Dominique Blache

Relationships between changes in plasma concentrations of leptin before and after parturition and the timing of first post-partum ovulation in high-producing Holstein dairy cows.

During early lactation, dairy cattle are in negative energy balance and the delay to first post-partum ovulation depends on the time taken to recover from this situation. Lactating cows rely heavily on body fat to meet their requirements, leading to the suggestion that leptin, a hormone secreted mainly by adipocytes, is acting as a metabolic signal to sites that control the reproductive axis. The relationship between plasma leptin concentrations and the timing of the first ovulation post partum in 20 high-producing Holstein dairy cows, using a radioimmunoassay based on recombinant bovine leptin was studied. Plasma leptin concentrations declined after parturition, reached a nadir of 0.74 +/- 0.17 ng mL(-1) on 10.1 +/- 2.2 days after parturition (all values are mean +/- SEM). They then increased and became stable near the time of ovulation. Leptin concentrations averaged 1.81 +/- 0.31 ng mL(-1) in the 14 days prepartum, 1.32 +/- 0.21 ng mL(-1) in the post-partum preovulatory period and 1.61 +/- 0.24 ng mL(-1) in the post-ovulatory period. The differences between periods were significant (P<0.01). The interval from parturition to first ovulation averaged 25.9 +/- 2.0 days and was not correlated with the prepartum, preovulatory or post-ovulatory leptin values. However, the interval to first ovulation correlated significantly (r = 0.83 P < 0.0001) with the interval from parturition to the leptin nadir. These results show that plasma concentrations of leptin decrease in dairy cows in the early post-partum period and suggest that a delay in the recovery of leptin secretion increases the delay to the first ovulation.

Long-term alterations in adiposity affect the expression of melanin-concentrating hormone and enkephalin but not proopiomelanocortin in the hypothalamus of ovariectomized ewes.

We have developed a ruminant model to study long-term alterations in adiposity on the expression of appetite-regulating peptides in the hypothalamus. In this model endocrine and metabolic status are fully defined as well as body composition. The current study sought to define the effects of altered adiposity on the expression of genes for neuropeptide Y (NPY), POMC, enkephalin (ENK), and melanin-concentrating hormone (MCH). Ovariectomized ewes with high (60 +/- 1 kg) (FAT) or low (37 +/- 3 kg) body weights (THIN) were blood sampled every 10 min for 8 h to determine metabolic and endocrine status. The animals were then killed and the brains perfused for in situ hybridization. Body composition analysis was performed on the carcass using dual energy x-ray absorptiometry; this indicated that the FAT animals were 36 +/- 1% fat, whereas the THIN animals were 15 +/- 2% fat. The LH interpulse interval was lower and mean GH concentrations were higher in the THIN animals; cortisol and TSH levels were not different between the two groups but free T4 and free T3 levels were lower; the FT3:FT4 ratio was higher in THIN ewes. Levels of insulin, lactate, and nonesterified fatty acids were lower in the THIN group, and plasma glucose and urea concentrations were similar in THIN and FAT animals. Levels of gene expression of NPY and MCH were higher in THIN ewes. POMC expression was similar in the two groups. In the THIN animals, ENK expression was lower in the paraventricular and ventromedial nuclei but higher in the periventricular region. In conclusion, we have shown that alterations in adiposity influence the expression of appetite-regulating peptides in the absence of ovarian steroids. The appetite stimulators, NPY and MCH, appear to be involved in the metabolic response to altered adiposity, whereas ENK in the periventricular region may be linked to the secretion of GH and possibly LH. Our results suggest that altered expression of appetite- regulating peptides can be linked with the endocrine and metabolic adaptations that occur with long-term changes in adiposity.

