School of Agriculture, Food and Ecosystem Sciences - Research Publications
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ItemHexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction(PUBLIC LIBRARY SCIENCE, 2022-12-01)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 nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001's bioactivity: MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal.
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Item275 Access to Shade Mitigate Heat Stress and Improves Growth Performance in Lambs During Summer(Oxford University Press (OUP), 2021-10-08)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 providing access to shade is an effective ameliorative strategy to mitigate heat-stress in sheep during summer heatwaves.
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ItemOnset of normal cycles in postpartum anovulatory dairy cattle treated with kisspeptin(BIOSCIENTIFICA LTD, 2022-03)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 and afternoon milking, while the last group of eight cows served as untreated controls. Kp treatment caused a desirable hormone response from the cows' brain. Normal oestrous cycles resulted, but only when a mature follicle was present in the ovary. Further study is required to analyse whether the use of a long-acting Kp drug could be used as an effective treatment for stimulating dairy cows to become more fertile after calving.
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ItemGlucagon-like peptide-1 control of GnRH secretion in female sheep(BIOSCIENTIFICA LTD, 2021-03)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.
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ItemHexapeptides from a mammalian inhibitory hormone activate and inactivate nematode reproduction( 2021-09-30)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 nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001’s bioactivity : MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal.
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ItemAccess to Shade Mitigate Heat Stress and Improves Growth Performance in Lambs During Summer(American Society of Animal Science, 2021)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.
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ItemKiss1 expression in the hypothalamic arcuate nucleus is lower in dairy cows of reduced fertility†(OXFORD UNIV PRESS INC, 2022-04-26)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.
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ItemResponse to kisspeptin and gonadotropin-releasing hormone agonist administration in Holstein-Friesian dairy heifers with positive or negative genetic merit for fertility traits(ELSEVIER SCIENCE INC, 2022-04)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.
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ItemThe melanocortin pathway and energy homeostasis: From discovery to obesity therapy(ELSEVIER, 2021-06)BACKGROUND: Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency. SCOPE OF REVIEW: Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target. MAJOR CONCLUSIONS: Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug.