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J. Anim. Sci. 2002. 80:2949-2952
© 2002 American Society of Animal Science

Seasonal serum concentrations of melatonin in cycling and noncycling mares1,2

M. A. Diekman3, W. Braun4, D. Peter5 and D. Cook6

Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-1151

3 Correspondence:
phone: (765) 494-4829; fax: (765) 494-9346; e-mail:
mdiekman{at}purdue.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
To determine whether secretory patterns of melatonin change throughout the seasons in mares, blood samples were drawn by venipuncture from nine mares at noon and midnight for five successive days at monthly intervals from August through July at the University of Missouri in Columbia, MO. In addition, during September, December, March, and June, blood samples were drawn from indwelling catheters at 2-h intervals for 48 or 72 h. Mares were predominantly Quarter Horses weighing approximately 450 kg and ranged from 3 to 12 yr of age. Mares were housed in outdoor paddocks with three-sided run-in sheds for shelter. During the noon and midnight bleeding period, mares were placed in a larger open-sided barn with outside runs. Mares remained outdoors with the barn being used as a shelter in the event of inclement weather. All lights in the shed were converted to red light. Often, moonlight provided enough illumination to collect blood samples. Mares were returned to their normal paddock after each sampling period. For analysis of data, a mare was considered to be cycling if serum concentrations of progesterone were greater than 1 ng/mL. For a mare to be classified as exhibiting a nocturnal rise of melatonin, serum concentrations of melatonin had to be at least two times greater at midnight than at noon. By month, a relationship did not exist ({chi}2; P > 0.05) among mares that were exhibiting estrous cycles and exhibiting nocturnal rises of melatonin. Likewise, examination of serum profiles of melatonin taken at 2-h intervals for 48 h revealed considerable variation among mares throughout the seasons. A nocturnal rise in serum melatonin was observed only in June (P < 0.02). In March and December, serum melatonin was greater in cycling mares than noncycling mares, but the elevation was not associated with light–dark periods (P < 0.01). Two of the mares exhibited estrous cycles throughout the seasons but melatonin secretion in these two mares were similar to that observed in the seven mares that demonstrated seasonal anestrous. From these results, it does not appear that changes in serum concentrations of melatonin are used as a cue to regulate cyclic activity in the mare throughout the seasons.

Key Words: Estrous Cycle • Mares • Melatonin • Photoperiod


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Approximately 30 yr ago it was established that the duration of the estrous cycle and ovarian activity in the mare was related to season of the year (Hughes et al., 1972b). Even though 10 to 20% of mares tend to exhibit estrous cycles through the year, the mare is considered to be a seasonal breeder with the ovulatory season extending from May to October (Ginther, 1974; Roberts, 1980). A long photoperiod applied during the anovulatory season hastened ovulation in pony mares by 2 mo (Kooistra and Ginther, 1975; Oxender et al., 1979; Freedman et al., 1979). Seasonal patterns in reproductive activity appear to be mediated by secretion of melatonin from the pineal gland. Superior cervical ganglionectomy or pinealectomy during the winter anestrous period did not alter the first ovulation of the subsequent breeding season in mares, but ovulation in mares was delayed by 2 mo in the second breeding season (Sharp et al., 1979; Grubaugh et al., 1982).

Under natural lighting conditions, serum concentrations of melatonin are higher in anestrus mares during the breeding season (Sharp et al., 1980). Serum concentrations of melatonin in mares exhibited a day-night rhythm, but the rhythm was abolished by superior cervical ganglionectomy (Kilmer et al., 1982). Mares exposed to continuous darkness had rhythmic but asynchronous fluctuations in serum melatonin, suggesting a circadian secretion (Kilmer et al., 1982). To determine whether secretory patterns of serum melatonin were dependent upon the season, serum concentrations of melatonin were measured every 2 h for two consecutive days in March, June, September, and December. After the cyclicity of each mare was determined throughout the seasons, it was of interest to compare serum melatonin patterns associated with each mare’s reproductive status. Knowledge of secretory profiles of melatonin may prove to be useful in controlling estrous activity of mares with a desire to advance or delay the breeding season.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animals.

Nine mares at the veterinary clinic at the University of Missouri were utilized in this study. Mares were predominantly Quarter Horses weighing approximately 450 kg and ranged from three to 12 yr of age. Mares were housed in outdoor paddocks with three-sided run-in sheds for shelter. They were fed alfalfa hay daily and periodically supplemented with a grain mixture.

Blood Collection.