Expression of orexin receptors in the brain and peripheral tissues of the male sheep

Orexins exert their effects through two specific receptors (OX1R and OX2R) that have been found mainly in the brain and also in peripheral tissues of rats and humans. Here, we demonstrate expression of mRNA encoding for ovine OX1R and OX2R in central and peripheral tissues of sheep. Gene expression for orexin receptors in the hypothalamus and the preoptic area was localised by in situ hybridisation. OX1R was detected in arcuate nuclei (ARC), median eminence (ME), the lateral hypothalamic nuclei and preoptic area (POA) and it was scattered along the third ventricle from the paraventricular (PVN) to the ventromedial hypothalamic nuclei (VMH). OX2R was localised in the PVN, ARC, ME, ventral VMH and a small region of the ventral POA. Gene expression for OX1R and OX2R in central and peripheral tissues was analysed using quantitative real time RT-PCR. Both orexin receptor genes were expressed in the hypothalamus, POA, hippocampus, amygdala, olfactory bulb, pineal gland and recess and pituitary gland, whereas only OX1R mRNA was detected in the testis, kidney and adrenal gland. The expression of the genes for orexin receptors in this range of ovine tissues suggests roles for orexins in multiple physiological functions, with actions at both central and peripheral levels.

Rapid Induction of Cell Proliferation in the Adult Female Ungulate Brain (Ovis aries) Associated with Activation of the Reproductive Axis by Exposure to Unfamiliar Males

Abstract In many species, the reproductive centers of the brain are profoundly affected by sociosexual stimuli. This is particularly evident in female ungulates such as sheep, in which exposure to males switches them from reproductively quiescent to fertile. In two experiments with female sheep, we tested whether the brain centers that control gonadotropin-releasing hormone (GnRH) neuronal activity respond differentially to “novel” vs. familiar males and whether the neuroendocrine response is associated with increased cell proliferation in the hippocampus, a site associated with memory formation. In experiment 1, groups of 10 female sheep that had previously been habituated to males for 3 mo were re-exposed to familiar males or were exposed to novel males. Only the novel males increased luteinizing hormone (LH) pulse frequency, indicating stimulation of GnRH neuronal activity. In experiment 2, groups of six female sheep were injected with bromodeoxyuridine (BrdU) and then maintained in isolation from males or exposed to novel males. Two days later, the hippocampus and hypothalamus were removed and processed for fluorescence immunohistochemistry. Again, exposure to males increased LH pulse frequency. Most important, male exposure also doubled the number of BrdU-positive cells in the dentate gyrus of the hippocampus. No BrdU-positive cells were detected in the hypothalamus. We conclude that the stimulus from novel males switches on the reproductive centers of the brain of female sheep and rapidly doubles the rate of cell proliferation in the hippocampus. The rapidity of this response contrasts with rodents, in which several days of exposure to male pheromones seem necessary for an effect on neurogenesis.

Insulin resistance in divergent strains of Holstein-Friesian dairy cows offered fresh pasture and increasing amounts of concentrate in early lactation.

The objective of this study was to determine whether the physiological response to an intravenous glucose challenge would be affected by genetic strain or concentrate supplementation in grazing Holstein-Friesian cows in early lactation. North American (NA; n = 30) or New Zealand (NZ; n = 30) cows were randomly allocated to 1 of 3 feeding treatments. All cows were offered a generous pasture allowance, and 4 of the 6 groups received either 3 or 6 kg of dry matter (DM)/cow per day of concentrates. During wk 5 of lactation, all cows underwent an intravenous glucose challenge. Cows of NA origin produced more milk than NZ cows, but there was no significant strain effect on milk fat or protein yield. Milk yield and the yield of individual components increased with increasing level of concentrate eaten, but there were no significant strain x diet interactions. During wk 1 to 6, mean body weight and body condition score decreased in all treatments. Average body weight was greater in NA cows, but body condition score was greater for NZ cows. There was no strain or diet effect on the length of the postpartum anovulatory interval, with cows ovulating before 40 d postpartum on average. Glucose fractional turnover rate was greater in NZ cows compared with those of NA origin and in all cows receiving 6 kg of DM concentrates, indicating a less severe insulin resistance in those treatments. Consistent with this, the time taken to dispose of half the peak glucose concentration was less when 6 kg of DM concentrate was fed, and tended to be less in NZ than in NA cows. There was no effect of genetic strain on glucose area under the curve (AUC) at 60 or 120 min, but AUC at both time points was less in cows receiving 6 kg of DM concentrates per day. Neither genetic strain nor nutrition affected basal or peak insulin concentrations, insulin increment, or insulin AUC, and there were no strain x diet interactions for any of the glucose challenge response variables measured. In conclusion, differences in milk production between NA and NZ cows in early lactation can, at least in part, be explained by the greater degree of insulin resistance in the NA cows, and this insulin resistance can be overcome by supplementing grazing cows with 6 kg of DM concentrates.

