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Triiodothyronine (T3) concentrations in plasma decrease during acute illness and it is unclear if this contributes to disease. Clinical and laboratory studies of T3 supplementation in disease have revealed little or no effect. It is uncertain if short term supplementation of T3 has any discernible effect in a healthy animals. Observational study of intravenous T3 (1 µg/kg/h) for 24 h in a healthy sheep model receiving protocol-guided intensive care supports (T3 group, n=5). A total of 45 endpoints were measured including hemodynamic, respiratory, renal, hematological, metabolic and endocrine parameters. Data were compared with previously published studies of sheep subject to the same support protocol without administered T3 (No T3 group, n=5). Plasma free T3 concentrations were elevated 8-fold by the infusion (pmol/l at 24 h; T3 group 34.9±9.9 vs. No T3 group 4.4±0.3, P<0.01, reference range 1.6 to 6.8). There was no significant physiological response to administration of T3 over the study duration. Supplementation of intravenous T3 for 24 h has no physiological effect on relevant physiological endpoints in healthy sheep. Further research is required to understand if the lack of effect of short-term T3 may be related to kinetics of T3 cellular uptake, metabolism and action, or acute counterbalancing hormone resistance. This information may be helpful in design of clinical T3 supplementation trials.

We tested the hypothesis that divergent genetic merit for fertility of dairy cows is due to aberrant reproductive neuroendocrine function. The kisspeptin status of non-pregnant cows of either positive (POS) or negative (NEG) breeding values (BVs) for fertility was studied in three groups (n = 8), based on their previous post-partum period: POS cows, which had spontaneous ovarian cycles (POS-CYC) and NEG cows, which either cycled (NEG-CYC) or did not cycle (NEG-NONCYC). Ovarian cycles were synchronized, blood samples were taken to define endocrine status, and the animals were slaughtered in an artificial follicular phase. The brains and the pituitary glands were collected for quantitative polymerase chain reaction (qPCR) and in situ hybridization of hypothalamic GNRH1, Kiss1, TAC3, and PDYN and pituitary expression of LHB and FSHB. Gonadotropin releasing hormone (GnRH) and kisspeptin levels were quantified in snap frozen median eminence (ME). GNRH1 expression and GnRH levels in the ME were similar across groups. Kiss1 expression in the preoptic area of the hypothalamus was also similar across groups, but Kiss1 in the arcuate nucleus was almost 2-fold higher in POS-CYC cows than in NEG groups. TAC3 expression was higher in POS-CYC cows. The number of pituitary gonadotropes and the level of expression of LHB and FSHB were similar across groups. We conclude that the lower levels of Kiss1 and TAC3 in NEG cows with low fertility status and may lead to deficient GnRH and gonadotropin secretion.

Previous research has identified that Holstein-Friesian dairy heifers with positive (POS) genetic merit for fertility traits (FertBV) reach puberty earlier than heifers with negative (NEG) FertBV. The hypothalamus-pituitary-gonadal (HPG) axis is functional in heifers before the onset of puberty, with increased LH release evident as heifers progress toward puberty. We investigated the functionality of the HPG axis in peripubertal Holstein-Friesian dairy heifers with divergent POS or NEG FertBV, hypothesizing that the earlier puberty onset of POS heifers is associated with earlier activation of the HPG axis than in NEG heifers. In experiment 1, we tested the dose responsiveness of POS heifers to an intravenous injection of either kisspeptin [Kiss; 2, 4, or 8 µg/kg of body weight (BW); n = 3 per dose] or a GnRH agonist (buserelin; 5, 10, or 20 ng/kg of BW; n = 3 per dose). The use of these 2 agonists investigates the status of the HPG axis in both the hypothalamus (Kiss) and pituitary (buserelin) glands. Doses of 4 µg/kg BW of Kiss and 10 ng/kg BW of buserelin produced submaximal LH responses and were used in experiment 2, in which previously unused POS (n = 22) and NEG (n = 18) FertBV heifers were challenged with both agonists at 10 and 12 mo of age in a partial crossover design. Heifers were randomly allocated to treatment groups, balanced for age and BW. The LH response to buserelin was greater in POS heifers than NEG heifers at 10 mo of age, with no difference in response at 12 mo. The FSH response to buserelin and the LH and FSH responses to Kiss did not differ between the POS and NEG heifers at either age. These results indicate an association between divergent genetic merit for fertility and the LH release to buserelin at 10 mo of age, supporting the hypothesis that gonadotropin responsiveness to a GnRH agonist is more advanced in POS heifers than in NEG heifers.

