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Department of Animal Science, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge 70803-4210
2 Correspondence:
phone: 225-578-3445; fax: 225-578-3279; E-mail:
dthompson{at}agctr.lsu.edu.
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Body Condition • Hormones • Leptin • Mares • Reproduction
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionImplicationsLiterature Cited |
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Leptin is a hormone suggested to be a signal between body fat and the hypothalamus (Houseknecht et al., 1998). In other species, leptin concentrations vary directly with percentage of body fat (Prolo et al., 1998; Chilliard et al., 2000). Administration of leptin to female mice induces early puberty (Chehab et al., 1997), and in leptin-deficient ob/ob mice, which are infertile, leptin restores LH secretion and gonadal function (Barash et al., 1996). McManus and Fitzgerald (2000) reported that mares deprived of feed for 24 h exhibited a decrease in plasma leptin concentrations, but had no change in gonadotropin or prolactin secretion. Fitzgerald and McManus (2000) described a profound seasonal variation in leptin concentrations in mature mares that was barely discernable in young mares.
The present experiment was designed to test the hypothesis that nutritional restriction leading to low body condition would intensify the depth and/or length of the seasonal anovulatory period in mares. Specifically, the effects of body condition on follicular patterns, pituitary hormone secretion, ovarian progesterone secretion, and leptin concentrations were studied during the autumnal transition and winter anovulatory period in mares either maintained in good condition or fed restricted diets starting in September.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionImplicationsLiterature Cited |
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Twenty-four light horse mares, 3 to 19 yr of age, with a mean BW of 496 kg and a mean body condition score (BCS) of 7 (6.5 to 8.0; Henneke et al., 1983) were separated into two groups of 12, such that breed type, age, and BW were evenly distributed across groups. One group was then randomly selected to be the high-BCS group and the other the low-BCS group. Beginning in September, mares in the high-BCS group were given (continued on) free choice access to pasture (dormant Alicia bermudagrass and winter ryegrass) and were supplemented with a good quality bermudagrass hay as needed. Mares in the low-BCS group were slowly limit-grazed for decreasing periods of time on equivalent pastures until they reached the desired BCS of 3.0 to 3.5, at which time they were grazing approximately 2 h/d. Good quality bermudagrass hay was supplemented as necessary. All mares had free access to water and a trace-mineralized salt block (Champions Choice, Cargill, Inc., Minneapolis, MN). Mares were housed at the Louisiana State University Agricultural Center, Idlewild Research Station in Clinton, LA, and were maintained on routine herd health and deworming regimens (Evans, 2001). During the experiment, mares in both groups were dewormed at least once with moxidectin dewormer (Quest Paste, Fort Dodge Animal Health, Overland Park, KS). Fecal egg counts were used to assess deworming requirements, and over the course of the experiment, mares in the low-BCS group had to be dewormed more frequently than those in the high-BCS group, likely due to copraphagy.
Determination of BCS and Stage of Cyclicity.
Body weights and BCS were recorded weekly throughout the experiment. Body condition score was determined each time by the same technician by visual appraisal and palpation of the six areas suggested by Henneke et al. (1983). Once the mares approached their target BCS (December), ovarian examinations by transrectal ultrasonography (Aloka 550V with 5-MHz linear-array transducer, Aloka Science and Humanity, Wallingford, CT) and estrous detection with a stallion was initiated. Mares were checked for estrus and their ovaries assessed for ovarian size, follicle number and size, and number of corpora lutea every 72 h. Follicles were assigned to categories based on their size (small, 10 mm or less; medium, 11 to 19 mm; large, 20 mm or larger). When a mare achieved a follicle of at least 30 mm, her ovaries were examined daily until the follicle ovulated or regressed to below 20 mm.
Blood Sampling and Challenges.
Beginning in September, weekly blood samples were collected at 0730 by jugular venipuncture into two 7-mL evacuated tubes (one containing potassium oxalate and sodium fluoride and one containing sodium heparin; Vacutainer, Becton and Dickinson, Franklin Lakes, NJ). These samples were centrifuged at 1,500 x g at 4°C for 15 min and the plasma harvested and stored at -15°C. Concentrations of LH (Thompson et al., 1983a), FSH (Thompson et al., 1983b), GH (Thompson et al., 1992), prolactin (Colborn et al., 1991), IGF-I (Sticker et al., 1995b), insulin (DePew et al., 1994), TSH (Sticker et al., 2001), and leptin (McManus and Fitzgerald, 2000) were determined by RIA previously validated for horse tissues. Intra- and interassay coefficients of variation and assay sensitivities were 6%, 9%, and 0.2 ng/mL for LH; 7%, 11%, and 1.4 ng/mL for FSH; 8%, 11%, and 0.5 ng/mL for GH; 7%, 12%, and 0.2 ng/mL for prolactin; 5%, 12%, and 8 ng/mL for IGF-I; 5%, 8%, and 0.1 ng/mL for insulin; 5% and 0.02 ng/mL for TSH; and 4%, 8%, and 0.8 ng/mL for leptin. Glucose concentrations were determined colorimetrically using commercial kits (Sigma Tech. Bull. No. 315, Sigma Chemical, St. Louis, MO).
