Responses of Adenohypophyseal Hormones to Substance P Administration in Geldings: Comparison to Responses After Brief Exercise and Sulpiride Administration

Nine adult geldings were used in three experiments to study the possible role of substance P in the prolactin responses to nondopaminergic stimuli. Experiment 1 was performed as an incomplete Latin square design to determine the secretory responses of prolactin, growth hormone (GH), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) to IV administration of substance P. Doses tested and compared to no peptide (0 dose, control) were 62, 125, 250, and 500 μg of substance P. The three highest doses of peptide caused an immediate rise in heart rate, sweating, salivation, rhinorrhea, stretching of hind legs, and defecation. The lowest dose (62 μg) caused minor sweating, some rhinorrhea, and a rise in heart rate. Recovery from these physical responses was complete in approximately 30 minutes. All doses of substance P caused an immediate rise (P < .01) in plasma prolactin concentrations, with the three highest doses producing similar responses, and the 62 μg dose producing a minimal response (P < .05). Concentrations of ACTH (P < .01) and GH (P = .05) also increased after substance P administration; concentrations of LH, FSH, and TSH were unaffected. Experiment 2 compared the effects of brief exercise on hormonal characteristics. Two minutes of trotting increased (P < .01) plasma concentrations of GH, ACTH, and prolactin, as well as LH (P = .055). Experiment 3 determined the relative responses of prolactin to a fixed dose of sulpiride (0.1 mg/kg of body weight). In general, the prolactin responses to substance P were similar to those after exercise, which were both generally less than after sulpiride. These data are consistent with a possible role of substance P in the prolactin response to stressful stimuli.     

Repeatability of Prolactin Responses to a Small Dose of Sulpiride in Estrogen-Primed Geldings in Spring and in Mares During the Estrous Cycle in Summer

Two experiments were conducted to assess the repeatability of prolactin responses to a small dose of sulpiride in estrogen-primed geldings in spring and in mares during the estrous cycle in summer. Six long-term geldings each received a single intramuscular injection of 100 mg of estradiol cypionate on March 31, 2011, and were then challenged with an intravenous injection of dl-sulpiride (5 μg/kg of body weight of the racemic mixture) every other day for a total of 8 days. Jugular blood was collected at 0, 10, 20, 40, and 60 minutes after the injection of sulpiride for prolactin measurement. The experiment was repeated with six mares during the summer (July), except that the number of challenges was extended to 15 over 30 days so that any effect of estrous cycle stage could be assessed. Prolactin responses in geldings during April were robust and were varied in a quadratic manner (P < .003) over the eight sulpiride injections, increasing linearly to a plateau by the fourth injection. Mares also displayed robust prolactin responses to sulpiride injections in July, and there was no effect (P > .1) of day of injection and no effect of stage of estrous cycle (follicular phase, early diestrus, or late diestrus). We concluded that prolactin responses to this dose of sulpiride were sufficiently robust and repeatable for use as a paradigm for studies of the relative competitive efficacy and duration of action of various dopaminergic compounds and their vehicular formulations.     

Inhibitory Effects of Pergolide and Cabergoline Formulations on Daily Plasma Prolactin Concentrations in Geldings and on the Daily Prolactin Responses to a Small Dose of Sulpiride in Mares

Two experiments were conducted to assess the efficacy and duration of action of two dopaminergic compounds, pergolide and cabergoline, on daily prolactin secretion in geldings and on prolactin responses to a small dose of sulpiride over 10 days. In the first experiment, oral administration of 2 mg of pergolide was compared to a single injection of 2 mg of pergolide in a slow-release vehicle and a single injection of 5 mg of cabergoline in slow-release vehicle. Controls received vehicle only. All drug treatments reduced (P < .05) prolactin concentrations relative to that in controls but differed substantially in duration of action (oral pergolide approximately 6 hours or less, injected pergolide 6 to 24 hours, and injected cabergoline at least 6 days). In the second experiment, repeated small doses of sulpiride (2 μg/kg of body weight intravenously) were used to stimulate prolactin release in mares, and the ability of seven daily injections of pergolide (2 mg each) and a single injection of cabergoline (5 mg) in slow-release vehicle to suppress this release were compared. Control mares receiving vehicle injections had robust prolactin responses to the sulpiride injections on all days of injection (days 1, 0, 1, 2, 3, 4, 6, 8, and 10 relative to treatment). Prolactin responses were muted (P < .05) by pergolide and cabergoline treatments on the first day of injection (day 0, 30 min after treatment) and were basically absent on days 1 to 8. The single injection of cabergoline continued to be suppressive through day 10, whereas mares previously treated with pergolide (through day 6) had begun to recover a prolactin response by day 10. We conclude that either daily 2-mg pergolide injections in slow-release vehicle or a single injection of 5 mg of cabergoline in slow-release vehicle is an effective way to apply dopaminergic activity to horses for approximately 7 to 10 days and may have application in the treatment of pituitary pars intermedia dysfunction in affected horses.     