UNLABELLED: The efficacy of a long-acting synthetic derivative of kisspeptin (Kp) to initiate normal oestrous cycles was tested in 24 mixed-aged, Holstein-Friesian cows that were 18-25 days postpartum on the day of treatment (D0). Groups of eight cows received saline (Sal) vehicle by intramuscular injection at 8:00 and 16:00 h (Sal-Sal), Kp at 8:00 h and vehicle at 16:00 h (Kp-Sal) or Kp on both occasions (Kp-Kp). The Kp dose was 15 nmol per 60 kg body weight. The ovaries of the cows were examined daily by ultrasonography between D4 and D14. Blood samples were collected from a tail vessel at 0, 2, 4, 8, 10 and 12 h relative to the time of the first injection for luteinizing hormone (LH) and follicle-stimulating hormone assay. Additional samples were collected daily from D4 until D14 and D19, 22, 26 and 29 for progesterone assay. LH surge-like responses were observed in cows treated with Kp at 8:00 h. Ovulation was consistently induced by Kp within 48 h when a dominant ovarian follicle of at least 10 mm in diameter was observed (8/14) but in no cases (6/14) during a new wave of ovarian follicular development comprising follicles <10 mm in diameter. The subsequent ovulatory cycle was of normal length in most cases as compared with short 8- to 12-day cycles observed in spontaneously ovulating cows. We conclude that Kp treatment can induce ovulation in postpartum dairy cows, with ensuing oestrous cycles of normal length, if administered when a mature dominant follicle is present in the ovaries. LAY SUMMARY: Cow fertility is important for efficient, profitable dairy farming. Cows that take too long after calving to become fertile are problematic. We tested a synthetically made, long-acting hormone called kisspeptin (Kp) to advance the time that cows become fertile after calving. Twenty-four dairy cows that had been calved for 3-4 weeks were used. One group of eight cows received an injection of Kp at the morning milking, another eight cows received Kp at both the morning

BACKGROUND: Biopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. METHODS: Reproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. RESULTS: Reproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nemato

The role of glucagon-like peptide-1 (GLP-1) on gonadotropin-releasing hormone (GnRH) secretion was investigated in ovariectomised (OVX) ewes, in which GnRH and luteinising hormone (LH) secretion had been restrained by treatment with oestrogen and progesterone. Guide tubes for microinjection were placed above the median eminence (ME) and the animals were allowed to recover for 1 month. Jugular venous blood samples were taken via cannulae at 10 min intervals. Vehicle (50 nL) was injected into the ME at 2 h, followed by injection of GLP-1 ((7-36)-amide - 0.5 or 1 nmol) or its receptor agonist, exendin-4 (0.5 nmol) at 4 h (n = 5). Plasma LH levels were quantified as a surrogate measure of GnRH secretion. GLP-1 microinjection into the ME elicited a large amplitude LH pulse in jugular plasma, the effect was greater at the higher dose. Exendin-4 microinjection caused a large, sustained increase in plasma LH levels. To determine how GLP-1 might exert an effect on GnRH secretion, we employed double labelled in situ hybridisation, with RNAScope, for co-localisation of the GLP-1 receptor (GLP-1R) in GnRH, Kisspeptin and NPY cells in the hypothalami of three ewes in the luteal phase of the estrous cycle. GLP1R expression was clearly visible but the receptor was not expressed in GNRH1 or NPY expressing neurons and was visualised in <5% of KISS1 expressing neurons. We conclude that GLP-1 may act at the level of the secretory terminals of GnRH neurons in the ME to stimulate GnRH secretion, the pathway through which such effect is manifested remains unknown.

