was observed, especially for dibenzofurans. These toxicokinetic data can inform recommendations to ensure the chemical safety of sheep food products.

The effect of human menopausal gonadotropin and equine chorionic gonadotropin on the reproductive performance of treated ewes with short-term progesterone injections and sponge during the non-breeding season.

This study investigated the effect of human menopausal gonadotropin (hMG) on reproductive efficiency of synchronized ewes with the sponge and progesterone (P4) injection-based protocols. In study 1, anoestrous ewes (n = 120) were used. Sixty ewes were treated with sponge (S) for 12 days. The injection of eCG (SeCG group, n = 30) or hMG (ShMG, n = 30) was given at the time of sponge removal. Thirty ewes received IM injection of P4, three times every 48 h and the injection of hMG was given 24 h after the third P4 injection (3PhMG group, n = 30), and 30 ewes were used as control group. Pregnancy was diagnosed on day 50 after the release of ram. In study 2, 60 ewes were randomly divided into two equal groups. In the treated group with antibiotics (n = 30), before inserting, the sponges were impregnated with the antibiotic penicillin G sodium (5,000,000 IU) and in the control group (n = 30), there was no added antibiotics. Before inserting and after removing sponges, a vaginal cytology sample was taken with a sterile cotton swab. The number of neutrophils in each sample was counted and analysed. The rate of oestrus and total pregnancy was greater in SeCG (96.7, 93.3%), ShMG (82.8, 93.1%) and 3PhMG (67.9, 89.3%) groups compared with the control group (13.8, 41.4%) (p < .05). No significant difference was found in single, twin and total lambing and pregnancy rates after injection of eCG and hMG during the non-breeding season (p > .05). A higher percentage of control ewes had the vaginal smear with neutrophils more than 50% (96.7% vs. 76.7%; p < .05). In conclusion, a single dose of hMG can induce fertile oestrus in synchronized ewes with P4 administered by either injection or intravaginally. Purulent discharge and percentage of neutrophils were significantly reduced in the synchronized ewes by the impregnated sponges with the antibiotic penicillin.

Should lambs born to one-year-old ewes and grown to heavy live weights be retained as replacements and mated at seven months of age?

ABSTRACT The lifetime productivity and efficiency of singleton- and twin-born ewe lambs born to one-year-old ewes were compared with twin-born ewe lambs born to mixed-age ewes. Romney ewe lambs either born as twins to mixed-age ewes (Heavier n = 135 and Control n = 135), or to one-year-old ewes as singletons (L1; n = 135) or twins (L2; n = 88) were included. The Heavier ewes weighed 47.9 ± 0.36 kg at ewe lamb breeding while Control ewes weighed 44.9 ± 0.38 kg. The lifetime number of lambs born and weaned, and the lifetime weight of lambs weaned were used to calculate both feed and ewe efficiency. Heavier ewes had greater lamb production as one-year-old ewes than L1, L2 and Control ewes, after which the yearly lamb production and efficiency did not differ between treatments. Heavier ewes had a greater lifetime number and weight of lambs weaned than L1 and L2 ewes but did not differ from Control ewes. The lower lifetime production of L1 and L2 ewes was driven by their low production as one-year-old ewes. Farmers, therefore, could select their replacements from ewes born to one-year-old ewes and have similar lamb production to ewes born to mixed-age ewes, providing they reach recommended pre-breeding live weights.

The effect of repeated lipopolysaccharide endotoxin challenge on immune response of breeding ewes and subsequent lamb performance.

