Twenty-four-hour profiles of metabolic and stress hormones in sheep selected for a calm or nervous temperament.

Even in the absence of stressors, temperament is associated with changes in the concentration of stress-responsive hormones and, possibly because of such changes, temperament can affect metabolism. We tested whether, in sheep bred for temperament for 14 generations, "nervous" females have greater concentrations of stress-responsive hormones in the absence of stressors than "calm" females, and whether these differences are associated with changes in the concentrations of metabolic hormones. In resting "calm" (n = 8) and "nervous" (n = 8) sheep, concentrations of cortisol, prolactin, leptin, and insulin were measured in blood plasma sampled via jugular catheter every 20 min for 24 h. The animals were individually penned, habituated to their housing and human handling over 7 wk, and fed before sampling began. Diurnal variation was evident for all hormones, but a 24-h cortisol pattern was detected in only 7 individuals. There was no effect of temperament on any aspect of concentrations of cortisol or prolactin, but "calm" animals had greater concentrations of insulin in the early afternoon than "nervous" animals (14.5 ± 1.1 vs 10.0 ± 1.6 µU/mL; P = 0.038), and a similar tendency was seen for leptin (P = 0.092). We conclude that selection for temperament affects the concentration of metabolic hormones in the absence of stressors, but this effect is independent of stress-responsive hormones.

Reproductive hormonal patterns in pregnant, pseudopregnant and acyclic captive African wild dogs (Lycaon pictus).

African wild dogs are one of the most endangered canid species, with free-living populations declining as a consequence of habitat loss, disease and human conflict. Captive breeding is considered an important conservation strategy, but is hampered by a poor overall understanding of the reproductive biology of the species. To improve our basic knowledge, we studied hormone patterns in 15 female wild dogs using non-invasive faecal collections. By comparing longitudinal hormone profiles with behavioural and anatomical changes, females could be allocated among three reproductive classes: pregnant (n=1), pseudopregnant (n=9) and acyclic (n=4). We also monitored a single female in which contraception was induced with a deslorelin implant. Comparison of pseudopregnant and acyclic females showed that, in both classes, faecal oestradiol concentrations increased from anoestrus to pro-oestrus then declined into the oestrous and dioestrous phases. Progestagen concentrations rose steadily from anoestrus to the dioestrous phase in both pseudopregnant and acyclic females and, pseudopregnant females had significantly higher concentrations of progestagens than acyclic females in all phases of the oestrous cycle. Most females classed as pseudopregnant were found in female-only groups, suggesting that wild dogs are spontaneous ovulators. Furthermore, only one adult female did not ovulate, so suppression of reproduction in subordinates is likely to be behavioural rather than physiological.

The use of a 'first-wave' model to study the effect of nutrition on ovarian follicular dynamics and ovulation rate in the sheep.

We have developed an experimental model in which groups of ewes are simultaneously experiencing the first ovarian follicular wave of their oestrous cycle. We used this 'first-wave model' in a 2 × 2 factorial experiment (ten ewes per group) to study the effect of body condition (BC) and a short-term supplement on follicular dynamics and ovulation rate. The 'first-wave' was established by giving ewes three injections of prostaglandin (PG), 7 days apart. The 6-day supplement (lupin grain) began 2 days after the second PG injection and continued until the third. Follicles were studied by ultrasound, and blood was sampled to measure glucose and hormones. The supplement increased (P<0.01) the concentrations of glucose, insulin and leptin, decreased FSH concentrations (P<0.01) and tended to increase oestradiol concentrations (P=0.06). The supplement tended to increase the number of 3 mm follicles (P=0.06). Compared with low-BC ewes, high-BC ewes had more follicular waves (P<0.05), higher concentrations of insulin, leptin and IGF1 (P<0.05) and tended to have higher FSH concentrations (P=0.09). Leptin and insulin concentrations remained high until the end of supplementation in high-BC ewes, whereas they decreased after the third day of supplementation in low-BC ewes. In conclusion, high concentrations of metabolic hormones in fat ewes are associated with the development of more follicular waves. When a supplement is superimposed on this situation, changes in glucose and metabolic hormones allow more follicles to be selected to ovulate.

Dose-response studies for pituitary and testicular function in male dogs treated with the GnRH superagonist, deslorelin.

