Photoperiodic versus metabolic signals as determinants of seasonal anestrus in the mare

The objectives of this study were to compare the timing and mechanisms controlling the onset of anestrus in young and mature mares treated either continuously with melatonin and in those that remained untreated. Changes in body weight, subcutaneous body fat measured to provide an estimate of total body fat, and circulating concentrations of leptin were compared throughout the 1-yr experimental period. The results demonstrate that in young mares the timing of anestrus occurs significantly earlier in the year than in mature mares and that mature mares are more likely to exhibit continuous reproductive activity during the nonbreeding season. The propensity of mature mares to exhibit this phenomenon is not modified by continuous treatment with melatonin but is associated with higher mean circulating concentrations of leptin, body weight, and estimated percent of body fat. In both young and mature mares, body weight, percent of body fat, and circulating concentrations of leptin are higher during summer than winter months. We conclude that, in the mare, the reproductive response to a decrease in photoperiod or a presumptive inhibitory melatonin signal is modified by energy availability, which may be signaled to the hypothalamus-pituitary axis via a change in the circulating concentration of leptin. An additional observation confirmed that the prolactin axis is responsive to continuous treatment with melatonin but that a suppression of prolactin secretion is limited to the spring months.     

Effects of cloprostenol sodium and clenbuterol HCl on reproductive performance in postpartum anestrous cows

Two experiments were conducted to study effects of cloprostenol sodium (cloprostenol) and clenbuterol HCl (clenbuterol) during postpartum anestrus on subsequent reproductive performance in cows. In Experiment I, 96 cows received either 0.5 mg cloprostenol (PGF, n = 25), 364 mg clenbuterol (CLEN, n = 24), 0.5 mg cloprostenol and 364 mg clenbuterol (CLEN+PGF, n = 21) or no treatment (Control, n = 26) on Day 20 post partum. Treatments failed to influence postpartum interval, pregnancy rate or the incidence of short estrous cycles preceding the first normal estrous cycle. In Experiment II, anestrous cows were administered cloprostenol (0.5 mg) on either Day 20 (PGF20, n = 27) or Day 35 post partum (PGF35, n = 25), or served as untreated controls (Control, n = 26). Neither postpartum interval nor pregnancy rate were affected by cloprostenol treatment. In conclusion, treatment of postpartum cows with PGF did not alter the resumption of normal estrous cycles following parturition.     

Influence of reproductive stage at PRID insertion on synchronization of estrus and ovulation in mares

In the present study, we investigated the effects of reproductive status, size of follicles and plasma progesterone concentrations of mares at PRID insertion on the efficacy of the treatment, estrous cycle patterns, plasma concentrations of progesterone and LH. The progesterone-releasing device (PRID) was administered intravaginally to 28 Haflinger mares for 11 days at different reproductive stages: anestrus (n=6), estrus (n=11) and diestrus (n=11). Plasma concentrations of progesterone at insertion (Day 1) of PRID differed among treatment groups (anestrus: 0.2-0.6 ng mL(-1), estrus: 0.2-0.5 and diestrus: 1.6-10.8 ng mL(-1); P<0.001). Total secretion of progesterone (area under curve (AUC)) during treatment period revealed highest values in diestrus (38.2+/-3.1 ng mL(-1)h(-1)) followed by estrus (25.1+/-2.7) and anestrus (21.0+/-0.4 ng mL(-1)h(-1); P<0.05). Progesterone area under curve (AUC) was positively correlated with initial progesterone concentrations (R=0.5; P<0.05), but it did not correlate with the interval from PRID removal to ovulation. Plasma concentrations of LH during treatment period, were significantly lower in anestrous mares (184.6+/-28.6 ng mL(-1)h(-1)) when compared to estrous and diestrous mares (349.7+/-53.3 and 370.5+/-40.3 ng mL(-1)h(-1); P<0.05). Follicular size at PRID insertion had no effects on the intervals from PRID removal to subsequent estrus and ovulation. Follicle diameters at removal of PRID were significantly correlated with the interval from coil removal to estrus (R=-0.55, P<0.05) and ovulation (R=-0.72, P<0.0004) in cyclic mares. In anestrus 0 of 6 (0%) mares, in estrus 5 of 11 (45.5%) and in diestrus 6 of 11 (54.5%) mares ovulated within a defined interval of 1 day before to 1 day after mean interval from PRID removal to ovulation. In cyclic mares, response to treatment was significantly higher when compared to anestrous mares: almost all mares responded with estrus and ovulation independent from the stage of the estrous cycle at the start of treatment. However, accuracy of synchronization was still unsatisfactory. In cyclic mares, the plasma progesterone concentrations at insertion of PRID seem to be more important for the efficacy of the treatment than the assignment to estrous cycle stages.     