During January, several hormonal challenges and a period of frequent sampling were conducted to determine the effects of high vs low BCS on hormonal profiles and secretagogue responses. On these days, each mare was fitted with an indwelling jugular catheter and allowed a brief (about 1 h) period of rest. Blood sampling on a set schedule was then started, and test substances, when appropriate, were administered via the jugular catheter. During the sampling periods, mares did not eat but were provided water at regular intervals.
The first challenge was on January 7, when all mares were administered sulpiride (Sigma), a dopamine receptor antagonist and prolactin secretagogue. Blood samples were collected at -15, 0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, and 240 min, relative to injection of sulpiride (0.1 mg/kg BW) in saline. Plasma was harvested for determination of prolactin concentrations.
On January 12, a period of frequent sampling was performed to assess basal patterns in plasma LH, FSH, prolactin, and GH concentrations. Blood samples were collected every 15 min for 12 h starting in the morning. Immediately following the last (12-h) blood sample, a GH secretagogue was administered to assess GH responsiveness. The secretagogue used was EP51389 (Deghenghi, 1997) at 10 µg/kg BW in saline, which previously has been used to induce GH secretion in horses (Bynum et al., 1998; Kennedy et al., 2002). Additional blood samples were drawn at 10, 20, 30, 40, 50, 60, 75, and 90 min after secretagogue injection for GH determination.
On January 19, mares were administered a combination of GnRH (1 µg/kg BW; Sigma) and TRH (4 µg/kg BW; Sigma) in saline to assess LH, FSH, TSH, and prolactin responsiveness to these secretagogues. Blood samples were collected at -10, 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min relative to injection.
Statistical Analyses.
Data were analyzed by the SAS GLM procedure (SAS Inst. Inc., Cary, NC). Follicle data in January were analyzed in a completely randomized ANOVA (Steele and Torrie, 1980). Hormonal and glucose data over time (weekly data and challenges) were analyzed in split-plot ANOVA (Gill and Hafs, 1971) for a completely randomized design. In these analyses, the main effect of treatment was tested with the mare within treatment as the error term; the time factor and its interaction with treatment were tested with residual error. Differences between groups in each time period in the treatment-time interactions were assessed by the LSD test (Steel and Torrie, 1980).
Analysis of leptin concentrations indicated a prerestriction difference (P < 0.05) between groups in September. Further examination of the individual mares on that date indicated there was a cluster of mares that had low (4 ng/mL or less; n = 15) leptin concentrations and others that had high (7 ng/mL or higher; n = 8) concentrations (one mare was missing the prerestriction blood sample). Due to chance, there was an uneven distribution of mares with low vs high leptin concentrations in the two groups. To account for this, separate analyses were performed with only the low-leptin and only the high-leptin mares, and means for all three analyses are presented. Two alternate approaches also were compared (use of the pre-experimental leptin concentration as a covariate and a natural log adjustment), and all approaches produced similar outcomes.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionImplicationsLiterature Cited |
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), whereas the BW of mares in the low-BCS group decreased (P < 0.005) by about 90 kg. By the end of December, all mares in the low group had BCS of 3 or 3.5, whereas mares in the high group ranged from 7.5 to 8.5 (P < 0.001; Figure 1b).
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). In spite of this, some of these mares did show strong signs of behavioral estrus, such as squatting, urinating, and eversion of the clitoris when exposed to the stallion. Seven of the mares with high BCS continued to ovulate and cycle normally throughout January. As a group, the mares in the high-BCS group had higher (P = 0.034) progesterone concentrations, a greater (P = 0.006) number of corpora lutea (7 vs 0; SEM = 0.04) and more large (P = 0.008) and medium (P = 0.07) follicles during January (Table 1) than did mares with low BCS. Individually, one mare with high BCS went quiescent (mid November until late spring), and four mares experienced a brief period with low progesterone concentrations (late December to mid February); however, they continued to have several large follicles on their ovaries at ultrasound examination.
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) were higher (P < 0.02) in mares of high BCS compared with mares in low BCS, however average concentrations in both groups decreased over time (P < 0.008). Plasma IGF-I concentrations also tended (P = 0.10) to be higher in mares with high BCS (Figure 2c).
