Adult Rams Research Articles

Effect of active immunization against testosterone on plasma gonadotrophin concentrations, spermatogenic function, testicular blood flow, epididymis mass and mating behaviour in adult rams.

The long-term effects of active immunization against testosterone were studied in rams, with particular reference to blood concentrations of gonadotrophin and testosterone, spermatogenesis, testis blood flow and mating behaviour. Ten 18-month-old Merino rams, kept on pasture, were studied for 1 year. Every 2 months, five rams received injections of BSA in Freund's adjuvant and five other rams were treated with testosterone-3(o-carboxymethyl)oxime-BSA as immunogen. Anti-testosterone antibodies (mean titre: 1:4484 +/- 582, after boosters) were maintained in the circulation, with the help of regular booster injections. In time, immunization reduced live mass in testosterone-immunized rams; however, there was no effect on testicular volume throughout the whole study. In testosterone-immunized rams, significantly higher concentrations of gonadotrophins were found in jugular venous plasma, as well as increased concentrations of total plasma testosterone. LH pulse frequency, amplitude and nadir were increased significantly in testosterone-immunized rams. After 12 months of immunization, no differences were found in the number of spermatozoa per ejaculate, in daily sperm production or in testis mass between the two groups of rams; however, testicular blood flow (per testis) and epididymis mass were significantly reduced in testosterone-immunized rams. Testosterone immuno-neutralization also resulted in a significant reduction in the number of mounts culminating in ejaculation performed during a 10 min trial carried out on a number of occasions during the experiment. Additional information on these rams was collected 3 months after castration. However, there were no significant differences in mean plasma LH and FSH concentrations, either before, or after, a single GnRH injection between the two groups of rams at this time.

A Comparison of LH Secretion and Brain Estradiol Receptors in Heterosexual and Homosexual Rams and Female Sheep

This study examined endocrine components of sexual orientation of male sheep. Sexual orientation of adult rams was identified through standardized sexual performance tests. Four rams that copulated with ewes, four rams that never mounted females and copulated with males, and eight ewes were used in the experiments. Exogenous estradiol benzoate (50 μg, i.m.) stimulated (P < .05) a preovulatory-like LH surge 16-22 hr after administration to females. Estradiol did not (P > .05) affect LH release of heterosexual or homosexual rams. Thirty days after the estradiol challenge, sheep were euthanized and areas of the amygdala (AMY), hypothalamus (HYP), anterior pituitary (AP), and preoptic area (POA) of the hypothalamus were collected. Occupied and unoccupied content of estradiol receptors (ER) was determined. The content of ER in the amygdala of both homosexual rams and ewes was similar, but less than (P < .05) the content of ER in heterosexual rams. The ER content measured in other brain regions did not differ by sex or orientation. In summary, results from these data show that the preovulatory LH surge mechanism that is a characteristic of the female does not occur in either homosexual or heterosexual rams. Conversely, the ER content of the AMY of homosexual rams is similar to that of ewes and differs from the heterosexual male. Differences in ER content between heterosexual and homosexual rams imply that the amygdala serves as a link for input from potential mates. These data suggest that the amygdala not only plays a role in sexual behavior but may be involved in sexual orientation of rams.

Bilateral lesions of the suprachiasmatic nuclei alter the nocturnal melatonin secretion in sheep.