We tested the effect of dose of GnRH superagonist on pituitary and testicular function in a study with four groups of four male dogs. The Controls received blank implants and the other three groups received implants containing 3, 6 or 12 mg deslorelin (D-Trp6-Pro9-des-Gly10-GnRH ethylamide). In all deslorelin-treated groups, there was initially an acute increase in plasma concentrations of LH and testosterone, followed by declines such that both hormones became undetectable after approximately 12 days. There was a dose-response in some of these early aspects of the hormone profiles. With respect to long-term effects of treatment, the 12-mg dose had significantly greater effects than the smaller doses for the duration of minimum testicular volume [366 +/- 77, mean +/- SEM (3 mg), 472 +/- 74 (6 mg), and 634 +/- 59 (12 mg) days], absence of ejaculate [416 +/- 88 (3 mg), 476 +/- 83 (6 mg), and 644 +/- 67 (12 mg) days], undetectable plasma concentrations of LH and testosterone [367 +/- 64 (3 mg), 419 +/- 72 (6 mg), and 607 +/- 69 (12 mg) days], the delay until complete recovery of LH and testosterone secretion [394 +/- 65 (3 mg), 484 +/- 72 (6 mg) and 668 +/- 47 (12 mg) days], and the delay until testes had regrown to normal volume [408 +/- 77 (3 mg), 514 +/- 74 (6 mg), 676 +/- 59 (12 mg) days]. The time taken to restore full ejaculates was also longest for the 12-mg dose: 716 +/- 67 (12 mg) days vs 440 +/- 66 (3 mg) and 538 +/- 83 (6 mg) days after implantation. There was no correlation between delay to recovery of normal ejaculate quality and body mass. We conclude that the dose-response relationship with deslorelin implants is not expressed with respect to the degree of suppression of reproduction, but on the maximum duration of suppression and thus to delay until recovery.

Reproductive capacity of Merino ewes fed a high-salt diet.

An option to increase the productivity of saline land is to graze sheep on salt-tolerant plants, which, during the summer/autumn period, can contain 20% to 25% of their dry matter as salt. This study assessed the impact of coping with high dietary salt loads on the reproductive performance of grazing ewes. From the time of artificial insemination until parturition, 2-year-old maiden Merino ewes were fed either a high-salt diet (NaCl 13% of dry matter) or control diet (NaCl 0.5% of dry matter). Pregnancy rates, lamb birth weights, milk composition and the plasma concentrations of hormones related to salt and water balance, and energy metabolism were measured. Leptin and insulin concentrations were lower (1.4 ± 0.09 v. 1.5 ± 0.12 ng/ml; (P < 0.05) and 7.2 ± 0.55 v. 8.2 ± 0.83 ng/ml; P < 0.02) in response to high-salt ingestion as was aldosterone concentration (27 ± 2.7 v. 49 ± 5.4 pg/ml; P < 0.05), presumably to achieve salt and water homeostasis. Arginine vasopressin concentration was not significantly affected by the diets, but plasma concentration of T3 differed during gestation (P < 0.02), resulting in lower concentrations in the high-salt group in the first third of gestation (1.2 ± 0.18 v. 1.3 ± 0.14 pmol/ml) and higher concentrations in the final third of gestation (0.8 ± 0.16 v. 0.6 ± 0.06 pmol/ml). T4 concentration was lower in ewes ingesting high salt for the first two-thirds of pregnancy (162 ± 8.6 v. 212 ± 13 ng/ml; P < 0.001). No substantial effects of high salt ingestion on pregnancy rates, lamb birth weights or milk composition were detected.

Pituitary and testicular endocrine responses to exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone in male dogs treated with GnRH agonist implants.

The present study tested whether exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) can stimulate LH and testosterone secretion in dogs chronically treated with a GnRH superagonist. Twenty male adult dogs were assigned to a completely randomised design comprising five groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other four groups received a 6-mg implant containing a slow-release formulation of deslorelin (d-Trp6-Pro9-des-Gly10-LH-releasing hormone ethylamide). The same four control dogs were used for all hormonal challenges, whereas a different deslorelin-implanted group was used for each challenge. Native GnRH (5 microg kg(-1) bodyweight, i.v.) was injected on Days 15, 25, 40 and 100 after implantation, whereas bovine LH (0.5 microg kg(-1) bodyweight, i.v.) was injected on Days 16, 26, 41 and 101. On all occasions after Day 25-26 postimplantation, exogenous GnRH and LH elicited higher plasma concentrations of LH and testosterone in control than deslorelin-treated animals (P < 0.05). It was concluded that, in male dogs, implantation of a GnRH superagonist desensitised the pituitary gonadotrophs to GnRH and also led to a desensitisation of the Leydig cells to LH. This explains, at least in part, the profound reduction in the production of androgen and spermatozoa in deslorelin-treated male dogs.