Effectiveness of exposure to longday followed by melatonin treatment on semen characteristics of Damascus male goats during breeding and non-breeding seasons

Eight mature Damascus male goats were randomly divided into two equal groups. The first group served as control and the second group was used to study the effect of exposure to longday (16L/8D) followed by melatonin treatment (2mg/(animal/day)) on semen characteristics and blood hormonal levels during breeding and non-breeding seasons. During breeding season, longday exposure followed by melatonin treatments resulted in significant increases (P<0.01) in means of sperm motility, ejaculate volume, sperm concentration, total sperm output, total functional sperm fraction and blood testosterone concentration, while means of reaction time, dead sperm, abnormal sperm and blood triiodothyronine (T(3)) concentration were significantly decreased (P<0.01). During non-breeding season, longday exposure followed by melatonin treatment exhibited improvements in some reproductive parameters by reducing (P<0.05) reaction time and percentage of dead sperm (P<0.01) and increasing (P<0.05) total functional sperm fraction. Furthermore, longday exposure followed by melatonin treatment resulted in significant increases (P<0.01) in both of blood testosterone and T(3) concentrations. It is concluded that longday exposure followed by melatonin treatment successfully improve semen characteristics of Damascus male goats during breeding and non-breeding seasons. However, the beneficial effects were more pronounced during the breeding season.     

Reproduction in high body condition mares with high versus low leptin concentrations

Previous results from our laboratory indicated that a majority of mares with high body condition scores (BCS) displayed estrous cycles or had considerable follicular activity during the winter. Among these high BCS mares, about 35% of them exhibited a persistent hyperleptinemia and hyperinsulinemia. The current experiment was designed to compare the reproductive characteristics of high BCS mares with hyperleptinemia to those with normal (low) plasma concentrations of leptin during the winter and the first estrous cycle (or the first full cycle encountered for those already cycling). Light horse mares with high BCS (6-8.5) were assigned to groups based on leptin concentrations (8/group): low (<5 ng/mL) and high (>10 ng/mL). Beginning 7 January, mares were assessed every 3d for follicular activity and then daily once a follicle >25 mm was detected. Mares were subsequently monitored through their first and second ovulations. Leptin concentrations remained higher (P<0.001) in mares in the high leptin group over the duration of the experiment. Also, high leptin mares had greater (P<0.0001) insulin response to glucose infusion and a faster (P<0.05) rate of glucose clearance. One mare with high leptin and three mares with low leptin had progesterone concentrations indicative of the presence of a corpus luteum at the onset of the experiment. Plasma concentrations of LH, FSH, and progesterone did not differ between groups (P>0.1) during the first estrous cycle occurring after 7 January. Date of first ovulation after 7 January and interovulatory interval were similar (P>0.1) for the two groups, as were estimates of follicular numbers on the ovaries (small, medium, and large; P>0.1). It is concluded that the perturbations in leptin and insulin secretion observed in some high BCS mares are not associated with alterations in ovarian activity or the estrous cycle during winter and into the period of vernal transition.     