The objective of the present study was to investigate the effects of provision of shade on behavior, physiology, and growth of Merino lambs exposed to natural Australian summer conditions. Sixty Merino lambs were randomly allocated to either pasture with shade (n = 30;paddock with trees) or a pasture without shade (n = 30;paddock without any trees) for one month during southern-Australian summer (February-2021). Sheep were grazing on the pastures as per standard protocols followed on the farm with ad libitum access to water. Lambs were monitored twice daily between 0900-1000h and 1400-1600h to record their behavior, and physiological parameters were recorded on hot days (environmental temperature (T) >30°C). Behavioral patterns were represented as the proportion of animals doing specific activities in each treatment group. Grazing was the most frequent activity observed in animals during morning measurement (> 60% in both groups). However, high temperature (T > 30°C) decreased grazing behavior in sheep to < 5% in both treatments. Standing behavior was significantly greater (65.2 vs 21.6%; P < 0.05) for animals under non-shade treatment, while lying behavior was more frequent (17.3 vs 76.3%; P < 0.01) in animals having access to shade. Significantly more animals were seeking water troughs (P < 0.05) in the non-shade group (33.2%) compared to the shade group (10.3%). On an average hot day (T >28°C), most lambs in the shade group were seeking shade (P < 0.01), and when the temperature exceeded >32°C, 90% of the lambs were seeking tree shade. Sheep with access to shade exhibited lower (P < 0.01) respiration rate (117breaths/min) and rectal temperature (39.7°C) than non-shade group (151breaths/min;40.2°C). Overall, access to shade improved (P < 0.001) average daily gain (+0.047kg/day) in sheep compared to the non-shade group (-0.028kg/day). In conclusion, our research confirms that provid

The objective of the present study was to investigate the effects of provision of shade on behavior, physiology, and growth of Merino lambs exposed to natural Australian summer conditions. Sixty Merino lambs were randomly allocated to either pasture with shade (n = 30;paddock with trees) or a pasture without shade (n = 30;paddock without any trees) for one month during southern-Australian summer (February-2021). Sheep were grazing on the pastures as per standard protocols followed on the farm with ad libitum access to water. Lambs were monitored twice daily between 0900-1000h and 1400-1600h to record their behavior, and physiological parameters were recorded on hot days (environmental temperature (T) >30°C). Behavioral patterns were represented as the proportion of animals doing specific activities in each treatment group.

Background: Biopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. Methods: Reproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. Results: Reproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nemato

Expression of particular genes in hypothami of ewes was measured across the natural pubertal transition by in situ hybridization. The ewes were allocated to three groups (n = 4); prepubertal, postpubertal and postpubertally gonadectomized (GDX). Prepubertal sheep were euthanized at 20 weeks of age and postpubertal animals at 32 weeks. GDX sheep were also euthanized at 32 weeks, 1 week after surgery. Expression of KISS1, TAC3, PDYN in the arcuate nucleus (ARC), RFRP in the dorsomedial hypothalamus and GNRH1 in the preoptic area was quantified on a cellular basis. KISS1R expression by GNRH1 cells was quantified by double-label in situ hybridization. Across puberty, detectable KISS1 cell number increased in the caudal ARC and whilst PDYN cell numbers were low, numbers increased in the rostral ARC. TAC3 expression did not change but RFRP expression/cell was reduced across puberty. There was no change across puberty in the number of GNRH1 cells that expressed the kisspeptin receptor (KISS1R). GDX shortly after puberty did not increase expression of any of the genes of interest. We conclude that KISS1 expression in the ARC increases during puberty in ewes and this may be a causative factor in the pubertal activation of the reproductive axis. A reduction in expression of RFRP may be a factor in the onset of puberty, removing negative tone on GNRH1 cells. The lack of changes in expression of genes following GDX suggest that the effects of gonadal hormones may differ in young and mature animals.