Infectious disease caused by exposure to Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is recognized to suppress female fertility. However, the effect of varying low-dose endotoxin exposure during distinct stages of follicle development on immune response, reproductive performance, and lamb performance has yet to be elucidated. Therefore, the objective of this study was to evaluate acute phase response, mRNA abundance of inflammatory markers, reproductive performance and lamb growth characteristics of ewes challenged with subclinical doses of LPS. Rambouillet ewes (n = 36; 68.2±1.1kg; age 3 to 7 yr) stratified by body weight (BW) and age were assigned to treatment groups. Ewes received subcutaneous injections of saline (CON, n = 12), 1.5 µg/kg BW LPS (LOW, n = 12), or 3.0 µg/kg BW LPS (HIGH, n = 12) on days 5, 10, and 15 of a synchronized follicular wave. Ewes were subsequently placed with a raddle-painted ram on day 16 for a 35-d breeding season. On treatment days 5 and 15, blood samples, peripheral blood leukocytes, and rectal temperature were collected before and at regular intervals for 12h after LPS challenge. Immune response to LPS was confirmed by increased temperature and serum cortisol concentrations on days 5 and 15. Endotoxin increased circulating plasma concentration of the acute phase protein, haptoglobin by greater than 15%, in both LPS-treated groups on days 5 and 15 at 12h compared with control (P≤ 0.05). Pro- and anti-inflammatory mRNA gene expression demonstrated no differences in expression for tumor necrosis factor-α or peroxisome proliferator-activated receptor gamma among treatment groups (P > 0.10). Likewise, Toll-like receptor 4 (TLR4), interleukin-8 (IL-8), and superoxide dismutase 2 (SOD2) expression was similar among treatment groups on day 5. However, ewes challenged with LPS on day 15 displayed greater mRNA expression for TLR4 from 2 to 6h (P < 0.05), a 7-fold increase for IL-8 from 1.5 to 2.5h (P < 0.05), and 8-fold induction for SOD2 from 2 to 6h (P < 0.05) as compared with controls. First service conception rates were 90% for control ewes and 75% for both treated groups (P = 0.84). Treated ewes demonstrated a reduction in lamb birth weight compared with controls (P ≤ 0.05) and a tendency for reduction of 60-d adjusted weaning weight (P = 0.09). Data suggest that subacute endotoxin exposure aligning with key follicle and oocyte maturation events results in detrimental growth performance of the subsequent lamb.

A Comparison of Two Estrus Synchronization Protocols Utilizing Progesterone Supplement 24 Hours before CIDR-G Removal in Sheep

Two experiments investigated the effect of progesterone administration 24 h prior to intravaginal CIDR-G withdrawal using two protocols in two geographical locations and different breeds of sheep. Ewes in both experiments were administered with intravaginal CIDR-G either for 8 days (Expt. 1, n=36 thin tail mixed-breed ewes) in early April or for 12 days (Expt. 2, n=28 fat tail Awassi ewes) in late May. Half of the ewes in each experiment were assigned randomly one day before CIDR-G removal and given 25 mg P4 (P4-treated) and the second half were given saline solution and served as controls. Fertile rams were allowed with the ewes of both experiments immediately following CIDR-G removal (day0, 0h) and estrus was monitored at 6-h intervals for 4 days. Blood samples were collected for analysis P4 and LH levels. Estrus responses differed between the two experiments. P4 profiles during the period of CIDR-G insertion behaved the same way among ewes of both experiments, but levels differed (P &lt; 0.05) on day 0 in response to P4 supplement. The preovulatory LH surge was greater among groups treated with P4 supplement than those that did not. In both experiments, the percentage of ewes exhibiting estrus was greater (P &lt; 0.05) and intervals to estrus were longer (P &lt; 0.05) in P4-treated than control ewes. In the 12-day protocol in Expt. 2, ewes treated with P4 supplement produced greater (P &lt; 0.05) incidence of estrus, longer (P &lt; 0.05) intervals to onset of estrus and LH surge and had higher (P &lt; 0.01) magnitude of LH surge and pregnancy rate than controls and those of the 8-day protocol. In conclusion, P4 supplement 24 h prior to CIDR-G removal in a 12-day protocol can be used successfully to improve reproductive performance of ewes.

Incorporation of L-Carnitine in the OvSynch protocol enhances the morphometrical and hemodynamic parameters of the ovarian structures and uterus in ewes under summer climatic conditions