Intracerebroventricular infusion of leptin into mature merino rams of different metabolic status: effects on blood concentrations of glucose and reproductive and metabolic hormones.

In mature Merino rams, nutrition is one of the external cues that most strongly affects the reproductive centres of the preoptic-hypothalamic continuum. The signalling pathways that link dietary status and the activity of the neurones that produce gonadotrophin-releasing hormone signals are thought to be partly hormonal in nature to reflect the amount of body reserves. Among the hormones thought to be involved are insulin and leptin. This study tested whether recombinant bovine leptin infused (0.4 microg/h) into the third cerebral ventricle would stimulate pulsatile luteinizing hormone (LH) secretion in mature Merino rams when their energy status was low or decreasing, during both chronic (fasting) and acute reductions of energy balance. Leptin may interact with other hormones that depend on energy availability, so we also monitored changes in circulating concentrations of insulin, thyroid hormones, growth hormone, prolactin and adrenocorticotrophin. Overall, our data do not support this hypothesis. The dietary regimes induced clear responses in the metabolic profiles of the animals but there was no clear effect of central leptin administration on LH pulse frequency. The relationships between the hormonal systems measured in the present study add weight to the contention that leptin plays only a permissive role in the nutritional control of the reproductive axis and that other hormonal signals (particularly insulin) or pathways are acting in concert with leptin to stimulate the reproductive axis.

Clarification of emu serum for peptide hormone assay using polyethylene glycol precipitation.

Interference in radioimmunoassays (RIAs) was frequently encountered during endocrinological studies of the emu (Dromaius novaehollandiae). Interference was greatest when serum was cloudy or opaque. Such samples appeared seasonally, in spring and summer during the phase of fat deposition, and in the winter when females were laying. These poor quality samples did not allow accurate measurement by RIAs of several peptide hormones for a full year. To prepare them for assay, these sera were clarified using a polyethylene glycol (PEG) solution at a final concentration of 7.5%. This treatment was effective in most cases. After treatment with the PEG, recoveries of LH, glucagon, and prolactin were greater than 75% and that for insulin was 40%. Regardless of the level of recovery, there was a high correlation of assay results between non-opaque native and PEG-treated sera. Serum samples containing large amounts of interfering high molecular weight components, such as lipoproteins, can be clarified with PEG, enabling their accurate measurement by RIA.

Reproduction and plasma concentrations of leptin, insulin and insulin-like growth factor 1 in growth-hormone-transgenic female sheep before and after artificial insemination.

The transgenic sheep used in this study expressed an additional copy of the gene for ovine growth hormone (GH), so they had continuously high plasma concentrations of GH. They were used to test whether the GH transgene affected plasma concentrations of the metabolic hormones leptin, insulin-like growth factor 1 (IGF-1) and insulin, and whether these effects were associated with changes in conception, pregnancy or parturition following artificial insemination. Compared with control animals, the GH-transgenic sheep had higher bodyweight, lower body condition score and less subcutaneous fat (P < 0.05). These sheep also had lower plasma concentrations of leptin, higher plasma concentrations of insulin, and higher plasma concentrations of IGF-1 (P < 0.001). A similar proportion of GH-transgenic and control ewes came into oestrus, but the conception rate to artificial insemination was lower in GH-transgenic ewes than in the controls. Only four live lambs were recovered from 12 GH-transgenic ewes (33%) compared with 38 lambs from 43 controls (88%). This outcome was not associated with any difference in plasma progesterone profile in the period leading up to artificial insemination (Day 0). The GH-transgenic ewes had lower concentrations of FSH at all times measured (Day -19, Day -2 and Day 19). These results indicate that appropriate regulation of GH secretion from pituitary or peripheral tissues is necessary for normal reproduction and normal levels of metabolic hormones. Chronically high concentrations of GH were associated with increased levels of IGF-1 and insulin, and decreased levels of leptin.