With increases in the frequency, intensity and duration of heat waves forecast plus expansion of tropical agriculture, heat stress (HS) is both a current and an emerging problem. As cinnamon has been shown to increase insulin sensitivity, which is part of the adaptive response to HS, the aim of this experiment was to determine if cinnamon could improve insulin sensitivity and ameliorate HS in grower pigs. In a 2 × 2 factorial design, 36 female Large White × Landrace pigs were fed control (0%) vs. cinnamon (1.5%) diets and housed for 7 day under thermoneutral (20 °C, TN) vs. HS conditions (8 h 35 °C/16 h 28 °C, 35% relative humidity). At the completion of the challenge, insulin sensitivity was assessed by an intravenous glucose tolerance test (IVGTT). Heat stress increased parameters such as respiration rate and rectal temperature. Furthermore, biochemical changes in blood and urine indicated the pigs were experiencing respiratory alkalosis. Minimal modelling of parameters of insulin sensitivity showed that HS pigs had a lower insulin response to the IVGTT and improved insulin sensitivity. Cinnamon had additive effects with heat stress, reflected in lowering the insulin area under curve (AUC) and elevated insulin sensitivity compared to TN. However, this apparent improvement in insulin sensitivity did not ameliorate any of the other physiological symptoms of HS.

The objective of this study was to compare the thermotolerance of second-cross (SC; Poll Dorset × Merino × Border Leicester) and Dorper lambs. Dorper and SC lambs (4-5 months of age) were subjected to cyclic heat stress (HS) (28-40 °C). The temperature was increased to 38-40 °C between 800 and 1700 h daily and maintained at 28 °C for the remainder of the day (30-60% relative humidity (RH)) in climatic chambers for 2 weeks (n = 12/group), with controls maintained in a thermoneutral (TN) (18-21 °C, 40-50% RH) environment (n = 12/group). Basal respiration rate (RR), rectal temperature (RT) and skin temperature (ST) were higher (p < 0.01) in SC lambs than in Dorpers. HS increased RR, RT and ST (p < 0.01) in both genotypes, but the levels reached during HS were lower (p < 0.01) in Dorpers. HS increased (p < 0.01) water intake to a greater extent in SC lambs, while feed intake was reduced (p < 0.05) by HS in SC lambs but not in Dorpers. HS increased (p < 0.01) blood urea nitrogen and creatinine in SC lambs only. Plasma non-esterified fatty acid concentrations were reduced (p < 0.05) by HS in SC lambs but increased (p < 0.05) in Dorpers. There was no effect of HS on pO2, cHCO3- and cSO2, but higher (p < 0.01) blood pH and lower (p < 0.01) pCO2 were recorded under HS in both genotypes. Blood electrolytes and base excess were reduced (p < 0.01) under HS, while a genotype difference (p < 0.05) was only observed in blood K+ and hemoglobin concentrations. Basal plasma prolactin concentrations were lower (p < 0.01) in Dorpers but were elevated at a similar level during HS (p < 0.01) in both genotypes. Dorper lambs are more resilient to HS than SC lambs. Future research should focus on confirming whether the better heat tolerance of Dorpers is translated to better returns in terms of growth performance and carcass traits over the summer months.

Climate change is a major global threat to the sustainability of livestock systems. Climatic factors such as ambient temperature, relative humidity, direct and indirect solar radiation and wind speed influence feed and water availability, fodder quality and disease occurrence, with production being most efficient in optimal environmental conditions. Among these climatic variables, ambient temperature fluctuations have the most impact on livestock production and animal welfare. Continuous exposure of the animals to heat stress compromises growth, milk and meat production and reproduction. The capacity of an animal to mitigate effects of increased environmental temperature, without progressing into stress response, differs within and between species. Comparatively, small ruminants are better adapted to hot environments than large ruminants and have better ability to survive, produce and reproduce in harsh climatic regions. Nevertheless, the physiological and behavioral changes in response to hot environments affect small ruminant production. It has been found that tropical breeds are more adaptive to hot climates than high-producing temperate breeds. The growing body of knowledge on the negative impact of heat stress on small ruminant production and welfare will assist in the development of suitable strategies to mitigate heat stress. Selection of thermotolerant breeds, through identification of genetic traits for adaption to extreme environmental conditions (high temperature, feed scarcity, water scarcity), is a viable strategy to combat climate change and minimize the impact on small ruminant production and welfare. This review highlights such adaption within and among different breeds of small ruminants challenged by heat stress.