The hypothalamic suprachiasmatic nuclei (SCN) constitute both the biological clock of many circadian rhythms, and the first relay in the transmission of light cues from the retina to the pineal gland, which releases, via nocturnal melatonin secretion, an endocrine expression of the daylength. The aim of the present work was to investigate the precise role of the SCN in the entrainment of the nocturnal rhythm of melatonin (MEL) in sheep. Bilateral lesions of the SCN were performed via a transsinusal surgical approach in 10 adult rams submitted to a constant photoperiod (16:8D). Lesioned rams were compared to 4 sham and 2 control animals. Blood samples were collected 8 days before, 8 days after, and one month after surgery. Plasma MEL levels were estimated using direct radioimmunoassay. At the end of the experiment, histology and immunohistochemistry of the suprachiasmatic area were performed, and the extent of lesions was evaluated using a computerized image analysis system. Six rams exhibited a complete lesion of the SCN, and in the four remaining animals, the lesions were restricted to the anterior part of the SCN. For all animals, the nocturnal melatonin secretion was altered, but depending on the extent of the lesion, two types of results were observed: Eight days after surgery, in 3 of the 4 rams bearing anterior lesions of the SCN (SCNx-), a nocturnal increase in melatonin secretion still occurred at dusk, but the duration of this secretion extended beyond the end of the night. One month after surgery, melatonin profiles were once again normal, as compared to sham animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of an albendazole controlled release capsule on immunity to internal parasites in sheep

Abstract Extract During a study into internal parasitism in two lines of adult rams (aged 16 months), it was discovered that treatment with an albendazole controlled release capsule (Nufarm) in an earlier trial had affected some parameters measured in the current trial. In the earlier trial, grazing lambs (aged 6 months) received an albendazole controlled release capsule (CRC) to prevent establishment of adult parasites, or were allowed to become subclinically infected. Full results from both trials are in preparation.

Asthénospermie immuno-induite par une protéine épididymaire, la préalbumine ovine (oPES)

The motility of ejaculated spermatozoa (% of progressive spermatozoa) has been evaluated visually on 26 adult rams. Half the animals were immunized with a secretory epididymal protein (prealbumin epididymal-specific (PES) ovine) injected according to immunizing protocols (Freund or Hunter adjuvant) intraperitoneally or intradermally (Freund or Hunter adjuvant) or intramuscularly (Hunter adjuvant or PES only). The other animals received the same product without the protein (controls). The product resulted in a strong asthenospermia which parallels the transient presence of anti-PES antibodies in the seminal plasma. A study with 64 ewes showed that the fertility of 10 immunized rams that had recovered sufficient forward motility to ensure fertilization, did not differ from that of control rams.

Effect of actively immunizing sheep against growth hormone-releasing hormone or somatostatin on spontaneous pulsatile and neostigmine-induced growth hormone secretion.

The physiological role of endogenous circulating GH-releasing hormone (GHRH) and somatostatin (SRIH) on spontaneous pulsatile and neostigmine-induced secretion of GH was investigated in adult rams actively immunized against each neuropeptide. All animals developed antibodies at concentrations sufficient for immunoneutralization of GHRH and SRIH levels in hypophysial portal blood. In the anti GHRH group, plasma GH levels were very low; the amplitude of GH pulses was strikingly reduced, although their number was unchanged. No stimulation of GH release was observed after neostigmine administration. The reduction of GH secretion was associated with a decreased body weight and a significant reduction in plasma IGF-I concentration. In the anti-SRIH group, no changes in basal and pulsatile GH secretion or the GH response to neostigmine were observed as compared to controls. Body weight was not significantly altered and plasma IGF-I levels were reduced in these animals. These results suggest that in sheep, circulating SRIH (in the systemic and hypophysial portal vasculature) does not play a significant role in pulsatile and neostigmine-induced secretion of GH. The mechanisms of its influence on body weight and production of IGF-I remain to be determined.

Seasonal variation in testes size in Polish Merino rams and its relationship to reproductive performance in spring

Observations relating testes size to body weight in growing and adult Polish Merino rams were performed during 3 consecutive years in a flock of 1700 pedigree ewes. October-born ram lambs were measured for testis diameter (TD), scrotal circumference (SC), and body weight (BW) at 100 (January), 150 (March), 180 (April), 240 (June), 365 (October), and 480 d (February) of age. In adult rams (n = 63 to 73 per year) BW and SC were measured in March, May (prior to breeding), August and October. Semen quality was evaluated before each breeding season, and the results of controlled reproductive performance (2 natural services to designated rams) were analyzed. Indices were applied to relate testis size to body weight: TD BW , SC BW and TW BW . Heritability estimates of testes indices in ram lambs at 100 to 240 d and at 480 d of age ranged from 0.21 to 0.36 (SE = 0.067 to 0.090). In adult rams SC measurements within months in consecutive years were repeatable (r = 0.55 to 0.68; P≤0.01). Single measurements of adult ram gonads were not related to other reproductive traits, but significant relations were found when dynamic changes in testes size were examined as the percentage of testes weight change from March to May. Consistent correlations (positive or negative r = 0.2 to 0.5; P ≤ 0.01) between seasonal changes in adult ram testes, semen quality, reproductive performance and in testes size in growing ram lambs were observed. The data suggest that the best time for selecting Polish Merino rams for aseasonality in reproduction is in March at 150 d of age.