Relationships between plasma concentrations of leptin and other metabolic hormones in GH-transgenic sheep infused with glucose.

To study the regulation of leptin secretion in sheep, we infused glucose (0.32 g/h/kg for 12 h) into GH-transgenic animals (n = 8) that have chronically high plasma concentrations of ovine GH and insulin, but low body condition and low plasma leptin concentrations, and compared the responses with those in controls (n = 8). In both groups, the infusion increased plasma concentrations of glucose and insulin within 1 h and maintained high levels throughout the infusion period (P < 0.0001). Compared with controls, GH-transgenics had higher concentrations of insulin, IGF-1, GH (all P < 0.0001) and cortisol (P < 0.05), but lower GH pulse frequency (P < 0.0001). Overall, leptin concentrations were lower in GH-transgenics than in controls (P < 0.01). A postprandial increase in leptin concentrations was observed in both groups, independently of glucose treatment, after which the values remained elevated in animals infused with glucose, but returned to basal levels in those infused with saline, independently of transgene status. In both GH-transgenics and controls, glucose infusion did not affect the concentrations of GH, IGF-1, or cortisol. In conclusion, GH-transgenic and control sheep show similar responses to glucose infusion for leptin and other metabolic hormones, despite differences between them in body condition and basal levels of these hormones. Glucose, insulin, GH, IGF-1 and cortisol are probably not major factors in the acute control of leptin secretion in sheep, although sustained high concentrations of GH and IGF-1 might reduce adipose tissue mass or inhibit leptin gene expression.

Use of a new drug delivery formulation of the gonadotrophin-releasing hormone analogue Deslorelin for reversible long-term contraception in male dogs.

In the present study, we tested the effect of treatment with a slow-release implant containing the gonadotrophin-releasing hormone agonist Deslorelin(TM) (Peptech Animal Health Australia, North Ryde, NSW, Australia) on pituitary and testicular function in mature male dogs. Four dogs were treated with Deslorelin (6-mg implant) and four were used as controls (blank implant). In control dogs, there were no significant changes over the 12 months of the study in plasma concentrations of luteinising hormone (LH) or testosterone, or in testicular volume, semen output or semen quality. In Deslorelin-treated dogs, plasma concentrations of LH and testosterone were undetectable after 21 and 27 days, testicular volume fell to 35% of pretreatment values after 14 weeks and no ejaculates could be obtained after 6 weeks. Concentrations returned to the detectable range for testosterone after 44 weeks and for LH after 51 weeks and both were within the normal range after 52 weeks. Semen characteristics had recovered completely by 60 weeks after implantation. At this time, the testes and prostate glands were similar histologically to those of control dogs. We conclude that a single slow-release implant containing 6 mg Deslorelin has potential as a long-term, reversible antifertility agent for male dogs.

Effect of nutritional supplementation on quantities of glucose transporters 1 and 4 in sheep granulosa and theca cells.

The stimulatory effect of nutritional supplementation on ovarian activity in sheep has been linked to an increase in glucose availability that, with insulin, directly decreases follicular steroidogenesis. Glucose uptake occurs by glucose transporters, but it is not known which glucose transporters are present in the sheep ovary or whether they are affected by nutritional stimulation. The aim of this study was to determine whether widely distributed glucose transporter 1 (GLUT1) or insulin-responsive GLUT4 are present in the granulosa or theca cells of sheep ovarian follicles, and whether their concentrations are affected by nutritional stimulation. Merino ewes (n = 49-51 per group) were stimulated nutritionally for 5 days before luteolysis with lupin grain or with one of two regimens of a glucogenic mixture, administered orally, which increases blood glucose concentrations towards the upper end of the normal range. Water was used as a control. Ovaries (n = 3 per group) were dissected and the granulosa cells and thecal shell from individual follicles were examined for glucose transporters using western blotting. GLUT1 concentration was 7-18 times higher in the granulosa than in the theca cells. GLUT4 was detected at a similar concentration in both types of cell. Nutritional treatment had no effect on the concentration of GLUT1 or GLUT4 in either tissue, and did not increase ovulation rate, despite increased concentrations of glucose and insulin. Concentrations of glucose transporters were not correlated with follicular concentrations of oestradiol or androstenedione. The presence of GLUT1 and GLUT4 in the granulosa and theca of sheep follicles indicates that the transporters have a role within the ovary in the modulation of follicular function.

