Circulating concentrations of leptin in sheep correlate with body fatness and are affected by level of food intake and photoperiod. The present objective was to elucidate the short-term dynamics of leptin secretion. Frequent blood samples were taken over 48 h from 12 Soay rams after 16 weeks in short-day photoperiod (SD, 16 h darkness:8 h light) with freely available food, and then after 16 weeks in long days (16 h light:8 h darkness) with food freely available (LD) or restricted to 90% maintenance (LDR) (n=6/ group). During the second 24 h of sampling, half were food deprived (n=6, SD and LD) and half had their meal times shifted (n=6, SD and LDR). A homologous RIA was developed, using antibodies raised in chicken against recombinant ovine leptin, to measure plasma concentrations. Simultaneous 24 h profiles of plasma insulin, glucose and non-esterified fatty acids (NEFA) were measured. Plasma leptin was higher in LD than SD, and in LD than LDR, associated with higher food intake, liveweight and body condition score (adiposity), but tended to be lower in LDR than SD, associated with lower food intake, liveweight and body condition score. There was no evidence for a circadian rhythm of plasma leptin, but clear evidence for post-prandial peaks of low amplitude (15-36%) 2-8 h after meals given at normal and shifted times. Complete food deprivation caused a dramatic fall in plasma leptin to basal levels within 24 h. There was a positive association of plasma leptin with plasma insulin, and negative association with NEFA, both between meals and during fasting. Thus, plasma leptin concentrations in sheep are sensitive to short-term changes in energy balance, as well as to long-term photoperiod-driven changes in food intake and adiposity.

Hypothalamic pathways involved in the regulation of energy balance have not been widely studied in ruminants to date. Here, we used in situ hybridisation to study the gene expression of a number of leptin-sensitive receptors and neuropeptides in the ovine hypothalamus. Gene expression was first localised for cocaine- and amphetamine-regulated transcript (CART) and agouti-related peptide (AGRP). We then examined in adult male castrated sheep the effects of acute negative energy balance induced by a 4-day fast on the amounts of these mRNAs and those for leptin receptor (OB-Rb), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC). CART mRNA was localised in the arcuate nucleus (ARC), paraventricular nucleus, median eminence and ventromedial hypothalamic nucleus, and extensive co-localisation with POMC mRNA was demonstrated in the ARC. AGRP mRNA was localised in the ARC. Fasting up-regulated gene expression for OB-Rb and for the orexigenic neuropeptides NPY and AGRP in the ARC. There was a trend towards down-regulation of gene expression for the anorexigenic neuropeptide CART and no effect on POMC in the ARC, although these results are inconclusive. The presence or absence of oestradiol-containing subcutaneous implants did not influence gene expression or the effects of fasting. The hypothalamic changes were consistent with responses to the observed reduction in circulation leptin and suggest that the peripheral feedback and central mechanisms for restoring the energy balance may be largely conserved across monogastric and ruminant species.

Intravenous infusion of ~-1~HjphenyIalanine (Phe) was carried out for 8 h in dry, non-pregnant and lactating dairy goats. Nitrogen balance was positive in the dry group and negative in the lactating group.Whole-body Phe flux was 50 % greater in lactating goats (P < 0.01). Fractional synthesis rates (K,) of tissue proteins were estimated from plasma-(K,) and tissue-(KJ specific radioactivities of Phe. In lactating goats, K , for mammary gland, duodenum and diaphragm was increased (P < 0.05). K, also tended to increase in liver, kidney and rumen (P < 0.08) of lactating goats, but was not different in uterus, spleen, caecum or heart. Values of Ksh were higher than Ksp; however, these measures agreed qualitatively. When absolute rates of protein synthesis were calculated, an increased contribution of mammary and visceral organs was seen in lactating goats. K, and absolute rates of protein synthesis of hind-limb skin were less in lactating goats (P < 0.05). A decreased proportion of skeletal muscle (P < 0.01) and decreased K, resulted in lower absolute synthesis of hind-limb muscle protein in lactating animals (P < 0.05). Decreased rates of muscle and skin protein synthesis would appear to participate in alterations of protein metabolism, permitting lactation to occur a t the expense of body reserves.Lactation : Phenylalanine flux : Protein synthesis : GoatIn high-producing dairy ruminants, early lactation is recognized as a period of negative nitrogen balance (Brun-Bellut et al. 1984). In order to maintain milk output, a utilization of maternal sources of protein and energy is necessary to supplement the dietary supply (Brun-Bellut et al. 1984;Giger, 1987;Barnes & Brown, 1990). Dairy goats have attracted some attention for studies of this phenomenon, because of their high milk output per unit body-weight and small body size (Wilkinson & Stark, 1987).Net body protein loss may result from an increase in protein degradation, a decrease in body synthesis, or both. However, the changes in protein metabolism which take place to permit high rates of milk synthesis in the face of negative N balance over-all remain to be fully characterized. Few studies of whole-body or tissue protein metabolism have been performed on ruminants early in lactation (Bryant & Smith 1982;Vincent & Lindsay, 1984;Oddy et al. 1988;Riis, 1988). Some attention has been paid to skeletal muscle (Smith et al. 1981;Bryant & Smith, 1982;Vincent & Lindsay, 1984; MiHican et al. 1987) because it is viewed as a primary site of protein mobilization. At the same time, the nature of any alterations of muscle protein content and metabolism occurring during lactation is not a topic of general agreement amongst these reports.There is little information concerning how the over-all flux of amino acids available for protein synthesis is divided amongst tissues and organs in large animals. Lobley et al.(1 980) characterized the percentage contribution of muscle, carcass, gastrointestinal tract, liver and skin to total protein synthesis in cattle. In lactation the dist...