Heat stress negatively impacts the reproductive performance of sheep including the efficiency of estrous synchronization regimens. This study aimed to investigate the potential effects of L-Carnitine (LC) administration on the efficacy of the OvSynch protocol in ewes under summer climatic conditions. Ewes were synchronized for estrus using the OvSynch protocol and a dose of LC (20 mg/kg body weight) was intravenously (IV) administered on the same day of PGF2α injection to one group (n = 8; LC group), while other ewes (n = 8; control group) received the same protocol without LC. Ultrasonographic evaluation (including B-mode, color, and pulsed Doppler) was used to assess the morphometrical and hemodynamic parameters of ovarian structures [number, size, and blood flow of follicles (GFs) and corpora lutea (CLs)] and uterus during the estrous phase (Day 0), and on Day 8 post ovulation (luteal phase). Uterine artery blood flow (MUA) was assessed by measuring the resistive index (RI) and pulsatility index (PI) at both stages. The serum samples were collected to measure the concentrations of estradiol (E2), progesterone (P4), and total antioxidant capacity (TAC) using commercial kits. Results revealed a significant (P<0.05) increase in the colored pixel area of GFs and uterus in the LC group (392.84 ± 31.86 and 712.50 ± 46.88, respectively) compared to the control one (226.25 ± 17.74 and 322 ± 18.78, respectively) during Day 0. Circulating E2 and TAC levels were significantly (P<0.05) higher in the LC-treated ewes (31.45 ± 1.53 pg/ml and 1.80 ± 0.13 mM/L, respectively) compared to those in the control ewes (21.20 ± 1.30 pg/ml and 0.98 ± 0.09 mM/L, respectively) during Day 0. Moreover, LC improved the colored pixel area of CLs (2038.14 ± 102.94 versus 1098 ± 82.39) and uterus (256.38 ± 39.28 versus 121.75 ± 11.36) and circulating P4 (2.99 ± 0.26 ng/ml versus1.67 ± 0.15 ng/ml) on Day 8. Values of RI of MUA were significantly lower in the LC group compared to the control one on Day 0 and Day 8 (0.48 ± 0.03 versus 0.72 ± 0.03 and 0.58 ± 0.03 versus 0.78 ± 0.02, respectively). In conclusion, LC incorporation in the OvSynch protocol enhanced the morphometrical and hemodynamic parameters of the ovarian structures and the uterus concomitantly with improvements in the TAC, E2, and P4 concentrations in ewes under hot summer conditions.

PSIV-17 Postnatal Obesity Amplifies Gnrh-Induced Lh Release in Prenatal Testosterone-Treated Female Sheep

Abstract Polycystic ovary syndrome (PCOS) is the most common hormonal disorder in females of reproductive age. Hypersecretion of luteinizing hormone (LH), partly due to increased pituitary sensitivity to gonadotrophin-releasing hormone (GnRH), is a significant cause of infertility in women with PCOS. Moreover, obesity amplifies the severity of reproductive dysfunction in PCOS. In sheep, testosterone (T) excess during gestational days (GD) 30 to 90 results in GnRH-mediated LH hypersecretion and recapitulates the PCOS phenotype. This study investigated 1) if mid-gestation (GD 60 to 90) is the susceptibility window for programming pituitary hypersensitivity to GnRH, and 2) the contribution of postnatal obesity in modulating LH responsiveness to GnRH. Suffolk ewes received T propionate (T; 100 mg i.m.) or corn oil (C; vehicle) twice weekly from GD 60 to 90 (term = 147 d). At 5 mo of age, T lambs were assigned randomly to either a maintenance (100% of NRC requirements) or overfed diet (130% of NRC requirements), and Control lambs were fed the maintenance diet. During the anestrous period (late summer), ewes (n = 8/group; ~17 mo of age) received a GnRH bolus injection (2 ng/kg) intravenously every 1.5 h and blood samples were collected at 15-min intervals for a 6-h period. Despite animals being in seasonal anestrous, few endogenous LH pulses were evident during the 6 h-sampling period. To avoid confounding, only clear responses to GnRH challenge that occurred within 15 to 45 min after each GnRH injection were considered for further analyses. Mean number of exogenous GnRH-induced LH responses that met these criteria did not differ between groups and averaged 1.81 ± 0.27 pulses/animal. The parameters of these GnRH-induced responses were analyzed as one-way ANOVA with Tukey’s HSD post-hoc analysis. Mean LH pulse peak was greater (P &amp;lt; 0.05) in T overfed ewes (4.59 ± 0.49 ng/mL) compared with T maintenance (2.19 ± 0.61 ng/mL) and Control ewes (2.58 ± 0.53 ng/mL). Additionally, amplitude of LH pulses was greater (P &amp;lt; 0.05) in T overfed ewes (2.66 ± 0.39 ng/mL) compared with T maintenance (1.03 ± 0.48 ng/mL) and tended (P = 0.089) to be greater in T overfed compared with Control ewes (1.41 ± 0.42 ng/mL). LH pulse peak and amplitude in T maintenance did not differ from Control ewes. In conclusion, failure of GD 60 to 90 T treatment to increase GnRH responsiveness as opposed to our earlier finding of increased responsiveness in GD 30 to 90 T-treaded sheep indicates that the susceptibility window for programming pituitary hypersensitivity to GnRH lies between GD 30 to 60. Postnatal obesity may act as an independent activational signal in amplifying the pituitary sensitivity to GnRH in female sheep. This premise is supported by observations that obesity exacerbates pituitary-mediated LH hypersecretion in women with PCOS. Research support: NIH-NICHD (R01HD099096).