Photoperiodic control of the concentration of luteinizing hormone, prolactin and testosterone in the male emu (Dromaius novaehollandiae), a bird that breeds on short days.

The objective of this study was to establish, for a short-day breeding bird, the male emu, whether the breeding season is principally controlled by changes in photoperiod, and to investigate the endocrine mechanisms involved. Two groups of adult males were subjected to three alternating periods of 150-185 days of 14 h light/day (LD) and 10 h light/day (SD) terminating in a 360-day period of LD or SD. Transfer from LD to SD led to increases in plasma concentrations of luteinizing hormone (LH) and testosterone, after 82 +/- 8 and 73 +/- 3 (SEM) days, and an increase in prolactin concentrations after 115 +/- 12 days. Concentrations of LH and testosterone began to decrease before transfer back to LD, at a time when prolactin concentrations were approaching peak values. Transfer from LD to 360 days of SD resulted in increases in LH and testosterone concentrations, and these terminated after an increase in prolactin concentrations. After transfer from SD to 360 days of LD, plasma concentrations of LH and testosterone began to increase, after delays of 222 +/- 24 and 225 +/- 13 days, and were high at the end of the study, while prolactin values remained depressed throughout. These observations clearly show that seasonal breeding in the emu is directly controlled by changes in photoperiod. The dynamics of the hormonal responses to change of photoperiod suggest that, despite being short-day breeders, the photoregulation of breeding in emus involves mechanisms that are currently accepted for birds, rather than mechanisms that have been proposed for short-day breeding mammals. The initiation of breeding in emus is due to dissipation of photorefractoriness by short days which leads to an increase in the secretion of gonadotrophins to levels that are sufficient to support full reproductive condition. The termination of breeding, while days are still short, is due to the antigonadotrophic action of prolactin which, unusually for birds, increases while the days are still short. In conclusion, breeding activity in male emus is strongly controlled by photoperiod. Emus are short-day breeders, but the central mechanisms that regulate the secretion of reproductive hormones seem to be similar to those previously proposed for long-day breeding birds. The pattern of prolactin secretion in emus suggests an important role for this hormone in the termination of the breeding cycle.

Plasma thyroid hormones and growth hormone in embryonic and growing emus (Dromaius novaehollandiae).

Growth hormone (GH), thyroxine (T4) and tri-iodothyronine (T3) are known to be involved in the regulation of growth and development in a variety of avian species. It has been suggested that an absence of GH and thyroid hormones in ostriches is the cause of their neoteny, a phenomenon in which juvenile characteristics are retained into adulthood. Neoteny is typical of all ratites, the single group of flightless birds that includes the ostrich, but similar endocrine studies have not been performed for other members of the group, such as the emu. To test the neoteny hypothesis further, in the present study we measured the plasma concentrations of T4, T3 and GH in emus during embryonic development and from hatching to 1 year of age. Concentrations of T4 and GH increased during the last weeks of incubation, whereas concentrations of T3 were highly variable. After hatching, the concentrations of both thyroid hormones were high during the first 3 days of life and then fell to a constant low level. Plasma concentrations of GH were high at the time of hatching and decreased gradually over the first 22 weeks of age; thereafter, the concentrations of GH were highly variable. No correlation was observed between hormone concentrations and live weight at any time. These results support the hypothesis that thyroid function is abnormally low in ratites, whereas patterns of GH secretion are similar to those observed in other birds. Dysfunction of the thyroid axis could explain, in part, the neotenous physical aspect of adult emus.

Metabolic factors affecting the reproductive axis in male sheep.