The influence of energy intake and percentage of body fat on the reproductive performance of nonpregnant mares

Sixty mares in transition from winter anestrus to normal cyclicity were assigned to a 3 x 2 factorial experiment to determine the effect of energy intake and percentage of body fat on the interval to first ovulation. The factors were 1) percentage of body fat--thin (<11.5), good (11.5 to 15), or fat (>15); and 2) energy intake--maintenance (100% of National Research Council (NRC) digestible energy requirement for maintenance) or high energy (150% of NRC digestible energy requirement for maintenance). Percentage of body fat was estimated by ultrasonographic scanning of rump fat thickness. Energy treatments began on April 2 and ended on June 4. Mares were teased daily with a stallion and their ovaries were palpated per rectum daily or every third day. A high energy intake was effective in hastening ovulation for mares in the thin group (P < 0.05) but not for mares in the moderate or fat groups. Mares in the fat group had a shorter (P < 0.05) interval from April 2 to ovulation (26.4 +/- 4.2 d) than those in the good or high energy-thin groups (48.7 +/- 2.8 and 49.1 +/- 4.2 d, respectively). Duration of the initial estrus was shorter (P < 0.05) for mares in the fat group (16.2 +/- 5.7 d) compared with mares in the good group (34.7 +/- 3.9 d) and tended (P<0.12) to be shorter than mares in the high energy-thin group (29.0 +/- 5.7 d).     

Circadian variations in plasma concentrations of melatonin and prolactin during breeding and non-breeding seasons in yak (Poephagus grunniens L.)

Circadian variations of plasma melatonin and prolactin concentrations were determined during breeding as well as non-breeding seasons in yak. Blood samples (5 ml) were collected during different phases of estrous cycle, viz. early (0-6 days), mid (7-12 days) and late luteal (13-19 days) at 2 h interval for 24 h from eight yaks during one breeding month (November); the same yaks were bled at 2 h interval during one non-breeding month (February) for 24 h. Plasma melatonin concentrations rose sharply (P < 0.01) after sunset to record peak concentrations between midnight and 2 a.m. declining sharply thereafter in both breeding as well as non-breeding seasons. Basal melatonin concentrations were recorded between 0600 and 1600 h. Stage of luteal phase did not influence the diurnal hormone change (P < 0.01). In the breeding season, mean plasma prolactin concentrations displayed circadian variations with maximum value at 0400 h (41.22+ /- 1.5 ng/ml) and minimum at 1400 h (12.0 +/- 4.02 ng/ml). In the non-breeding season plasma prolactin concentrations showed circadian variation with maximum value at 0000 h (59.9 +/- 10.5 ng/ml) and minimum at 1200 h (32.13 +/- 3.2 ng/ml). A positive correlation in breeding (r = 0.75) and in non-breeding season (r = 0.65) between circadian changes in mean plasma prolactin and melatonin concentrations were seen. Circadian changes of mean plasma melatonin concentrations during breeding and non-breeding seasons were not different (P > 0.05). However, mean plasma prolactin concentrations were found to be higher (P < 0.01) in the non-breeding season. Three conclusions were drawn from the study: (i) melatonin and prolactin concentrations followed a circadian pattern of secretion (ii) melatonin and prolactin secretion may be closely interrelated and (iii) higher prolactin concentrations during the non-breeding season could be due to nutritional and environmental stress and hence might be contributing to lack of cyclicity.     

Treatment of seasonally anestrous Romney ewes with continuous infusion of low doses of GnRH: effects on estrus, ovulation and plasma progesterone concentration

The aim of this study was to assess the suitability of a GnRH infusion regimen (125 ng/h or 250 ng/h) to induce estrous behaviour, ovulation and normal corpus luteum function in progesterone-primed Romney ewes each month of seasonal anestrus (i.e. September to February inclusive) over two years. None of the progesterone-primed control ewes (i.e. no GnRH treatment; N = 120 observations) ovulated, showed normal corpus luteum function or displayed estrous behaviour at any time during anestrus. Approximately 27 and 50% of the respective 125 ng/h and 250 ng/h GnRH-treated ewes (N = 120 observations per GnRH treatment) ovulated and showed normal luteal function. Of those which ovulated 59.2% and 52.4% in the respective 125 ng/h and 250 ng/h GnRH treatment groups showed estrous behaviour. There was a significant effect of GnRH dose on the median number of ovulations (250 ng/h > 125 ng/h; P<0.01) but no overall difference (when both treatment years and GnRH doses were pooled) in the median number of ovulations per month of anestrus. The frequency of ewes with an ovulation rate >2 was low with only 4/95 treated ewes with more than 2 corpora lutea (CL). Treatment of progesterone-primed ewes with 250 ng/h GnRH increased plasma LH (P<0.01) but not FSH concentrations; a significant increase in LH pulse amplitude (P<0.05) but not LH pulse frequency was observed. The plasma gonadotropin levels in the 125 ng/h GnRH treatment groups were not studied. We suggest that in breeds such as the Romney which have a strict (i.e. 5-6 month) anovulatory interval, the GnRH-infusion technique may be of limited practical use for inducing pregnancies during the non-breeding season.     