396 Implications of Repeated Subacute Lipopolysaccharide Exposure During Synchronized Estrous

Abstract Acute inflammation induced by chronic infection or a single intravenous administration of the bacterial endotoxin, lipopolysaccharide (LPS) induces immune responses capable of depressing female fertility. However, the effect of dose and repeated endotoxin exposure during the estrous cycle on immune response and reproductive performance has yet to be investigated. To investigate the effects of varying doses of LPS delivered at distinct stages of follicular maturation, thirty-six Ramboulliet ewes (68.2 ± 1.1 kg; age 3 to 7 yr) were stratified by weight and age and randomly assigned to vehicle (n = 12), low (n = 12) or high (n = 12) LPS-treated groups to evaluate febrile response, serum progesterone and cortisol concentrations, and reproductive performance. Ewes received subcutaneous injections of saline (CON), 1.5 µg/kg BW LPS (LOW), or 3.0 µg/kg BW LPS (HIGH) on d 5, 10, and 15 after onset of synchronized estrus. Estrous synchronization was determined by serum progesterone (P4) analysis. All ewes demonstrated similar P4 profiles, but HIGH-LPS ewes tended to have elevated values (P = 0.1). On treatment d 5 and 15, blood samples and rectal temperature were collected before and intermittently throughout the duration of the trial. Lipopolysaccharide increased rectal temperature of LOW and HIGH groups at 2, 3, and 6 h, respectively after initial LPS challenge (d 5) when compared with vehicle-treated controls (0.67, and 0.70 °C, P &amp;lt; 0.05), but no significance was observed at d 10 or 15. Serum concentrations of cortisol were similar among treatment groups at 0 h (P &amp;gt; 0.05). However, a rapid and sustained cortisol increase was demonstrated within 1 h of LPS exposure in both LPS-treatment groups compared with controls (P = 0.001). A similar pattern of cortisol release among treatment groups was demonstrated on d 15 (P &amp;lt; 0.05). While there was no significant difference in services per cycle by treatment, there was a marked difference (16.67%) in remarks during the second cycle for HIGH and LOW groups (P = 0.23) as compared with controls. Control ewes had d 100 pregnancy rate of 90.91%, while low and high LPS-treated groups had pregnancy rates of 91.67% (P = 0.96) and 83.33% (P = 0.59) respectively. The administration of LPS resulted in significant increases in serum cortisol in treated ewes, and serum progesterone was significantly increased in LOW LPS-treated ewes compared with CON or HIGH LPS-treated ewes. Thus, alterations of temperature, steroid hormone concentration, and time to estrus in response to LPS may ultimately implicate female fertility and prevent economic losses for producers.

376 Effects of Mineral Supplementation During Range Grazing on Ovine Performance

Abstract Western US sheep production systems are dependent on rangeland forages to meet nutritional needs for animal production. While many native rangeland soils and forages are deficient in minerals like Cu, Se, and Zn, it is unknown whether ewes will alter forage selection if they receive a mineral supplemented diet and if alternative forages will affect performance on range. The objectives of this study are to quantify the effects of mineral supplementation on ewe forage selectivity on rangeland, range utilization, and animal performance. We hypothesized ewes who receive a trace mineral supplement will have a more diverse diet, greater rangeland utilization, and improved performance characteristics than their non-supplemented contemporaries. In this study, (n = 59) ewes were grazed across four summer pastures on Cedar Mountain near Cedar City, Utah. Ewes between the ages of 1 and 6 with an average weight of 87 Kg ± 13 Kg were stratified by initial weight and breed into one of two different treatment groups: 1) trace mineral supplement ad libitum (TRT; n = 30), or 2) no mineral supplement (CON; n = 29). Ewe breed types consisted of black face type breeds (BLF; Suffolk) and white face type breeds (WF; Targhee, Rambouillet, and their crosses). All ewes received a similar diet of alfalfa hay and triticale without access to ad libitum mineral supplementation 30 days before the start of the trial. A solar powered C-lock inc. pro was used to track individual animal mineral intake. Back fat (BF) and loin eye depth (LED) were measured at day 0 and day 116 of the trial via ultrasound. Additionally, fecal samples were collected and used for fecal metabarcoding (fDNA) at approximately 30% pasture utilization, which measures the proportion of plant DNA in the feces to determine forage species consumed. Plant species were sorted into functional groups (forbs, grass likes, grasses, shrubs, and trees). The GLM procedure of SAS was used to analyze BF, LED, and fDNA. The mixed procedure of SAS with day as a repeated measure was used to analyze percentage change in body weight. Control ewes had greater BF by 0.08 cm (P = 0.02). No difference in BF was observed by breed (P = 0.8). Loin eye depth was greater by 0.2 cm (P = 0.01) in BLF ewes than WF ewes. Supplemented ewes maintained 1% more of their initial body weight than CON ewes (P = 0.01). No forage selection differences were observed by treatment or breed (P&amp;gt; 0.2). The current study results indicate that mineral supplementation on range may not affect forage selectivity but can benefit animals by maintaining body weight over the grazing season.