Evidence that testosterone and follicular fluid do not interact in the control of FSH secretion in rams

The hypothesis that testosterone and inhibin interact in the control of FSH secretion in rams was tested. Adult rams were castrated and were simultaneously given testosterone implants and 3-times daily sc injections of 0, 0.4, 0.8 or 1.6 ml charcoal-treated bovine follicular fluid (bFF). After 1 wk, the implants were removed, and the bFF injections continued as before. Blood samples were taken daily for mean LH, FSH and testosterone concentrations, and every 10 min for 12 h in the presence and in the absence of testosterone for assessment of pulsatile LH release. The bFF specifically inhibited FSH secretion from rat pituitary cells in culture. In the presence of testosterone, there were no main effects of bFF on mean plasma FSH or LH concentrations, nor were these values different from their pre-treatment means (P>0.05). Treatment with bFF did not affect LH pulse frequency or amplitude, but the number of rams showing LH pulses was reduced in the 0.8 and 1.6-ml dose groups (P<0.05). Removal of testosterone increased (P<0.05) both gonadotropins. In the absence of testosterone, no main effect of bFF on mean LH or FSH concentrations was observed, although the 1.6-ml dose suppressed the postcastration rise of both LH and FSH. These data suggest that inhibin does not interact with testosterone and that a physiological level of testosterone is sufficient for the regulation of FSH secretion in adult rams.

Inhibition of FSH but not LH affects spermatogenesis in the mature ram

We examined the effect of passive immunisation for 20 days with antisera to follicle stimulating hormone (FSH) and luteinising hormone (LH) on testicular function in adult rams in the breeding season in a 2 × 2 factorial experiment with six rams per cell. There was no main effect of immunisation with the anti-LH antiserum nor was there any interaction. However, there was a significant main effect of immunisation with the anti-FSH antiserum which decreased the daily production of B 2 spermatogonia and of leptotene and pachytene spermatocytes by 21.5%, 29.6% ad 33.9% respectively. There was no significant main effect of anti-FSH on the numbers of Sertoli cells or A 0 spermatogonia per testis nor on the daily production of A 1 spermatogonia or of round or elongated spermatids. Testosterone levels were measured in serial bleeds before the start and at the end of the experiment as well as in single bleeds prior to each immunisation. There was only one instance of an effect of immunisation with the anti-FSH antiserum on the secretion of testosterone which occurred prior to the third immunisation. This experiment has shown that FSH is necessary for normal spermatogenesis in the adult ram and that FSH inhibition leads to a decrease in the daily production of late spermatogonia. This is because there is a step between the development from A 1 to B 2 spermatogonia which is sensitive to FSH deprivation. As a consequence, the eventual production of later germinal cells is decreased.

Immunocytochemical demonstration of cytoskeletal proteins in seminiferous tubules of adult rams and bulls.

The immunohistochemical localization of actin, vimentin and tubulin in adult ovine and bovine seminiferous tubules was studied at both the light and electron microscopical levels, using an improved methodical protocol. alpha-smooth muscle actin and structural F-actin were found to be present in myoid peritubular cells. Structural F-actin also occurs in Sertoli cells at three localizations; 1) As part of the Sertoli-Sertoli-junctions in a stage-dependent manner; 2) where Sertoli cells are in contact with spermatocytes; 3) in ectoplasmic specializations lining the recesses that support elongating spermatid heads. Vimentin is localized in peritubular myoid and fibroblastic cells and in Sertoli cells. In the latter, vimentin is concentrated in a perinuclear position. alpha-tubulin is observed in the cytoplasm of spermatogonia, in axonema and manchette of spermatids and in Sertoli cells with their abundant microtubules. Since the alpha-tubulin reaction decorates even the narrowest lateral and apical cell processes, it is an excellent marker to study changes in Sertoli cell shape and size during the seminiferous epithelial cycle in rams and bulls.