Blood samples were drawn by venipuncture from nine mares at noon and midnight for five successive days at monthly intervals from August through July. During September, December, March, and June, blood samples were drawn from indwelling catheters at 2-h intervals for 48 or 72 h. Blood samples were placed on ice during collection and stored at 4°C overnight. Serum was decanted after centrifugation at 1,500 x g for 30 min and stored at -20°C. Sera samples were shipped to Purdue University on dry ice and stored at -20°C until melatonin and progesterone concentrations were determined by RIA.

During the noon and midnight bleeding periods, mares were placed in a larger open-sided barn with outside runs. The mares remained outdoors with the barn being used as a shelter in the event of inclement weather. All lights in the shed were converted to red light to prevent any artificial light from affecting serum concentrations of melatonin. Often, moonlight provided enough illumination to collect blood samples. Mares were returned to their normal paddocks after each night’s bleeding. For the intensive bleeding periods, mares with indwelling jugular catheters were kept in the large shed described previously. At the end of the intensive bleeding period, the catheters were removed, and the mares were returned to their normal paddocks.

Serum Melatonin RIA.

Serum concentrations of melatonin were measured in each serum sample (250 µL) by RIA using Guildhay antisera (G/S/704-6483, Guildhay Antisera LTD. Surrey, UK; 1:5,000 initial dilution). All samples from a mare collected during an intensive bleeding period were assayed in the same assay. For noon and midnight bleedings, samples from all mares during the month were assayed in a single assay. Assays were conducted in our laboratory as described previously for pig and sheep sera (Green et al., 1996), except that mare sera were extracted with 3 mL of ethyl ether to eliminate interference in the assay. The slope of the inhibition curve for 4 vol of extracted mare sera (50, 100, 200, and 400 µL) was parallel to the standard curve that was prepared in 250 µL of melatonin-free sera (sera were mixed twice with 2% charcoal). Recovery of unlabeled melatonin added to sera that was extracted averaged 96.2% ± 2.1. The average mass of melatonin required to displace 50% of the tritiated melatonin was 38.7 pg ± 1.5 (28 assays). Sensitivity of the assays was 6 pg/mL. Intra- and interassay coefficient of variation was 10.1 and 12.2%, respectively.

Serum Progesterone RIA.

Serum concentrations of progesterone were measured by RIA in samples collected at the beginning and end of a bleeding period to determine the reproductive status (cycling vs noncycling) in each month for each mare. Utilizing a progesterone assay validated previously in our laboratory for sheep (Bollinger et al., 1997) and pig sera (Diekman and Hoagland, 1983), antisera GDN-337 was used to measure serum concentrations of progesterone in mares. Recovery of [3H] progesterone added to serum before extraction with 3 mL of ethyl ether averaged 91%. The slope of the inhibition curve for 3 volumes of extracted mare sera (50, 100, and 200 µL) was parallel to the standard curve. Sensitivity of the assays was 10 pg/tube (100 pg/mL). Intra- and interassay CV were 7.8 and 10.6%, respectively.

Statistical Analyses.

For analysis of data, a mare was considered to be cycling if serum concentrations of progesterone were greater than 1 ng/mL and followed a serum profile typically observed with a functional corpus luteum. For a mare to be classified as exhibiting a nocturnal rise of melatonin, serum concentrations of melatonin had to be at least two times greater at midnight than at noon. Differences in serum concentrations of melatonin were examined between the light–dark periods in cycling and noncycling mares throughout the seasons by General Linear Model (GLM) procedures of the SAS (SAS Inst. Inc., Cary, NC). Mares within light–dark period x cycling status x month was used as the error term to test for treatment effects. A probability value of 0.05 was used as an indicator of statistical significance among comparisons. Chi-square analysis was conducted to determine whether mares were exhibiting nocturnal rises of melatonin during the months that mares were classified as cycling or noncycling (Steel and Torrie, 1980).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
A relationship did not exist (P > 0.05) between mares that were exhibiting estrous cycles and exhibiting nocturnal rises of melatonin throughout the year (Table 1Go). Two of the mares exhibited estrous cycles through the year, but melatonin secretion in these two mares were similar to that observed in the seven mares that demonstrated seasonal anestrus (data not shown). For the seven mares that demonstrated anestrus, the mean number of months that the mares were cycling was 6.0 ± 0.7. All seven mares cycled during the summer months (May, June, and July), whereas four of the mares also exhibited estrus cycles during April, August, and September. Three of the mares also cycled in March and October, two mares also cycled in November, and one mare also cycled in February. Of the seven mares that demonstrated anestrus, none of the mares cycled during the short days of December and January. Likewise, examination of serum profiles of melatonin in blood samples taken at 2-h intervals for 48 h revealed considerable variation among mares throughout the seasons (Table 2Go). A nocturnal rise is serum melatonin was observed only in June (P < 0.02). In March and December, serum melatonin was greater in cycling mares than noncycling mares (P < 0.01), but the elevation was not associated with the light–dark periods (Table 2Go).