We compared the thermotolerance of Dorper (D) and second cross (SC) (Poll Dorset x Merino/Border Leicester) lambs by assessing physiological and biochemical responses. After acclimatization, 4–5 month old lambs of each breed were exposed to either thermo-neutral (18 °C–21 °C, 40%–50% RH, n = 12/group) or cyclic heat stress (HS) (28 °C–40 °C; 40%–60% RH, n = 12/group) for 2 weeks in climatic chambers. The HS involved exposure to temperatures of 38 °C–40 °C between 0800 and 17.00 h daily; otherwise the temperature was maintained at 28 °C. Elevated temperature increased rectal temperature (p < 0.01), respiration rate (p < 0.01) and skin temperature (p < 0.01) in both breeds, (data for 12.00 and 16.00 h pooled), but to a lesser extent in D than in SC lambs (p < 0.01). The HS increased (p < 0.01) water intake to a greater extent in SC than in D lambs and HS reduced (p < 0.05) food intake in SC lambs but not in D lambs. There were no treatment effects on blood glucose and lactate levels in either breed. Significant effects of breed (p < 0.01) and treatment (p < 0.01) were observed in blood creatinine levels, being higher in SC lambs. Higher pH (p < 0.01) and lower pCO2 (p < 0.01) were recorded under HS in both breeds. Among blood electrolytes, Cl−, Na+ and base excess were significantly (all p < 0.01) reduced under HS, with no breed differences. In conclusion, the attenuated physiological responses to HS in Dorper lambs indicates better adaptation of this breed to high environmental temperature.

Sheep in semi-arid environments are likely to be affected by warming environment due to climate change. To identify genotypes best suited to warmer climates we assessed thermotolerance and meat quality of 5 female sheep of each of 4 breeds (Merino, Wiltshire, Dorper, and Southdown) under simulated summer conditions. The sheep were housed in metabolic crates within climate-controlled rooms and were exposed to thermo-neutral (TN; 18–21 °C and 40–50% relative humidity) or cyclic heat stress (HS; 28–40 °C and 30–40% RH) conditions for two weeks. Physiological responses were recorded 3 times daily, and brown fat tissue temperature was measured by data loggers inserted into the brisket. Sheep were slaughtered at an abattoir as per standard commercial procedure and samples obtained for carcass and meat quality attributes. When exposed to HS, Dorpers and Merinos exhibited lower respiration rate (151, 142 breaths/min, respectively) and rectal temperature (39.39, 39.32 °C, respectively) (P < 0.05) than Southdowns (192 breaths/min, 40.05 °C) and Wiltshires (200 breaths/min, 39.91 °C). Dorper and Wiltshire (n = 3) showed lowest sternal fat temperatures during HS indicating inherent differences in thermogenesis. HS had significant effect (P < 0.05) on post mortem muscle pH decline which was slower than TN sheep, except in Dorper again indicating better thermotolerance. There were significant (P = 0.03) breed effects on meat cooking loss % such that Southdown showed minimum cooking loss (17%) while Merino showed the greatest loss (24%). These results suggest that there are genetic (breed) differences in thermotolerance and meat quality of sheep, providing an opportunity to select best sheep suited to a warming climate.