Changes in food intake affect the reproductive axis in both sexes, and the nutritional signals involved and the sites that receive those signals are now beginning to be unravelled. Our studies have focussed on the mature male sheep, a model in which high food intake stimulates GnRH-LH pulse frequency for only 10-20 days but continues to promote testicular growth over several months. Different signals and different target organs seem to be responsible for these short- and long-term responses. Short-term dietary treatments lead to changes in blood concentrations of glucose, fatty acids, insulin and leptin, and concentrations of glucose, insulin, leptin and some amino acids in cerebrospinal fluid. It seems unlikely that amino acids affect GnRH-LH secretion directly in sheep. Intracerebroventricular infusions of insulin specifically increase LH pulse frequency, but intravenous, intra-abomasal or intracerebroventricular infusions of glucose have no effect, despite their effects on cerebrospinal fluid insulin concentrations. The addition of fatty acids to the diet also increases LH pulse frequency, but does not affect the concentrations of insulin or leptin in the cerebrospinal fluid. It appears that acute responses to changes in nutrition involve a range of alternative pathways, possibly including interactions among insulin, leptin and energy substrates. Effects of long-term dietary treatments on testicular size are only partly dependent on the GnRH-LH system (that is, on brain control) and so must also depend on other, as yet unknown, pathways. Concepts of 'metabolic sensing and integration' are being developed from the basis of existing knowledge of the central control of appetite and reproduction.

Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep.

In mature male sheep, the level of nutrition acutely influences the secretion of reproductive hormones. The mechanism involved is not fully understood but findings in humans and laboratory rodents would suggest a major role for leptin that is secreted from adipose tissue and then travels via the circulation to the central nervous system. Before we can begin to test this hypothesis, we need to be able to measure leptin concentrations in blood plasma and cerebrospinal fluid. We have therefore developed a radioimmunoassay using antibodies raised against biologically active recombinant bovine-ovine leptin. Using this assay, we found that plasma concentrations of leptin were highly correlated to back-fat thickness and to the ratio of back-fat thickness to liveweight, in female and castrated male sheep. Plasma concentrations of leptin were higher in female sheep than in castrated or intact male sheep. Serial samples (every 5 min) suggested that the secretion of leptin in male sheep is episodic but it does not appear to show clear pulsatility, increases post-prandially, or a diurnal rhythm. Leptin concentrations in both plasma and cerebrospinal fluid increased within 5 days in male sheep fed a diet with a high content of energy and protein that also stimulates the secretion of LH pulses. These data suggest that in sheep, as in other species, leptin production is correlated with the mass of adipose tissue and that the hormone passes from the circulation to the cerebrospinal fluid and then to hypothalamic sites. There, it may affect appetite and perhaps GnRH secretion. The role of leptin in the link between nutrition and reproduction needs further investigation.

Determinants of the annual pattern of reproduction in mature male merino and Suffolk sheep: modification of endogenous rhythms by photoperiod.

In mediterranean environments, pastures are very poor during the autumn and consequently small ruminants, such as sheep, would have been losing body mass for many months so, during mating, gamete production would be depressed in both sexes. Effectively, the nutritive requirements for a photoperiod-driven, annual reproductive cycle are out of phase with seasonal changes in food availability. The problem could be overcome through more flexible timing of reproduction, perhaps explaining variations in seasonality between breeds that originate from differing latitudes. To study these concepts and the mechanisms involved, the endogenous rhythms and responses to photoperiod were compared in rams of 'mediterranean origin' (Merino) and 'temperate origin' (Suffolk). Groups of 16 rams of each breed were given a constant food supply and subjected to 16 months of constant equinoctial photoperiod (12L : 12D) or simulated 'mediterranean' changes in daylength (from 14L : 10D to 14D : 10L). With nutritional and photoperiodic inputs held constant, Merino and Suffolk rams showed similar endogenous rhythms in reproductive activity. Under constant nutritional inputs and a mediterranean photoperiodic cycle, the endogenous rhythms were modified differently in the two breeds, with the Merinos starting and finishing their seasons about 2 months earlier than the Suffolks. These observations partially explain the patterns observed in rams kept under field conditions. It is now necessary to test whether the rhythms of reproduction in these breeds are also modified by changes in nutrition and social cues.

Role of peripheral and central aromatization in the control of gonadotrophin secretion in the male sheep.