Seasonal relationships between dopamine D1 and D2 receptor and equine FSH receptor mRNA in equine ovarian epithelium

Dopamine (DA) blockade during anestrus or early spring transition can facilitate ovarian recrudescence and advance the timing of the first ovulation of the season. Some laboratories have reported variable results using DA antagonists to stimulate follicular growth during the mid-portion of the anestrual period. Differences in DA antagonist efficacy may be due to the FSH secretory status of the anestrous mare and the presence or absence of functional ovarian FSH receptors. We hypothesize that direct ovarian dopaminergic input can affect follicular growth through regulation of FSH receptor (FSHr) populations. To investigate this, the amount of DA D1 and D2 receptor (D1r, D2r) and FSHr mRNA was quantified in ovarian tissues in anestrous and mares expressing estrus at typical intervals that are detected during the breeding season. Ovaries (n=26) were collected from 10 anestrous mares and 13 mares that had initiated estrous cycles (n=8 luteal; n=5 follicular phase). The quantity of D1r and D2r mRNA and FSHr mRNA was determined in cortex of both groups and granulosa/theca (those having initiated estrous cycles) tissues by semi-quantitative polymerase chain reaction using the comparative cycle time method. The reference gene was glyceraldehyde-3-phosphate dehydrogenase. The fold-change for each sample was calculated based on a calibrator sample. Fold-change values for D1r and D2r were the dependent variable and tissue was the independent variable in a one-way ANOVA. Results of fold-change in FSHr were compared by ANCOVA due to unequal sample sizes from each mare. Correlations between receptors within each tissue type were determined. For each receptor type and tissue, correlations between follicular and luteal phases were determined. The fold-change of D1r mRNA was less than D2r mRNA in all tissue types and between seasons. The quantity of D2r message in ovarian cortex was greater (p<0.05) during anestrus than after estrous cycles had been initiated. Fold-change in D2r in granulosa/theca was not different dependant on estrous cycle phase or follicle size. Quantity of FSHr mRNA was less in anestrous ovarian cortex and greater after estrous cycles had been initiated. FSHr mRNA fold-change in the ovarian cortex after estrous cycle initiation was not different between estrous cycle phases, but was greater in smaller (<30mm) follicles compared with larger (>/=30mm) follicles. We have demonstrated an inverse temporal relation between ovarian D2r and FSHr in mares dependant upon season. The functional significance of this relationship deserves further study.     

Leptin Indirectly Affects Estrous Cycles by Increasing Metabolic Fuel Oxidation

In previous experiments, lean Syrian hamsters fasted on days 1 and 2 of the estrous cycle failed to show sex behavior and ovulation normally expected to occur on the evening of day 4. The first goal of the present experiment was to determine whether systemic treatment with theob(obese) protein leptin could reverse the effects of fasting on estrous cyclicity, social behaviors, and ovulation rate. Fasting-induced anestrus was reversed and normal sex and social behavior and ovulation rate were restored in hamsters injected intraperitoneally with 5 mg/kg leptin every 12 h during fasting on days 1 and 2 of the estrous cycle. A second goal was to test whether the effects of leptin could be prevented by treatment with pharmacological agents that block the oxidation of metabolic fuels. Glucose oxidation was blocked by treatment with 2-deoxy- -glucose (2DG) and fatty acid oxidation was blocked by treatment with methyl palmoxirate (MP). 2DG (1000 mg/kg) or MP (20 mg/kg) was administered at doses that did not induce anestrus in hamsters fedad libitum.As in the first experiment, fasting-induced anestrus was reversed by leptin treatment. However, when each injection of leptin was preceded by an injection of 2DG or MP, leptin treatment did not reverse fasting-induced anestrus. In summary, estrous cyclicity was not restored when oxidation of metabolic fuels was blocked, despite high endogenous levels of leptin. These results are consistent with the hypothesis that leptin acts indirectly on the reproductive system by increasing fuel oxidation.     