Objective Polycystic ovary syndrome (PCOS) is associated with increased obesity with a greater propensity to weight gain and a lack of sustainable lifestyle interventions. Altered brown adipose tissue (BAT) thermogenesis is a potential contributor to obesity in PCOS. BAT activity and modulation have not been studied in PCOS. This observational study explored BAT thermogenesis and its associations in women with and without PCOS. Participants and methods Cutaneous temperature was recorded from supraclavicular (indicator of BAT activity) and upper arm regions using dataloggers (SubCue, Calgary, Canada) in a cross-sectional substudy, nested within a randomized control trial, of community-recruited premenopausal women with (n = 47, Rotterdam diagnostic criteria) and without (n = 11) PCOS. Results Complete temperature data were available in 44 PCOS (mean age: 30.0 ± 6.2, mean BMI: 29.3 ± 5.5) and 11 non-PCOS (mean age: 33.0 ± 7.0, mean BMI: 25 ± 3) women. Women with PCOS had lower supraclavicular skin temperature compared to controls overall (33.9 ± 0.7 vs 34.5 ± 1, P < 0.05) and during sleep (34.5 ± 0.6 vs 35.2 ± 0.9, P < 0.001). In the PCOS group, supraclavicular skin temperature overall and over sleep and waking hours correlated inversely with testosterone (r = −0.41 P < 0.05, r = −0.485 P < 0.01 and r = −0.450 P < 0.01 respectively). Testosterone levels explained approximately 15%, 30% and 20% of the variability in supraclavicular skin temperature overall and over sleep and waking hours in women with PCOS, respectively. Conclusion Women with PCOS have lower BAT activity compared to controls. BAT thermogenesis is negatively associated with androgen levels in PCOS.

Variations in climatic variables (temperature, humidity and solar radiation) negatively impact livestock growth, reproduction, and production. Heat stress, for instance, is a source of huge financial loss to livestock production globally. There have been significant advances in physical modifications of animal environment and nutritional interventions as tools of heat stress mitigation. Unfortunately, these are short-term solutions and may be unsustainable, costly, and not applicable to all production systems. Accordingly, there is a need for innovative, practical, and sustainable approaches to overcome the challenges posed by global warming and climate change-induced heat stress. This review highlights attempts to genetically select and breed ruminants for thermotolerance and thereby sustain production in the face of changing climates. One effective way is to incorporate sustainable heat abatement strategies in ruminant production. Improved knowledge of the physiology of ruminant acclimation to harsh environments, the opportunities and tools available for selecting and breeding thermotolerant ruminants, and the matching of animals to appropriate environments should help to minimise the effect of heat stress on sustainable animal genetic resource growth, production, and reproduction to ensure protein food security.

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI 14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI 14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparentl

The activity of the hypothalamic-pituitary gonadal axis is influenced by energy reserves, such that an increase or a decrease in adiposity may perturb the secretion and action of gonadotrophin-releasing hormone (GnRH). This is considered to be a result of the signalling of hormones such as leptin, which act upon neuronal systems controlling GnRH secretion. Other work shows plasticity in the relationship between tanycytes and GnRH neurosecretory terminals in the median eminence across the oestrous cycle and we hypothesised that a similar plasticity may occur with altered metabolic status. We studied Lean, Normal and Fat ovariectomised ewes, which displayed differences in gonadotrophin status, and investigated the relationship between tanycytes and GnRH neuroterminals. Under both Lean and Fat conditions, an altered anatomical arrangement between these two elements was observed in the vicinity of the blood vessels of the primary plexus of the hypophysial portal blood system. These data suggest that such plasticity is an important determinant of the rate of secretion of GnRH in animals of differing metabolic status and that this also contributes to the relative hypogonadotrophic condition prevailing with metabolic extremes.

In the context of a hunt for a postulated hormone that is tissue-mass inhibiting and reproductively associated, there is described probable relatedness to a granin protein. A 7-8 kDa polypeptide candidate (gels/MS) appeared in a bioassay-guided fractionation campaign involving sheep plasma. An N-terminal sequence of 14 amino acids was obtained for the polypeptide by Edman degradation. Bioinformatics and molecular biology failed to illuminate any ovine or non-ovine protein which might relate to this sequence. The N-terminal sequence was synthesized as the 14mer EPL001 peptide and surprisingly found to be inhibitory in an assay in vivo of compensatory renal growth in the rat and modulatory of nematode fecundity, in line with the inhibitory hormone hypothesis. Antibodies were raised to EPL001 and their deployment upheld the hypothesis that the EPL001 amino acid sequence is meaningful and relevant, notwithstanding bioinformatic obscurity. Immunohistochemistry (IHC) in sheep, rodents and humans yielded staining of seeming endocrine relevance (e.g. hypothalamus, gonads and neuroendocrine cells in diverse tissues), with apparent upregulation in certain human tumours (e.g. pheochromocytoma). Discrete IHC staining in Drosophila melanogaster embryo brain was seen in glia and in neuroendocrine cells, with staining likely in the corpus cardiacum. The search for the endogenous antigen involved immunoprecipitation (IP) followed by liquid chromatography and mass spectrometry (LC-MS). Feedstocks were PC12 conditioned medium and aqueous extract of rat hypothalamus-both of which had anti-proliferative and pro-apoptotic effects in an assay in vitro involving rat bone marrow cells, which inhibition was subject to prior immunodepletion with an anti-EPL001 antibody-together with fruit fly embryo material. It is concluded that the mammalian antigen is likely secretogranin II (SgII) related. The originally seen 7-8 kDa polypeptide is suggested to be a new proteoform of secretogranin II of