There was an interaction (P < 0.0001) between treatment and week as revealed by the overall ANOVA for plasma leptin concentrations in all mares (Figure 3a) and in mares that had low (Figure 3b) or high (Figure 3c) leptin concentrations before onset of restriction in September. Leptin concentrations for both groups showed a downward trend from September to January. Regardless of pretreatment leptin status, the leptin concentrations in mares restricted to obtain low BCS dropped lower (P < 0.01) than those fed to maintain high BCS.
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. Prolactin concentration increased (P < 0.0001) within 15 min in both groups, but there was no difference (P > 0.1) in response between the mares with high vs low BCS. In contrast, the prolactin response to TRH administered on January 19 (Figure 4b) was greater (P < 0.01) in mares with high BCS relative to those with low BCS.
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) concentrations were greater (P < 0.0001) and GH concentrations (Figure 5b) were lower (P < 0.0001) in mares with high BCS relative to those with low BCS. Plasma FSH and prolactin concentrations were not affected (P > 0.10; data not shown). The GH response to EP51389 that immediately followed the 12-h period of frequent sampling (Figure 6) was also lower (P < 0.03) in mares with high vs low BCS.
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) was greater (P < 0.02) in mares with high BCS relative to those with low BCS. The FSH response to GnRH and the TSH response to TRH on the same date were not affected (P > 0.10) by BCS (data not shown).
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Discussion |
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Henneke et al. (1984) showed that breeding efficiency was enhanced in mares entering the breeding season or foaling at a BCS of 5.0 or greater. Those researchers also reported that initial excess stores of body fat enhance fertility. Also, nonfoaling mares with BCS >5 had average intervals from February 1 to first estrus and to first ovulation of 26 and 37 d, respectively; corresponding intervals for mares with BCS <5 were much longer (39 and 63 d, respectively). Ovarian activity in mares in the present experiment was actually followed with ultrasound, teasing, and blood sample collection for progesterone analysis until all mares went through vernal transition and ovulated or until the end of May. The anovulatory period in the mares with low BCS extended from around mid-October to April or May, a period of 6 to 7 mo. In Henneke’s study, whether mares were a BCS >5 or <5, ovulation still occurred by April 1. The time frame differences between the two experiments may be due to the fact that mares in the present experiment were at the two extremes of BCS from approximately late October. Although some mares in the experiment of Henneke et al. (1984) started the experimental period at a BCS of 3.0 to 3.5, they were fed to gain weight. Similar to these data and those of Henneke et al. (1984), Fitzgerald and McManus (2000) reported that young (2 to 5 yr of age) mares, with inherently less body fat than their mature (>10 yr of age) counterparts, entered the seasonal anestrous period earlier than the mature mares, and the mature mares were more likely to continue estrous cycles throughout the winter.
Leptin is an adipocyte-derived protein that has been shown to vary directly with body mass index and percentage body fat in other species (Prolo et al., 1998; Chilliard et al., 2000). Fitzgerald and McManus (2000) reported that their young mares with inherently less body fat also had lower leptin concentrations in summer and fall than did their older, fatter mares. Our results in the present experiment indicate that lower nutrient intake and hence lower BCS does indeed result in reduced leptin concentrations, regardless of the mare’s age. Moreover, this was the case for mares with relatively high leptin concentrations in September (7 ng/mL or greater) as well as for those with low concentrations in September (4 ng/mL or less). Because leptin concentrations were not assessed until the experiment was over, it was not possible to detect the unequal distribution of mares with low vs high leptin in September, before nutrient restriction was started. As it turned out, only two of the 12 mares destined to be restricted had high leptin concentrations in September, whereas five of the mares not destined to be restricted had high leptin concentrations in September, even though all mares had BCS between 6.5 and 8.0 at that time. This may indicate that there is a genetic component to leptin production and secretion that determines relative levels (low vs high) under conditions of good body condition. Under conditions of poor body condition, leptin concentrations were consistently low, regardless of their initial level in September.
The decrease in leptin concentrations in mares of high BCS in the winter also was consistent, even though BCS and BW were relatively constant throughout the experiment. January concentrations averaged about 50% of those in September. Fitzgerald and McManus (2000) also reported a profound seasonal variation in leptin concentrations in mature mares, with average concentrations increasing from July through September and then decreasing precipitously from October to December. In contrast, leptin concentrations in young mares did not increase during the summer, and dropped only slightly in the winter. Examination of the individuals in the present experiment and their leptin data indicated that age did not account for the differences in pretreatment leptin concentrations among mares in September.
Although the mares with low BCS were reproductively inactive from the ovarian standpoint, three of them did show a behavioral (paradoxical) estrus when exposed to the stallion. These mares showed all the outward signs of estrus (squatting, urinating, clitoral eversion); however, upon examination of the ovaries and analysis of blood for progesterone, it was determined that they were reproductively inactive. Ginther (1992) described similar estrous displays in seasonally inactive mares, and suggested that the term "seasonally anovulatory" be used rather than seasonally anestrous for that reason. Likewise, Nequin et al. (2000) observed estrous behavior during periods of low progesterone and very low estrogen concentrations. Therefore, although estrous detection with a stallion is helpful for determining estrus in cyclic mares, behavioral displays of estrus are possible in the absence of significant follicular activity.