Changes in insulin, glucose and ketone bodies, but not leptin or body fat content precede restoration of luteinising hormone secretion in ewes.

The reproductive system, including pulsatile luteinising hormone (LH) secretion, is inhibited by deficits in energy availability and restored by energy surfeits. Plasma LH, insulin, leptin, ghrelin, glucose, ketone body, and nonesterified fatty acid concentrations were measured in ovariectomised, food‐restricted ewes before and after return to ad libitum feeding to determine the factors that change in time to account for the restoration of pulsatile LH secretion. At 07.00 h, blood was sampled every 10 min for 5 h from ovariectomised, hypogonadotrophic, chronically food‐restricted and ad libitum‐fed ewes (Fed). At 12.00 h, four of the food‐restricted sheep were given ad libitum access to food (Re‐Fed), while three ewes continued to be food restricted (Restricted). Sampling continued for 5 h and resumed again on the mornings of days 2, 4, and 9. A pulse of LH was seen within 1 h of re‐feeding in all Re‐Fed ewes, and interpulse interval (IPI) was significantly shorter in Re‐Fed compared to Restricted ewes and longer than in Fed ewes during the period after re‐feeding. Re‐Fed LH IPI was not restored to that of Fed ewes until sometime between days 4 and 9. The first pulse occurred within minutes, whereas restoration of IPI occurred after 4–8 days. Prior to the initial LH pulses seen in Re‐Fed ewes, plasma ketone bodies first fell and then rose to levels significantly above those in Restricted ewes. Significant changes in circulating insulin, ghrelin, glucose, and total ketone body concentrations, daily food intake and lean body mass preceded restoration of Re‐Fed LH IPI some time between days 4 and 9, but there were no significant changes in adiposity or circulating leptin concentrations, consistent with the hypothesis that LH pulses are reinitiated by changes in the availability of oxidisable metabolic fuels and possibly insulin, but not leptin concentrations.

Leptin‐Mediated Effects of Undernutrition or Fasting on Luteinizing Hormone and Growth Hormone Secretion in Ovariectomized Ewes Depend on the Duration of Metabolic Perturbation

We aimed to determine the importance of leptin in the regulation of luteinizing hormone (LH) and growth hormone (GH) secretion in ovariectomized (OVX) ewes. Lean and fat sheep were produced by dietary manipulation over 8 months and were then fasted for 32 h. Plasma concentrations of glucose, insulin and leptin were higher in the fat group. Fasting decreased plasma concentrations of glucose and insulin and increased concentrations of nonesterified fatty acids (NEFA) in fat and lean ewes, but leptin concentrations were reduced in the fat group only. Plasma GH concentrations were higher in the lean group and LH concentrations were lower; there was no effect of fasting. These data suggested that long‐term changes in plasma leptin concentrations might affect LH and GH secretion, but acute changes with fasting had no effect. OVX ewes of normal body weight were fasted for 72 h with or without intracerebroventricular (i.c.v.) infusion of leptin (4 µg/h), achieving similar metabolic effects to the 32 h fast. The 72‐h fast increased LH pulse amplitude, mean GH and cortisol concentrations, but these changes were corrected towards normal by leptin treatment. Thus, leptin could attenuate fasting‐induced alterations in the secretion of LH, GH and cortisol. Finally, we food‐restricted OVX ewes for 4 months (lean), leading to a 20‐kg reduction in body weight. Plasma concentrations of leptin and insulin were decreased, and plasma GH concentrations increased, but there was no effect on plasma concentrations of LH, glucose or NEFA. Icv infusion of leptin did not affect any endocrine or metabolic parameter in these ewes. In summary, maintenance of a lean or fat condition for a prolonged period (8 months) or an extended fasting (72 h) can affect LH and GH secretion, but short‐term food restriction (4 months) affected only GH secretion and short‐term fasting (32 h) had no effect on either LH or GH secretion. This is in spite of altered plasma leptin concentrations in all circumstances studied. Although leptin treatment can restore plasma concentrations of LH, GH and cortisol towards normal in sheep fasted for 72 h, some other factor(s) must signal to the brain to cause shifts in neuroendocrine function in other conditions where nutritional/metabolic status is altered.