Inhibition of FSH affects spermatogenesis in the mature ram

We examined the effect of immunosuppression of follicle stimulating hormone (FSH) on spermatogenesis in adult rams in the breeding season. Rams were immunised for 20 days with one of two anti-FSH antisera, AS1 ( n=5) or AS2 ( n=5) and spermatogenesis compared with that of control rams ( n=4) treated with equine γ-globulins. Treatment with either AS1 or AS2 had no effect on the number of Sertoli cells or A 0 spermatogonia per testis nor on the daily production of A 1 spermatogonia or of spermatids. However, treatment with either AS1 or AS2 significantly decreased the nuclear size of the Sertoli cells, the daily production of B 2 spermatogonia and of leptotene spermatocytes. Treatment with AS1 also decreased the daily production of pachytene spermatocytes. Neither anti-serum affected the secretion of testosterone or luteinising hormone (LH). This experiment has shown that FSH is necessary for normal spermatogenesis in the adult ram because, between the development from A 1 to B 2 spermatogonia, there exists a step which is sensitive to FSH deprivation. This effect is probably mediated through an impairment of Sertoli cell function.

Effect of high-protein feed supplements on concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 in plasma and on the amounts of GH and messenger RNA for GH in the pituitary glands of adult rams.

Three groups of mature rams were maintained on diets of hay, hay + 2% lupin or hay + 2% cowpea for 11 weeks. Serial blood samples were taken at 15-min intervals for 12 h for the determination of GH and IGF-I content by radioimmunoassay and for IGF-binding protein-3 (IGFBP-3) levels by Western blotting. The rams were killed after 77 days of supplementary feeding and their pituitary glands analysed for content of GH and GH mRNA. Mean plasma GH and baseline GH levels were significantly (P < 0.01) decreased in the rams fed lupin and cowpea compared with controls fed hay and GH pulse amplitude was significantly (P < 0.001) decreased in the group fed the cowpea diet. The frequency of GH pulses was not significantly altered by either treatment. Plasma concentrations of IGF-I were elevated in rams fed lupin (P < 0.001) or cowpea (P < 0.05). IGFBP-3 levels were not significantly (P > 0.05) altered by either treatment. There were no significant differences in pituitary content of GH mRNA but pituitary content of GH was increased in rams fed lupin (P < 0.05) and cowpea (P = 0.07). In conclusion, a high-protein diet decreases plasma GH levels and increases IGF-I without changing plasma IGFBP-3 levels in rams. Thus ongoing synthesis of GH, as indicated by the mRNA levels, may cause a build up of GH stores in the pituitary gland.

Photoperiod-mediated increases in serum concentrations of inhibin, follicle-stimulating hormone, and luteinizing hormone are accentuated in adult shortened-scrotum rams without corresponding decreases in testosterone and estradiol.