Both testosterone and its aromatized metabolite, oestradiol-17beta, are known to act centrally on the secretion of GnRH, but the major site of aromatization is not clear as aromatase activities are found in numerous tissues including brain and testis. Here, we tested the importance of central aromatization of testosterone using a non-steroidal aromatase inhibitor, fadrozole. To distinguish between testicular and non-testicular sites, five intact and five testosterone-infused castrated rams (600 microg kg(-1) per 24 h for 3 days) were given four injections of fadrozole (i.m.; 500 microg kg(-1)) at 48, 52, 64 and 68 h relative to the start of testosterone infusion. Control rams (n = 5) received vehicle only. Fadrozole treatment decreased plasma oestradiol-17beta concentrations and increased the LH pulse frequency in both intact rams and testosterone-treated castrates, suggesting that non-testicular sites of aromatization are important in the control of pulsatile LH secretion. To test the importance of central aromatization, intact rams (n = 5) were infused into the third ventricle with vehicle (artificial cerebrospinal fluid) or with fadrozole (20 and 200 microg kg(-1) per day). After two weeks, the same two doses of fadrozole were infused intravenously instead of intracerebrally. Central infusion of fadrozole did not affect plasma oestradiol concentrations but increased LH pulse frequency. Only the highest dose increased LH pulse frequency when infused intravenously. In conclusion, central aromatization is involved in the control of pulsatile LH secretion in male sheep.

Morphometric and endocrine analyses of the effects of nutrition on the testis of mature Merino rams.

The effects of nutrition on the testis were investigated in groups of five mature Merino rams that were fed either a sub-maintenance (low) diet or a supra-maintenance (high) diet for 69 days. Testosterone, oestradiol and inhibin were measured in blood plasma sampled simultaneously from jugular and testicular veins after an i.v. injection of 200 ng ovine LH kg-1. Plasma concentrations of testosterone, inhibin and oestradiol were higher in testicular than in jugular vein plasma for both diets (P < 0.01). After the LH injection, jugular plasma testosterone increased more rapidly (P < 0.01) in rams fed the high diet than in rams fed the low diet. This was not seen in the testicular vein. Oestradiol concentrations were higher in rams on the high diet than in those on the low diet, in both the testicular (P < 0.0001) and the jugular vein (P < 0.02). Diet did not affect inhibin concentrations. Testes were surgically removed and processed for light microscopy. Testicular mass and seminiferous tubule length and diameter were higher with the high diet than the low diet (P < 0.01). The number of Sertoli cell nuclei per testis was also affected (high diet: 120 +/- 6 x 10(8); low diet: 77 +/- 7 x 10(8); P < 0.001), whereas the proportion of testis occupied by Sertoli cell nuclei was not affected. The number of Leydig cells per testis was not affected by diet, but Leydig cells occupied a greater volume of testis in rams on the high diet than in those on the low diet (P < 0.001). The effects of nutrition on Leydig and Sertoli cells are consistent with changes in the endocrine and exocrine functions of the testis. The finding that Sertoli cell population was altered in adult rams may be explained by the GnRH-independent effects of nutrition.

Level of nutrition modulates the dynamics of oestradiol feedback on plasma FSH in ovariectomized ewes.

The frequency of multiple ovulations in mature, cyclic ewes is strongly influenced by the level of nutrition, but it is difficult to demonstrate concurrent changes in plasma concentrations of gonadotropins. The failure to do so might be a consequence of rapid compensation by the homeostatic feedback mechanism linking secretion by the hypothalamus/pituitary gland and ovarian hormones. Most experimental models have examined the components of the homeostatic feedback system after steady state relationships had been established. We hypothesised that the effects of nutrition might be observed more readily if the system were disrupted and then examined while equilibrium was being re-established. This hypothesis was tested in three experiments in Merino ewes by allowing gonadotropin secretion to escape feedback for 5 days after ovariectomy and then replacing ovarian hormones and examining effects of feeding regimen on the return of plasma concentrations of FSH to baseline values. In all three experiments, oestrogen replacement caused plasma concentrations of FSH to decline more rapidly (P < 0.05) in ewes fed at 0.5x maintenance, than in ewes fed at 1.4x maintenance, with groups fed at maintenance being intermediate. No effect of diet was observed on plasma FSH concentrations in the absence of oestradiol, and neither progesterone nor charcoal-treated bovine follicular fluid influenced the effect of nutrition. Plasma concentrations of oestradiol were 9.8% lower on average (NS) in ewes fed above maintenance than in the sheep fed below maintenance over the three experiments, suggesting that there may have been a reduced clearance of oestradiol which contributed to the result. We conclude that feeding regimen affects the secretion or clearance of gonadotropins in mature ewes, as in the mature ram, and that this is one mechanism by which ovulation rate may be affected.