25 Role of Kndy and Arcuate Kiss1r-Containing Neurons in the Preovulatory Luteinizing Hormone Surge and Puberty Onset of Female sheep

Abstract Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p &amp;lt; 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.

The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

An extreme model of fertility in sheep demonstrates the basis of controversies surrounding antral follicle count and circulating concentrations of anti-Müllerian hormone as predictors of fertility in ruminants

Ovarian ultrasonography and measurement of circulating concentrations of anti-Müllerian hormone (AMH) have been used to estimate follicle number and predict fertility in mammalian females, but no study has evaluated follicle number and circulating concentrations of AMH in ewes known to differ in fertility. We tested the hypothesis that ewes that had failed to lamb (BARREN) in four consecutive annual breeding seasons of 21–35 d have fewer follicles and diminished circulating concentrations of AMH compared to closely matched ewes that consistently produced lambs (FERTILE) under the same breeding protocols. Once identified, BARREN ewes (n = 19) were paired by breed and sire to a FERTILE control ewe (n = 19) and reproductive tracts were recovered at necropsy. Visible antral follicles in both ovaries were counted and a representative cross section of one ovary was embedded for histological evaluation of pre-antral follicle numbers. Paired t-tests indicated that BARREN ewes had fewer antral follicles, fewer primordial follicles, and decreased circulating concentrations of AMH compared to FERTILE ewes (P ≤ 0.01), but there were ewes in each fertility classification that had ovarian phenotypes like the opposite fertility classification. The best technologies we have currently for estimating follicle numbers are ultrasonography and measurement of circulating concentrations of AMH, but no single technique is perfect for predicting fertility. A better understanding of the under-lying biological mechanisms linking AMH, folliculogenesis, and fertility is required to improve the use of measurements of follicle number for predicting fertility in livestock.

Parity and housing effects on the behavioural and hypothalamic–pituitary–adrenal axis responses of pregnant ewes

It is common in many countries for sheep to be housed during winter from mid-gestation until lambing to protect ewes and lambs from adverse conditions and improve late gestation nutritional management. Keeping ewes indoors, however, has its own challenges as the animals may be mixed with unfamiliar conspecifics, have limited floor and feeding space, experience changes to their diet and increased handling by humans. Therefore, the objective of this study was to investigate the effect of variation in housing management (space allowance and social stability) on the behaviour and hypothalamic–pituitary–adrenal (HPA) axis responses of pregnant ewes from mid-to-late gestation (weeks 11–18 of pregnancy). Seventy-seven ewes (41 primiparous, 36 multiparous) were divided into two groups: ‘Control’ and ‘Restricted space and mixed’ (RS-Mix), where RS-Mix ewes were allocated half the amount of space (1.27 vs 2.5 m2 for RS-Mix and Control, respectively) and feedface (concentrate feeder space) allowance (36 vs 71 cm per ewe) given to the Control group and were also subjected to two social mixing events. Aggressive behaviour at the feedface and time spent standing, lying, walking, feeding and ruminating were recorded and faecal samples were collected for assessment of faecal glucocorticoid metabolite (FGM) concentrations. Higher aggression was observed in RS-Mix ewes during the first week of observation (P = 0.044), which gradually declined to the same level as Control ewes by the end of the study (P = 0.045). RS-Mix ewes were significantly less likely to be able to freely join the feedface compared to Controls (P = 0.022). No other significant treatment effects on aggressive behaviour or FGM during gestation were found. RS-Mix ewes displayed significantly higher ruminating behaviour at week 18 of gestation compared to Control ewes (P < 0.001), but no other effects were seen on general pen behaviour. However, the effect of indoor housing had a significant impact on primiparous ewes, who had lower weight gain (P = 0.015) and higher FGM concentrations (P = 0.014) compared to multiparous ewes regardless of treatment group. The data suggest that, although no sustained effects on behaviour or HPA axis responses were seen with the differences in space and feeder allowance or social stability at the levels used in this study, inexperienced (primiparous) ewes may find indoor housing more stressful; and are less able to adapt compared to multiparous ewes. These effects may influence the behaviour of the ewe at lambing time, and her offspring.