Effect of a single subcutaneous injection of melatonin on estrous response and conception rate in goats

The objectives of this study were to evaluate the effect of a single subcutaneous injection of melatonin, on estrous induction and conception rate during the non-breeding season at different times of the year. In Experiment 1 the melatonin powder was dissolved in an oily vitamin A, D, E solution and injected subcutaneously randomly to goats in two dose treatment groups of 20 mg (MLT-20; n = 20) and 40 mg (MLT-40; n = 20) in January (winter). Twenty does were injected with 1 ml vitamin A and served as the control. In the MLT-20 treated goats 70% of the does were in estrus within 20.0 ± 2.0 days, whereas in the MLT-40 group 85% of the does were in estrus within 15.5 ± 2.5 days and 100% and 80% of the does, respectively, conceived on mating with the bucks. Only 10% of the untreated control does exhibited estrus, but none conceived. The breeding season was thus initiated earlier by 1–1.5 months in the treated goats. In Experiment 2, goats were treated with similar MLT-20 and MLT-40 treatments in May (spring), with 20 goats in each treatment group and 20 control goats. The proportion of goats that responded to the melatonin treatments was 80% and 90% in the MLT-20 and MLT-40 treatments, respectively, with no significant differences recorded regarding the estrous response. However, in the MLT-20 treatment group the estrous induction interval was significantly longer (P < 0.05), compared to the MLT-40 treatment. The pregnancy rates were not significantly different for the MLT-20 and MLT-40 treatments, with melatonin resulting in significantly higher pregnancy rates than in the control (88.4% versus 33.3%) and the breeding season initiated 2 months earlier. It could be concluded that a single subcutaneous injection of melatonin can initiate the breeding season (irrespective of the season of the year) earlier by 1–2 months in goats and this could be advantageous when using accelerated breeding systems.     

Body condition during pregnancy and lactation and reproductive efficiency of mares

In Experiment I, 32 mares were equally allotted to the following treatments: mares fed to A) high body condition from 90 days prepartum to foaling and maintained in high body condition to 90 days postpartum, B) high body condition from 90 days prepartum to foaling and allowed to lose body condition to 90 days postpartum, C) lose body condition from 90 days prepartum to foaling and maintained in low body condition to 90 days postpartum, and D) lose body condition from 90 days prepartum to foaling and allowed to gain weight after foaling to attain a high level of body condition by 90 days postpartum. After three cycles, pregnancy rate at 30 days postovulation was lower (P<0.05) in C mares (50%) than in those in the other three groups (100%). Maintenance of pregnancy to 90 days was also reduced (P<0.05) in C mares (25%) when compared with A, D (both 100%) and B mares (88%). Foal growth to 90 days of age was similar in all treatments. In Experiment II, 927 mares were evaluated for body condition and monitored for reproductive performance. Pregnancy rate was lower (P<0.05) and number of cycles/conception was higher (P<0.05) for barren and maiden mares entering the breeding season in thin condition and for pregnant mares foaling in thin condition (condition score less than 5.0) when compared with mares with a higher level of condition. Also, onset of estrus and ovulation appeared to be delayed in barren and maiden mares entering the breeding season in thin condition. Breeding efficiency was enhanced in mares entering the breeding season or foaling at a condition of 5.0 or above. Initial excess stores of body fat enhanced fertility. There were no detrimental effects of excess body fat stored in late gestation.     