In the present study, no differences were detected in weekly plasma samples for LH, FSH, GH, TSH, glucose, or insulin concentrations. Plasma prolactin, however, was greater in mares with high BCS and decreased over time from September to January. This is in agreement with studies that have shown prolactin to be lower during the winter months (Thompson et al., 1986a,b; Johnson, 1987). DePew et al. (1994) reported that plasma prolactin increased relative to feeding in both mares and stallions, which may explain why concentrations were greater in mares with high BCS. Similarly, Bryant et al. (1970) reported that plasma concentrations of prolactin were higher after feeding and were lower during feed restriction in goats; McAtee and Trenkle (1971) reported similar results in cattle. In contrast, McManus and Fitzgerald (2000) reported that circulating concentrations of prolactin were no different in feed-restricted mares than in controls; however, the restricted period was short term (24 h).
Although no differences were detected in GH concentrations, plasma IGF-I concentrations tended to be higher in mares of high vs low BCS. Leptin may be a hormonal signal indicating body fat stores; however, IGF-I seems to be more responsive to short-term changes in nutrition, and decreases within 48 h of feed restriction (Sticker et al., 1995a). As in cattle (Granger et al., 1989), the decrease in plasma IGF-I concentrations with long-term nutrient restriction is accompanied by an increase in GH characteristics in horses (Sticker et al., 1995a,b), although the GH changes in horses tend to be minor relative to other species. In a report by Breier (1999), decreased nutrition led to elevated GH secretion but reduced hepatic GH receptor number and plasma concentrations of IGF-I. The increased GH concentrations in the 12-h period of frequent sampling and the increased GH response to secretagogue in the mares with low BCS in the present experiment are consistent with those previous reports, and may be indicative of nutritional stress induced by low BCS.
In the present experiment, prolactin was secreted in both groups in response to sulpiride on January 7 and TRH on January 19; however, effects of BCS (lower response in mares with low BCS) were only evident after TRH injection. In general, the magnitude of the secretagogue-induced secretion is proportional to pituitary stores, which usually reflect pituitary synthetic activity. We have reported seasonal differences in pituitary prolactin content (Thompson et al., 1986a) and responsiveness to TRH (Thompson et al., 1986b) in vivo, as others have in vitro (Evans et al., 1991), and like plasma concentrations, both are lowest in the winter. The greater prolactin response to TRH in mares with high BCS is consistent with their higher average prolactin concentrations in weekly plasma samples, and together they may indicate a greater prolactin production and/or storage rate in these mares. It is not known whether the elevated prolactin concentrations contributed to the more active reproductive status in mares with high BCS, but previous research in our laboratory (Thompson et al., 1997) indicated that daily injection of 4 mg of recombinant porcine prolactin into seasonally anovulatory pony mares resulted in ovulation and/or luteal formation within 28 d. In contrast, Fitzgerald and Davison (1997) reported that prolactin concentrations did not differ between mares that underwent seasonal anestrus and mares that continued to cycle during the winter.
Relative to mares of low BCS, mares with high BCS secreted more LH in response to GnRH on January 19. The greater LH response was indicative of cyclic vs seasonally anovulatory mares (Hines et al., 1991; Ginther, 1992), which was consistent with the mares with high BCS continuing to grow follicles and ovulate. Several investigators have shown that both LH and FSH (Evans and Irvine, 1976; Alexander and Irvine, 1986; Johnson, 1987) are released from the pituitary when seasonally anestrous mares are given exogenous GnRH; however, those studies used mares of average body condition and the GnRH was given over several week’s time. The mares with high BCS in the present experiment also had higher LH concentrations during the 12-h sampling period on January 12 compared with mares with low BCS, which was likely due to those in the follicular phase when LH concentrations are elevated (Ginther, 1992).
In conclusion, nutrient restriction resulting in low BCS in mares resulted in a profound seasonal anovulatory period that was accompanied by lower leptin, IGF-I, and prolactin concentrations. All but one mare with high BCS continued to ovulate throughout the winter or had significant follicular activity on the ovaries. Although leptin concentrations on average are very low in mares with low BCS and higher in well-fed mares, there is a wide variation in concentrations among well-fed mares, indicating that some other factor(s) may determine leptin concentrations under conditions of high BCS.
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Implications |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication February 18, 2002. Accepted for publication June 25, 2002.
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Literature Cited |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionImplicationsLiterature Cited |
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