Sheep were fed ad libitum and killed at 6 and 18 days of lactation; ad libitum-fed nonlactating sheep were killed as controls. Despite increased food intake, lactating ewes were in negative energy balance. Lactation decreased plasma leptin and adipose tissue leptin mRNA concentrations. OB-Rb gene expression, determined by in situ hybridization, was increased in the hypothalamic arcuate nucleus (ARC) and ventromedial hypothalamic nucleus (VMH) at both stages of lactation. Neuropeptide Y (NPY) was increased by lactation in both the ARC and dorsomedial hypothalamus (DMH), although increased gene expression in the DMH was only apparent at day 18 of lactation. Gene expression was decreased for cocaine-and amphetamine-regulated transcript (CART) in the ARC and VMH and for proopiomelanocortin in ARC during lactation. Agouti-related peptide gene expression was increased in the ARC, and melanocortin receptor expression was unchanged in both the ARC and VMH with lactation. Thus the hypoleptinemia of lactation may activate NPY orexigenic pathways and attenuate anorexigenic melanocortin and CART pathways in the hypothalamus to promote the hyperphagia of lactation. hypothalamic neuropeptides; appetite NUTRIENT REQUIREMENTS ARE markedly increased during lactation to meet the demands for milk production (7,70). In most mammals, these additional requirements are met primarily by increasing food intake (7,20,70), but factors regulating the hyperphagia of lactation are not well understood. In addition, in many species, the increased intake is insufficient to meet the metabolic demands, resulting in a state of negative energy balance during early lactation when body lipid reserves are mobilized (7,69). In some species such as the rat, the degree of negative energy balance is usually slight, with animals mobilizing ϳ1 g fat/day (7). Domestic ruminants, however, usually exhibit a greater degree of negative energy balance during early lactation (7, 11), which can impact on their welfare and productivity (28), yet the regulatory mechanisms have not been elucidated.It is postulated that leptin, a peptide hormone secreted by adipose tissue (75), could play a role in the hyperphagia of lactation. Leptin acts on hypothalamic neuronal systems to regulate energy balance and neuroendocrine function; in particular, a reduction in circulating leptin is a potent signal of negative energy balance, activating compensatory orexigenic pathways in the hypothalamus (3-5, 35, 64). Key targets are neurons in the hypothalamic arcuate nucleus (ARC) expressing the signaling form of the leptin receptor (OB-Rb); these are the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AGRP) and the anorexigenic peptides proopiomelanocortin (POMC, precursor for melanocortins) and cocaine-and amphetamine-regulated transcript (CART) (4,5,31,62,64,72). Adipose leptin gene expression and serum leptin concentrations are reported to be reduced during lactation in the monogastric rat in most (16,36,39,56,65,74), but not all (17,19,61), studies. NPY (18,21,...

Body reserves (long-term) and food intake (short-term) both contribute nutritional feedback to the hypothalamus. Reproductive neuroendocrine output (GnRH/LH) is stimulated by increased food intake and not by high adiposity in sheep, but it is unknown whether appetiteregulating hypothalamic neurons show this differential response. Castrated male sheep (Scottish Blackface) with oestradiol implants were studied in two 4 week experiments. In Experiment 1, sheep were fed to maintain the initial body condition (BC) score of 2·0 0·00 (lower BC (LBC), n=7) or 2·9 0·09 (higher BC (HBC), n=9), and liveweight of 43 1·1 and 59 1·6 kg respectively. LBC and HBC sheep had similar mean plasma LH concentration, pulse frequency and amplitude, but HBC animals had higher mean plasma concentrations of insulin (P<0·01), leptin (P<0·01) and glucose (P<0·01). Gene expression (measured by in situ hybridisation) in the hypothalamic arcuate nucleus (ARC) was higher in LBC than HBC sheep for neuropeptide Y (NPY; 486% of HBC, P<0·01), agouti-related peptide (AGRP; 467%, P<0·05) and leptin receptor (OB-Rb; 141%, P<0·05), but lower for cocaine-and amphetamine-regulated transcript (CART; 92%, P<0·05) and similar between groups for pro-opiomelanocortin (POMC). In Experiment 2, sheep with initial mean BC score 2·4 0·03 and liveweight 55 0·8 kg were fed a liveweight-maintenance ration (low intake, LI, n=7) while sheep with initial mean BC score 2·0 0·03 and liveweight 43 1·4 kg were fed freely so that BC score increased to 2·5 0·00 and liveweight increased to 54 1·4 kg (high intake, HI, n=9). Compared with LI, HI sheep had higher mean plasma LH (P<0·05), baseline LH (P<0·01) and pulse amplitude (P<0·01) and showed a trend towards higher pulse frequency. Although there were no differences in final mean plasma concentrations, there were significant increases over time in mean concentrations of insulin (P<0·001), leptin (P<0·05) and glucose (P<0·001) in HI sheep. Gene expression for AGRP in the ARC was higher in HI than LI animals (453% of LI; P<0·05), but expression levels were similar for NPY, OB-Rb, CART and POMC. Thus, the hypothalamus shows differential responses to steady-state adiposity as opposed to an increase in food intake, in terms of both reproductive neuroendocrine activity and hypothalamic appetiteregulating pathways. Differences in hypothalamic gene expression were largely consistent with contemporary levels of systemic leptin and insulin feedback; however, increased nutritional feedback was stimulatory to GnRH/LH whereas constant high feedback was not. The hypothalamus therefore has the ability to retain a nutritional memory that can influence subsequent responses.