Effects of melatonin implants in pony mares. 2. Long-term effects

The effects of melatonin implant treatment over a 4 wk period at the summer solstice on the transition into and out of the following anovulatory season were evaluated in ovary-intact and ovariectomized mares. Melatonin implants tended to delay the timing of the final ovulation of the breeding season (P = 0.0797) in the ovary-intact mares. Although the decline in LH secretion associated with the end of the breeding season was parallel between treatments and ovarian statuses, the rate of LH secretion, as expressed by its mathematical accumulation, was lower in ovariectomized, melatonin-treated mares than in ovariectomized, control mares suggesting that melatonin administration advanced the offset of the breeding season in ovariectomized mares (P = 0.0001). The first ovulation of the subsequent breeding season was significantly delayed in the melatonin-treated mares as compared with that of control mares (P = 0.0031). During reproductive recrudescence, the time of the onset of the increase in LH secretion was similar among all 4 groups but the patterns of LH secretion were different for each treatment and ovarian status combination (P = 0.0112). Mares with melatonin implants had a slower rate of increase in LH secretion than control mares (P = 0.0001), and ovariectomized mares had a faster rate of LH increase than intact mares (P = 0.0001). These results suggest that melatonin implants during the summer solstice can alter the annual reproductive rhythm in mares and support the concept that endocrine patterns of reproductive recrudescence are not entirely independent of the ovary.     

Metabolic control of food intake and estrous cycles in syrian hamsters. I. Plasma insulin and leptin

The "adipostat hypothesis" refers to the idea that circulating hormone concentrations reflect levels of body adiposity and act as signals to control food intake and reproduction. Implicit in the adipostatic hypothesis are the following two assumptions: 1) plasma levels of adipostatic hormones accurately reflect body fat content and 2) decreased plasma concentrations of adipostatic hormones necessarily result in increased food intake and inhibited reproductive processes. The present experiments are designed to test these assumptions. Fat and lean Syrian hamsters were either fasted for 12, 24, 36, or 48 h or allowed ad libitum access to food. Contrary to the first assumption, plasma leptin and insulin levels in fat hamsters dropped dramatically by 12 h after the start of a fast, with no significant change in body fat content and no postfast hyperphagia. Lean hamsters showed anestrus after a 48-h fast but not after a 24-h fast. Contrary to the second assumption of the lipostatic hypothesis, lean hamsters fasted for 24 h and then refed for the next 24 h had leptin levels that were not significantly elevated compared with those of 48-h-fasted hamsters. Thus, in adult female Syrian hamsters, plasma leptin concentrations do not accurately reflect body fat content under all conditions; normal estrous cyclicity does not necessarily require plasma leptin concentrations higher than those of fasted hamsters; and decreased plasma leptin levels do not result in increased food intake.     

Effect of frontal hypothalamic deafferentation on duration of breeding season and melatonin secretion in the ewe

The objectives of this study were to determine if ewes subjected to frontal hypothalamic deafferentation (FHD) during anestrus remained anestrus or began to have estrous cycles, and if melatonin secretion was disrupted by FHD. Ovary-intact ewes in Group 1 were subjected to either FHD (n = 10) or sham FHD (n = 5) in early July 1983. Estrous cycles were monitored by measuring circulating progesterone concentrations from before FHD until September 1985. Group 2 ewes (n = 4) were subjected to FHD in October 1984. In late April 1985, blood samples were taken from all ewes at 1- to 4-h intervals from 1100 h to 0700 h of the following day to monitor diurnal changes of melatonin. Hypothalami were collected for histological evaluation of lesions. All Group 1 ewes (sham FHD and FHD) initiated normal estrous cycles in August and September 1983, and all ceased cycles by mid-February 1984. All sham FHD and 4 FHD ewes remained anestrus until August or September of 1984 and then resumed normal cycles. In contrast, 5 FHD ewes resumed cycles as early as April 1984 and then cycled intermittently or almost continuously. Two Group 2 ewes cycled continuously after FHD and 2 cycled infrequently. FHD ewes that showed prolonged breeding seasons had cuts that damaged the suprachiasmatic nucleus (SCN) and adjacent structures. Mean nocturnal (2000 h-0500 h) melatonin concentrations did not differ (p greater than 0.05) between sham FHD, FHD "normal season," and FHD "continuous cycle" ewes. In summary, damage to the SCN region by FHD during anestrus had no detectable effect on either onset or cessation of the next breeding season but greatly prolonged subsequent breeding seasons. Thus, the environmental signals that both initiated and terminated the 1983 breeding season apparently had been given before FHD was performed in midsummer. Damage to the SCN region during the breeding season caused some ewes to cycle continuously. The effects of FHD apparently were not due to disruption of melatonin secretion. FHD ewes that showed prolonged breeding seasons had normal seasonal changes of plasma prolactin concentrations. This suggests that different neural structures control seasonal patterns of gonadotropin and prolactin secretion.     