Relationships between "seasonal" changes in the secretion of gonadotropic (FSH, LH) and gonadal feedback (inhibin, testosterone, estradiol) hormones were examined in the adult ram. Eight Dorset x Leicester x Suffolk rams were kept in photoperiod-controlled rooms for 2 yr and subjected to alternating 4-mo periods of stimulatory (8 h light) and inhibitory (16 h light) day-lengths. The scrota of four rams were shortened at the outset to elevate testicular temperature and presumably impair inhibin secretion by Sertoli cells. Blood samples were collected serially from the jugular vein every 3 wk, and scrotal circumference of the four control rams were measured every 2 wk. Testicular recrudescence and regression were completed in the second month of the short- and long-day periods, respectively. Mean serum concentrations of testosterone and estradiol varied in synchrony across months (r = 0.42) and were similar between the groups of rams, except early in the short-day periods when testosterone was 50% lower in the shortened-scrotum rams because of smaller "seasonal" increases in testosterone pulse amplitude and baseline concentration. Increases in the secretion of gonadotropic hormones during testicular recrudescence were greater in magnitude (FSH) and/or of longer duration (FSH, LH pulse amplitude, and baseline LH) in rams with shortened scrota. In fact, mean FSH concentration was an average of 250% higher in the shortened-scrotum vs. the control rams throughout the last two photoperiod cycles. Mean inhibin concentration varied with month in accordance with FSH (r = 0.52) and either remained normal or was higher than usual (e.g., 55% during testicular regression) in the shortened-scrotum rams.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Copper from Industrial Emission on the Seminiferous Epithelium in Rams

Contents: The changes in the testes of adult rams have been followed by methods of light‐and electronmicroscopy after intoxication with copper emitted from a copper plant. In the toxic phase of the disease primarily the germinative epithelium was damaged. The breaking or decay of the cellular membrane with consequent devastation or stagnation of the spermiogenesis was observed. The toxic effect of the copper affects irreversibly not only all the cells of spermiogenesis in the developmental stages, but it damages also the Sertoli cells including the components of the interstitium. These changes participated in the impairment of reproduction in the flock under study.

Effects of metyrapone infusion on corticotropin-releasing factor and arginine vasopressin secretion into the hypophysial portal blood of conscious, unrestrained rams.

The effects of rapid changes of circulating cortisol levels on ACTH secretion and on corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) concentrations into hypophysial portal blood were studied in six adult rams. Pharmacological adrenalectomy was obtained by 3 h metyrapone infusion (100 mg.kg-1.h-1). Blockade of cortisol synthesis induced a tenfold increase of plasma ACTH levels accompanied by a moderate increase of CRF secretion (150% vs preinjection levels) and a large increment of AVP secretion (535% vs preinjection levels). ACTH levels remained high during the 3 h following the end of metyrapone infusion. During the same period, CRF secretion was still elevated (231% vs preinjection levels), while AVP secretion was further stimulated (2,151% vs preinjection levels). Subsequent hydrocortisone infusion (66 micrograms.kg-1.h-1) for 2 h induced a rapid decrease of both ACTH and AVP secretion, while CRF levels in hypophysial portal blood still remained elevated. These data suggest that changes in ACTH secretion induced by acute modifications of the negative glucocorticoid feedback are, in addition to the well documented direct effect of cortisol on the corticotropes, mainly mediated by variations of hypothalamic AVP secretion.

Combined effects of diet and cold exposure on insulin responsiveness to glucose and tissue responsiveness to insulin in sheep.

Hyperglycemic clamp and hyperinsulinemic euglycemic clamp techniques were used to investigate effects of diet and cold exposure on insulin responsiveness to glucose and tissue responsiveness and sensitivity to insulin in adult rams. The sheep were fed a high-concentrate diet (80% concentrate and 20% roughage) and a high-roughage diet (20% concentrate and 80% roughage), both 70% above the ME requirement for maintenance, and were exposed to a thermoneutral environment (20 degrees C) and a cold environment (0 degrees C) for 2 wk. The estimated ME intake was greater (P < .01) for the high-concentrate diet than for the high-roughage diet. In the hyperglycemic clamp experiment, the ratio of the plasma insulin increment to the glucose infusion rate (insulin responsiveness to glucose) was lower during cold exposure than in the thermoneutral environment in sheep fed the high-concentrate diet but was unchanged in sheep fed the high-roughage diet. In the hyperinsulinemic euglycemic clamp experiment, the glucose infusion rate (tissue responsiveness to insulin) was higher (P < .01) for the high-concentrate diet than for the high-roughage diet, and it was also higher (P < .01) during cold exposure than in the thermoneutral environment, indicating that tissue responsiveness to insulin was intensified in sheep fed the high-concentrate diet during cold exposure associated with the higher energy intake. These results suggest that both reduced insulin responsiveness and enhanced tissue responsiveness to insulin in sheep exposed to cold were dependent on the type of diet.