Review of sheep body condition score in relation to production characteristics

Body condition scoring of sheep was first developed as a technique in the 1960s. Unlike live weight, it circumvents the issues of skeletal size, breed and physiological state and is not influenced by gut fill or the length and wetness of the fleece. This review outlines the use of the technique and the relationships between body condition score and other physical measures. In addition, it summarises the literature, across a range of breeds and environments, on the effects of body condition score on reproductive and lactational performance, and the growth and survival of the offspring to weaning. We have proposed that while the relationship between body condition and production traits is positive, it is unlikely to be linear. Where appropriate, the review outlines areas that would benefit from further research. Finally, it outlines what a suitable body condition score profile might be for a ewe over the entire breeding cycle.

Role of leptin in the regulation of growth and carbohydrate metabolism in the ovine fetus during late gestation

Leptin is an important regulator of appetite and energy expenditure in adulthood, although its role as a nutritional signal in the control of growth and metabolism before birth is poorly understood. This study investigated the effects of leptin on growth, carbohydrate metabolism and insulin signalling in fetal sheep. Crown–rump length‐measuring devices and vascular catheters were implanted in 12 sheep fetuses at 105–110 days of gestation (term 145 ± 2 days). The fetuses were infused i.v. either with saline (0.9% NaCl; n= 6) or recombinant ovine leptin (0.5–1.0 mg kg−1 day−1; n= 6) for 5 days from 125 to 130 days when they were humanely killed and tissues collected. Leptin receptor mRNA and protein were expressed in fetal liver, skeletal muscle and perirenal adipose tissue. Throughout infusion, plasma leptin in the leptin‐infused fetuses was 3‐ to 5‐fold higher than in the saline‐infused fetuses, although plasma concentrations of insulin, glucose, lactate, cortisol, catecholamines and thyroid hormones did not differ between the groups. Leptin infusion did not affect linear skeletal growth or body, placental and organ weights in utero. Hepatic glycogen content and activities of the gluconeogenic enzymes glucose‐6‐phosphatase and phosphoenolpyruvate carboxykinase in the leptin‐infused fetuses were lower than in the saline‐infused fetuses by 44, 48 and 36%, respectively; however, there were no differences in hepatic glycogen synthase activity or insulin signalling protein levels. Therefore, before birth, leptin may inhibit endogenous glucose production by the fetal liver when adipose energy stores and transplacental nutrient delivery are sufficient for the metabolic needs of the fetus. These actions of leptin in utero may contribute to the development of neonatal hypoglycaemia in macrosomic babies of diabetic mothers.

Decrease in voluntary feed intake and pulsatile luteinizing hormone secretion after intracerebroventricular infusion of recombinant bovine leptin in mature male sheep.

The aim of the present study was to determine whether leptin might play a role in the gonadotrophic response of mature merino rams to changes in the level of nutrition in rams fed ad libitum. Recombinant bovine leptin was infused intracerebroventricularly and voluntary food intake (VFI) and luteinizing hormone (LH) pulse frequency were measured. In Experiment 1, rams (n = 5) were infused for 24 h per day for 5 days with vehicle or with leptin (0.04, 0.4 and 4.0 microg h(-1)). All doses decreased both VFI and LH pulse frequency. In Experiment 2, rams were infused for 24 h per day for 5 days with vehicle (n = 10) or leptin (4 pg h(-1); n = 5); a sub-group of 5 controls was pair-fed to the leptin-infused group to control for effects of changes in feed intake. LH pulse frequency was reduced equally in both the leptin-infused and pair-fed groups. Leptin did not affect other systems controlled by the hypothalamic-pituitary axis. Thus, rather than stimulate LH secretion, intracerebral leptin specifically inhibits it by reducing food intake, so it is unlikely that effects of nutrition on the reproductive axis in mature rams involves leptin as a single blood-borne signal. A range of nutritional or metabolic inputs may be needed, and perhaps interconnections between neural centres that control appetite and reproduction.