Changes in the hypothalamic-hypophyseal axis of mares associated with seasonal reproductive recrudescence

Four groups of mares, representing anestrus (AN; n = 8), early transition (ET; n = 7), late transition (LT; n = 8) and estrus (EST; n = 12) were used to examine changes in the hypothalamus and anterior pituitary during the period of transition from winter anestrus into the breeding season. Mares were of mixed breeding, between the ages of 3 and 20 years, and had shown normal patterns of estrous behavior and ovulation during the breeding season previous to this experiment. Hypothalamic content of gonadotropin-releasing hormone (GnRH) and anterior pituitary content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. The number of receptors for GnRH in anterior pituitary tissue was also determined. There was no effect of stage of transition into the breeding season on receptors for GnRH or content of FSH (p greater than 0.05). Likewise, content of GnRH in the hypothalamus did not differ between the four groups (p greater than 0.05). However, pituitary content of LH increased progressively from anestrus to the breeding season (p less than 0.05). Means for the AN, ET, LT and EST groups were 1.1 +/- 0.2, 2.2 +/- 0.3, 6.3 +/- 1.4 and 15.2 +/- 1.8 micrograms LH/mg pituitary, respectively. In addition, serum concentrations of LH associated with the first ovulation of the year for 5 of the EST mares were significantly lower (p less than 0.01) than those associated with the second ovulation of the year.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oxytocin, prostaglandin F2 alpha, and clenbuterol on uterine dynamics in mares

The effects of oxytocin, prostaglandin F2 alpha (PGF2 alpha), and clenbuterol on uterine contractility and tone during anestrus and diestrus, and during mobility and postfixation of the embryonic vesicle were studied in 51 pony mares. Contractility was assessed by scoring real-time ultrasound images, and tone was assessed by transrectal digital compression. Scoring was done by an operator who had no knowledge of treatment assignments. In anovulatory mares primed with progesterone for 16 d, oxytocin did not significantly alter contractility but did stimulate an increase in tone, whereas clenbuterol depressed both contractility and tone. The PGF2 alpha given on Days 12, 15, and 18 did not significantly alter uterine contractility in pregnant mares, but it increased contractility on all days in nonpregnant mares. Clenbuterol decreased both tone and contractility when given to pregnant mares on the day of embryonic-vesicle fixation, while it decreased tone but not contractility when given on Day 19. Clenbuterol treatment was associated with dislodgment of the fixed embryo in only 1 of 5 mares. However, on Day 19, clenbuterol treatment was associated with a change in shape of the conceptus when viewed in a cross section of the uterine horn. The conceptus shape became more circular rather than irregular or triangular, as indicated by a significant decrease in the variation in the distances between adjacent walls measured in 4 different directions. Results indicated that: 1) oxytocin increased uterine tone but did not alter contractility in progesterone-primed anestrous mares; 2) on Days 12, 15 and 18, PGF2 alpha increased uterine contractility in nonpregnant mares but not in pregnant mares; 3) clenbuterol decreased both tone and contractility at all reproductive states except for a lack of a decrease in contractility on Day 19 of pregnancy; and 4) reduction in uterine tone from clenbuterol treatment on Day 19 was associated with a change in the two-dimensional shape of the in situ conceptus from irregular to a more circular form.     

Effects of nutritional cues on the duration of the winter anovulatory phase and on associated hormone levels in adult female Welsh pony horses (Equus caballus)

Background

Mares have an annual reproductive rhythm, with a phase of inactivity in midwinter. The aim of this study was to determine the impact of food restriction on physiological and metabolic hallmarks of this rhythm.