Control of sheep and goat reproduction: Use of light and melatonin

Breeds of sheep and goats from temperate latitudes exhibit seasonal variations of breeding activity which are controlled by annual photoperiodic changes. Short days (SD) stimulate sexual activity, but prolonged exposure results in refractoriness to short days and subsequent cessation of reproductive activity. Refractoriness can be broken by exposing the animals to long days (LD), thus alternations between LD and SD are essential for the photoperiodic control of seasonal reproduction. Light pulses during the dark period can mimic a long day (‘LD’) and melatonin treatments can mimic a short day. For out-of-season control of sexual activity, treatments using the succession ‘LD’-decreasing days or ‘LD’-melatonin were very effective in advancing puberty in young rams in which sperm production was increased, permitting these animals to be submitted earlier for progeny testing, using artificial insemination (AI). In adult rams, such treatments also caused an important increase in testicular weight and sperm production in the spring. In the female goat, the succession ‘LD’-melatonin treatment efficiently induces and maintains oestrous and ovulatory activities in spring, leading to high fecundity after natural mating. This treatment so far appears to be less effective in the seasonal sheep breeds of Northern Europe than in goats. However, melatonin alone can be used after the end of May to advance the breeding season and to increase fecundity. Induction of permanent reproductive activity in rams and bucks was made possible by the observation that monthly alternations between LD and SD (short light cycles) abolished seasonality of behavioural and spermatogenic activities. These males could be used all the year round to produce a high number of AI doses without variations in sperm quality and with no variation in fertility. Short light cycles can be used in open barns by alternating ‘LD’ and melatonin. In contrast, in the ewe, such short light cycles were unable to abolish seasonality of ovulatory activity. Knowledge of the different effects of photoperiod on neuroendocrine pathways and the reproductive activity in sheep and goats has therefore allowed us to successfully apply light treatments to control seasonal reproductive activity in field conditions and in males raised in AI centres.

Moderate increases in peripheral blood estradiol concentration in the adult ram do not directly inhibit testosterone secretion.

Two experiments were conducted in July with adult Dorset x Leicester x Suffolk rams to determine whether increases of 150 or 300% in estradiol (E2) concentration in peripheral blood (from 6.3 +/- 0.8 pg/mL in control rams) would affect testosterone secretion directly as well as indirectly via the hypothalamic-pituitary axis. After 4 days of estradiol treatment (experiment 1) provided with subcutaneous polydimethylsiloxane implants filled with crystalline estradiol, luteinizing hormone (LH) and testosterone secretions were reduced by 50% (p < 0.05) in both groups of rams because of subtle decreases in pulse frequencies and amplitudes. Estradiol treatments were also associated with decreases in mean follicle-stimulating hormone (FSH) concentration (30-50% in both groups, p < 0.05) and increases in mean prolactin concentration (35% in low-E2 group; 105% in high-E2 group, p < 0.05), but testicular responsiveness to an LH challenge (single intravenous dose, 10 micrograms NIH-LH-S25) remained normal. When along with estradiol treatment, 10-micrograms doses LH were given every 80 min (experiment 2), testosterone secretion increased by 265% (p < 0.05) in both treated and control rams. Relative to day -1, secretion on day 4 was characterized by higher (p < 0.05) pulse frequencies and baseline concentrations and lower (p < 0.05) pulse amplitudes; values for all characteristics were similar to those for Dorset x Leicester x Suffolk rams in the breeding season. Interestingly, the decreases in mean FSH concentration brought about by estradiol and (or) LH treatments were not any greater than in experiment 1, and estradiol's ability to elevate mean prolactin concentration was blocked completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of follicle-stimulating hormone on inhibin release by different testicular compartments in the adult ram.