Maternal hepatic adaptations during obese pregnancy encompass lobe-specific mitochondrial alterations and oxidative stress.

Maternal obesity(MO) is rising worldwide, affecting half of all gestations, constituting a risk-factor for pregnancy-associated liver diseases (PALD) and hepatic diseases. PALD occur in approximately 3% of pregnancies and are characterized by maternal hepatic oxidative stress(OS) and mitochondrial dysfunction. Understanding the role of MO on liver function and pathophysiology could be crucial for understanding the altered pathways leading to PALD and liver disease. We investigated specific hepatic metabolic alterations in mitochondria and oxidative stress during MO at late-gestation. Maternal hepatic tissue was collected at 90% gestation in Control and MO ewes. Maternal hepatic redox state, mitochondrial Respiratory Chain(MRC) and OS markers were investigated. MO decreased MRC complex-II activity and SDHA and SDHB protein, increased complex-I and complex-IV activities despite reduced mtCO1 protein, and increased ATP5a protein. Hepatic MO-metabolic remodeling was characterized by decreased ANT-1/2 and VDAC protein expression and PKA activity, and augmented NAD+/NADH ratio due to reduced NADH levels. MO showed an altered redox state with increased OS, increased lipid peroxidation, decreased GSH/GSSG ratio, increased SOD and decreased catalase antioxidant enzymatic activities, lower catalase, glutathione peroxidase (Gpx)-4 and glutathione reductase protein expression, and increased Gpx-1 abundance. MO-related hepatic changes were more evident in the right lobe, corroborated by the integrative data analysis. Hepatic tissue from obese pregnant ewes showed alterations in the redox state, consistent with OS and MRC and metabolism remodeling. These are hallmarks of PALD and hepatic disease, supporting MO as a risk-factor and highlighting OS and mitochondrial dysfunction as mechanisms responsible for liver disease predisposition.

Effect of varying amounts of linseed oil or saturated fatty acids around insemination on reproductive and blood parameters of ewes.

The current study was designed to evaluate the effect of sequential low and high dietary linseed oil (LO; as omega-3 enriched fatty acid; FA) before and post insemination, respectively, on different plasma variables of ewes. Fat-tailed Qezel ewes were assigned randomly to be fed a diet enriched with 3% LO (n = 30) or the saturated FA (SFA; n = 30) three weeks before insemination (Day 0). The lipogenic diet supplemented with 6% LO or SFA was fed after insemination until Day +21. The control ewes were fed an isocaloric and isonitrogenous diet with no additional FA during the study. Estrus was synchronized by inserting a vaginal sponge (Spongavet®) for 12 days + 500 IU equine chorionic gonadotropin (eCG; Gonaser®), and ewes were inseminated via laparoscopic approach 56-59 h after eCG injection. The size of ovarian structures was assessed by transvaginal ultrasonography at -21, -14, -2, 0, and +10 days. Blood samples were collected weekly to measure the plasma's different biochemical variables and FA profile. Treatment did not affect the amounts of glucose, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, interleukin-10, interleukin-2, and non-esterified FA (p > 0.05). Conversely, concentrations of triglyceride, cholesterol, tumor necrosis factor-alpha, and insulin-like growth factor-1 were higher in SFA-fed ewes relative to control animals (p < 0.05). LO feeding resulted in greater amounts of n-3 FA isomers in plasma, while higher amounts of stearic acid were detected in SFA fed group 0 and +21 (p < 0.05). The number of ovarian follicles and corpora lutea also were not affected by treatment. Other reproductive variables were not affected by treatment except for the reproductive rate. It seems that LO or SFA feeding of fat-tailed ewes peri-insemination period was not superior to the isocaloric non-additional fat diet provided for the control group during the non-breeding season.