Methods

Over three successive years, 3 groups of 10 mares were kept under natural photoperiod. A 'well-fed' group was fed to maintain the mares in good body condition; a 'restricted' group received a diet calculated to keep the mares thin and a 'variable' group was fed during some periods like the 'restricted' group and during some other periods like the 'well-fed' group, with the aim of mimicking the natural seasonal variation of pasture availability, but a few months in advance of this natural rhythm.

Results

Winter ovarian inactivity always occurred and was long in the restricted group. In contrast, in the 'well-fed' group, 40% of mares showed this inactivity, which was shorter than in the other groups. Re-feeding the 'variable' group in autumn and winter did not advance the first ovulation in spring, compared with the 'restricted' group. Measurements of glucose and insulin concentrations in mares from the 'restricted' group during two 24 h periods of blood sampling, revealed no post-prandial peaks. For GH (Growth hormone), IGF-1 and leptin levels, large differences were found between the 'well-fed' group and the other groups. The glucose, insulin, GH and leptin levels but not melatonin level are highly correlated with the duration of ovulatory activity.

Conclusions

The annual rhythm driven by melatonin secretion is only responsible for the timing of the breeding season. The occurrence and length of winter ovarian inactivity is defined by metabolic hormones.     

Role of progesterone and estrogen in development of uterine tone in mares

In two experiments (30 mares/experiment), the uterus was recorded as having flaccid tone characteristic of estrus or seasonal anestrus (tone score 1), intermediate tone characteristic of diestrus (tone score 2), or increased or maximal tone characteristic of early pregnancy (tone score 3 or 4). In Experiment I (five mares/group), uterine tone in seasonally anovulatory mares was not altered significantly from the flaccid state by daily administration of 100 mg progesterone plus 1 mg estradiol 17beta or 1 mg estradiol 17beta alone. Uterine tone in seasonally anovulatory mares receiving 100 mg progesterone alone increased to intermediate level (score 2; P<0.05) and remained there throughout the treatment period. Tone scores in the group receiving a 14-d progesterone priming period followed by progesterone plus estradiol were higher (P<0.02) on Days 16 to 28 than scores in the group receiving progesterone alone throughout the treatment period. In Experiment II, (five mares/group), steroid treatments were begun on Day 10 postovulation. The combination of 1 mg exogenous estradiol plus progesterone produced greater uterine tone than exogenous progesterone alone. There were no significant differences between the pregnant control group and the group receiving progesterone plus 1 mg estradiol. There were no significant differences between the group receiving progesterone alone and the group receiving progesterone plus 5 mg estradiol. Results supported the hypothesis that the maximum uterine tone of early pregnancy is caused by progesterone priming followed by exposure to low levels of estradiol plus continued exposure to progesterone.     

Exposure to stallion accelerates the onset of mares' cyclicity

Horses (Equus caballus) belong to the group of seasonally polyestrous mammals. Estrous cycles typically start with increasing daylight length after winter, but mares can differ greatly in the timing of onset of regular estrus cycles. Here, we test whether spatial proximity to a stallion also plays a role. Twenty-two anestrous mares were either exposed to one of two stallions (without direct physical contact) or not exposed (controls) under experimental conditions during two consecutive springs (February to April). Ovarian activity was monitored via transrectal ultrasound and stallion's direct contact time with each mare was determined three times per week for one hour each. We found that mares exposed to a stallion ovulated earlier and more often during the observational period than mares that were not exposed to stallions. Neither stallion identity nor direct contact time, mare age, body condition, size of her largest follicle at the onset of the experiment, or parasite burden significantly affected the onset of cyclicity. In conclusion, the timing of estrous cycles and cycle frequency, i.e., crucial aspects of female reproductive strategy, strongly depend on how the mares perceive their social environment. Exposing mares to the proximity of a stallion can therefore be an alternative to, for example, light programs or elaborated hormonal therapies to start the breeding season earlier and increase the number of estrous cycles in horses.