Dose-Response of Prolactin to Increasing Doses of the Dopamine Receptor Antagonist,Sulpiride, in Horses: Effect of Season in Mares and Stallions of Estradiol Pretreatment in Geldings

Two experiments were performed to determine whether dopaminergic input to the adenohypophysis (1) differs across seasons in mares and stallions proportionally with changes in prolactin secretion and (2) is altered by estradiol administration in geldings. In experiment 1, prolactin responses to increasing doses of l-sulpiride in eight mares and eight stallions in March, June, September, and December were used to estimate the theoretical dose equivalent to 50% of maximal response. Prolactin areas increased (P < .001) with increasing doses of sulpiride and were greatest (P < .05) in March for stallions, but in June for mares. Mean half-maximal dose, which was assumed to be proportional to the dopaminergic input to the pituitary, was lowest (P < .05) in June and greatest in September. Experiment 2 used the same approach to determine whether the stimulatory effect of estradiol pretreatment on prolactin secretion was associated with an alteration of the half-maximal response. Geldings (n = 6/group) were administered 100 mg of estradiol cypionate in oil, or oil alone, on day 0 (October 3) and increasing doses of l-sulpiride starting on day 6. Estradiol treatment increased (P < .08) the prolactin response to l-sulpiride at 0.41 μg/kg body weight and all higher doses (P < .05); mean half-maximal dose did not differ (P > .1) between groups. We conclude that dopaminergic input to the adenohypophysis of mares and stallions varies with season and that the stimulatory effect of estradiol on prolactin secretion is not associated with a decrease in dopaminergic input to the adenohypophysis.     

Prolactin and Gonadotropin Responses in Geldings to Injections of Estradiol Benzoate in Oil, Estradiol Benzoate in Biodegradable Microspheres, and Estradiol Cypionate

We previously reported success in inducing early ovulation in seasonally anovulatory mares with a combination of estradiol pretreatment followed by daily administration of a dopamine antagonist (sulpiride). Although every-other-day injections of estradiol benzoate (EB) were effective in that experiment, practical application of this technology would require simplification of the treatment regimen. The current experiment was designed to compare, in a gelding model, the biologic responses of two alternative, one-injection regimens for estradiol delivery to the established EB treatment used previously. Fifteen long-term geldings were sampled via jugular venipuncture from November 5 to 7, 2006, and were then administered intramuscular injections of vegetable oil (n = 4); EB, 11 mg in oil (n = 4; controls); EB in biodegradable microspheres (300 mg; n = 3); or estradiol cypionate, 100 mg in oil (n = 4). Injections of EB in oil were repeated every other day for a total of 10 injections, as was done in our previous experiment. Jugular blood samples were drawn from all geldings at 3, 6, 12, 24, 36, and 48 hours relative to injections, and then on the mornings of days 3, 4, 6, 8, 10 to 18, 22, 26, and 30. On days 10 through 13, all geldings received subcutaneous injections of 125 mg sulpiride, a dopamine receptor antagonist, to stimulate prolactin secretion. On day 12, each gelding received an intravenous injection of 30 μg gonadotropin-releasing hormone (GnRH) analog and 3 mg thyrotropin-releasing hormone (TRH); frequent blood samples were drawn to characterize the luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin responses. Relative to geldings receiving oil, all geldings receiving estradiol injections had a rise (P < .05) in estradiol concentrations lasting at least 12 days. Daily LH concentrations increased (P < 0.01) in all treated groups, but the response was delayed approximately 14 days in the geldings receiving EB in microspheres. Daily FSH concentrations decreased (P < .01) in all treated groups, with the greatest response in the geldings receiving EB in microspheres. Prolactin in daily samples increased (P < .01) similarly in all estradiol-treated groups after injection of sulpiride. The LH response to GnRH analog was greatest (P < .05) in geldings receiving EB in oil and estradiol cypionate; the FSH response was not altered by treatment. The prolactin response to TRH was greater (P < .01) in estradiol-treated geldings relative to controls, but did not differ among groups. Compared with the responses to every-other-day EB injections in oil, as we used previously, a single injection of 100 mg estradiol cypionate gave the most similar and consistent responses. Because of these similar responses in this gelding model, it is likely that a single injection of 100 mg estradiol cypionate can be used in lieu of every-other-day injections of EB in oil in the treatment regimen we reported previously for stimulating ovarian activity in seasonally anovulatory mares.