OBJECTIVES: Triiodothyronine concentration in plasma decreases during septic shock and may contribute to multiple organ dysfunction. We sought to determine the safety and efficacy of administering triiodothyronine, with and without hydrocortisone, in a model of septic shock. DESIGN: Randomized blinded placebo-controlled trial. SETTING: Preclinical research laboratory. SUBJECTS: Thirty-two sheep rendered septic with IV Escherichia coli and receiving protocol-guided sedation, ventilation, IV fluids, and norepinephrine infusion. INTERVENTIONS: Two hours following induction of sepsis, 32 sheep received a 24-hour IV infusion of 1) placebo + placebo, 2) triiodothyronine + placebo, 3) hydrocortisone + placebo, or 4) triiodothyronine + hydrocortisone. MEASUREMENTS AND MAIN RESULTS: Primary outcome was the total amount of norepinephrine required to maintain a target mean arterial pressure; secondary outcomes included hemodynamic and metabolic indices. Plasma triiodothyronine levels increased to supraphysiological concentrations with hormonal therapy. Following 24 hours of study drug infusion, the amount of norepinephrine required was no different between the study groups (mean ± SD μg/kg; placebo + placebo group 208 ± 392; triiodothyronine + placebo group 501 ± 370; hydrocortisone + placebo group 167 ± 286; triiodothyronine + hydrocortisone group 466 ± 495; p = 0.20). There was no significant treatment effect on any hemodynamic variable, metabolic parameter, or measure of organ function. CONCLUSIONS: A 24-hour infusion of triiodothyronine, with or without hydrocortisone, in an ovine model of septic shock did not markedly alter norepinephrine requirement or any other physiological parameter.

The introduction of a novel male stimulates the hypothalamic-pituitary-gonadal axis of female sheep during seasonal anestrus, leading to the resumption of follicle maturation and ovulation. How this pheromone cue activates pulsatile secretion of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) is unknown. We hypothesised that pheromones activate kisspeptin neurons, the product of which is critical for the stimulation of GnRH neurons and fertility. During the non-breeding season, female sheep were exposed to novel males and blood samples collected for analysis of plasma LH profiles. Females without exposure to males served as controls. In addition, one hour before male exposure, a kisspeptin antagonist (P-271) or vehicle was infused into the lateral ventricle and continued for the entire period of male exposure. Introduction of a male led to elevated mean LH levels, due to increased LH pulse amplitude and pulse frequency in females, when compared to females not exposed to a male. Infusion of P-271 abolished this effect of male exposure. Brains were collected after the male effect stimulus and we observed an increase in the percentage of kisspeptin neurons co-expressing Fos, by immunohistochemistry. In addition, the per-cell expression of Kiss1 mRNA was increased in the rostral and mid (but not the caudal) arcuate nucleus (ARC) after male exposure in both aCSF and P-271 treated ewes, but the per-cell content of neurokinin B mRNA was decreased. There was also a generalized increase in Fos positive cells in the rostral and mid ARC as well as the ventromedial hypothalamus of females exposed to males. We conclude that introduction of male sheep to seasonally anestrous female sheep activates kisspeptin neurons and other cells in the hypothalamus, leading to increased GnRH/LH secretion.