The aim of this study was to determine the bidirectional release of immunoreactive inhibin-alpha (irINH-alpha) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-alpha was measured by RIA in fluid samples collected concurrently from the three testicular compartments--the seminiferous tubules, the interstitium, and the vascular system--through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively. Overall, the concentration of irINH-alpha in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-alpha concentration in rete testis fluid was inversely related to that in testicular lymph, but i.v. administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibin levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-alpha concentration so that, overall, the total output of inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-alpha in the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an FSH-induced release of a series of proteins in the M(r) range of 30,000-32,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that Dopaminergic Neurons are not Involved in the Negative Feedback Effect of Testosterone on Luteinizing Hormone in Rams in the Non‐Breeding Season

In this study we tested the hypothesis that the negative feedback effects by testosterone on the secretion of luteinizing hormone (LH) in rams involves dopaminergic afferents to gonadotrophin-releasing hormone neurons operating via D(2) receptors in the non-breeding season. In the first experiment, three groups (n = 5) of rams were treated with an intravenous injection of vehicle or 10 or 20 mg of the dopaminergic D(2) antagonist pimozide and jugular venous samples were collected every 10 min for 3 h before and 3 h following treatment. The plasma was assayed for LH. Three groups of ewes (n = 4 to 5) were similarly treated. There were no significant effects of treatment of the rams with pimozide on the plasma concentrations of LH or LH pulse frequency or pulse amplitude and the response of individual rams in each group was inconsistent. In contrast, treatment of the ewes with 20 mg pimozide significantly (P<0.001) increased the mean (± SEM) plasma LH concentrations (pretreatment 0.37 ± 0.04; post-treatment 2.42±0.25 ng/ml) and decreased (P<0.001) the LH inter-pulse interval (pretreatment 180.0; post-treatment 88.0±11.1 min); the 10 mg dose of pimozide did not affect these parameters. In the second experiment, two groups of rams (n = 5) and ewes (n = 7) were treated with an intravenous injection of vehicle or 0.33 mg pimozide/kg liveweight and jugular venous samples were collected every 10 min for 2 h before and 6 h following treatment. As in the first experiment, the mean (± SEM) concentrations of plasma LH were not affected by treatment with pimozide in the rams (pretreatment 0.18 ± 0.25; post-treatment 0.43 ± 0.14 ng/ml) but were significantly (P<0.05) increased in the ewes (pretreatment 1.12±0.22; post-treatment 1.93 ± 0.23 ng/ml). In the third experiment, four adult rams were castrated and 3 weeks later each animal had two cannulae inserted to allow injection into the lateral cerebral ventricles. Vehicle or 100 μg pimozide was injected intracerebroventricularly and blood samples were collected as in the other experiments. A Latin Square design was used so that each animal received each treatment (n = 4). This procedure was repeated after the animals had been injected (intramuscularly) with 16 mg testosterone propionate twice daily for at least 7 days. Treatment with testosterone propionate significantly decreased (P < 0.001) the plasma concentrations of LH (pre-treatment 7.71±0.27; post-treatment 0.75 ± 0.27 ng/ml; mean ± SEM) and follicle-stimulating hormone (pre-treatment 79.61±8.47; post-treatment 42.53 ± 6.08 ng/ml; mean ± SEM) and increased the mean (± SEM) LH inter-pulse interval (53.14 ± 3.58 min pre-treatment and 292.5 ± 32.94 min post-treatment) but had no effect on the amplitude of LH pulses (pre-treatment 3.61 ± 0.36; post-treatment 1.86±1.76 ng; mean ± SEM). Pimozide had no effect on the plasma concentrations of gonadotrophins. These results suggest that, in the ram, dopaminergic neurons do not influence the gonadotrophin-releasing hormone neurons via D(2) receptors in the non-breeding season and are not involved in the negative feedback effect of testosterone on the secretion of gonadotrophins. Conversely, our data suggest that such a mechanism is integral to the negative feedback effects of oestradiol on LH in anoestrous ewes. Finally, it also appears that the steroid-independent suppression of the secretion of gonadotrophins during the non-breeding season in rams is not mediated via D(2) receptors.