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  		Table 1. Presence of a nocturnal rise in serum melatonin in cycling or noncycling mares
 

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  		Table 2. Serum concentrations of melatonin (pg/mL) in cycling and noncycling mares during the light and dark periods throughout the seasons
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Photoperiodism has been implicated as a visual cue in regulating reproduction in seasonal breeders such as sheep, horses, hamsters, ferrets, deer, mink, skunks, voles, and wallabies (Short, 1985). The percentage of mares that cycled throughout the year in the present study (22%) was similar to previous reports of mares that do not exhibit seasonal anestrous (Hughes et al., 1972a; Thompson et al., 1983; Colquhoun et al., 1987). Of the seasonally breeding farm animals, modification of reproductive activity via photoperiodic cues mediated by the secretion of melatonin from the pineal gland is well documented in ewes (Bittmann et al., 1983; Matthews et al., 1993), but the relationship is less clear in mares (Kilmer et al., 1982). In the highly photoperiodic responsive ewe, the duration of the nocturnal rise of melatonin was identical to the length of nighttime (8.2 h of dark to 2 h of dark; Malpaux et al., 1989). Pinealectomy of mares (Grubaugh et al., 1982) or exposure of mares to continuous darkness (Kilmer et al., 1982) disrupted normal day/night secretion of melatonin. Insertion of intravaginal polyurethane sponges containing melatonin induced ovarian activity and estrus during the winter months in Quarter Horse mares (Thompson et al., 1983). The effectiveness of exogenous melatonin in restoring ovarian activity in Welsh pony mares was influenced by the timing of administration (Guillaume and Palmer, 1992). Foals younger than 7 wk of age failed to show significant time trends in serum melatonin concentrations, whereas foals more than 7 wk of age did, suggesting the circadian secretion in the horse may be an acquired phenomenon (Kilmer et al., 1982).

In the present experiment, a consistent nocturnal rise in serum melatonin was not evident in any of the mares. Certainly, the mares examined by Guerin et al. (1995) exhibited duration secretion of melatonin that closely matched the direction of neutral scotophase. The duration of the nocturnal rise in melatonin varied from 13.8 h in the winter to 10 h in the summer with serum concentrations of melatonin at 1 pg/mL during the day and ranging from 7 to 16 pg/mL at night. Higher nocturnal serum concentrations of melatonin were reported by Guillaume and Palmer (1991) with nighttime levels ranging from 26 to 93 pg/mL in 6.5 h and 13.5 h of dark, respectively. When mares were housed in constant darkness, serum concentrations of melatonin increased from 130 to 340 pg/mL (Kilmer et al., 1982) and from 47 to 186 pg/mL (Berglund et al., 1981). In the present experiment, serum concentrations of melatonin over the four sampling months ranged from 10 to 60 pg/mL, but mean values were similar in light and dark periods (16.6 vs 17.5 pg/mL). Similarly in the study conducted by Sharp et al. (1980), we observed that serum concentrations of melatonin were less during the months in the spring than the months in the fall. In contrast, Sharp et al. (1980) observed that serum melatonin was highest during anestrous periods, whereas we observed that melatonin secretions were greater during the night than the day in mares that were cycling in June.

In our mares, melatonin secretion was not influenced by elevated serum concentrations of progesterone as the number of cases where the midnight sample was two times greater than the noon sample occurred with equal frequency in cycling and noncycling mares. No discernable difference between serum melatonin secretion was observed between the two mares that cycled throughout the seasons and those mares that demonstrated seasonal anestrus. From these data, it does not appear that changes in serum concentrations of melatonin are used as a cue to regulate cyclic activity in the mare throughout the seasons.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
This study failed to establish any relationship between serum concentrations of melatonin and seasonal anestrus in the mare. The means whereby the mare uses photoperiod as a cue to regulate cyclic activity throughout the seasons remain unclear.


   Footnotes
 
1 Journal paper no. 16,664 of the Purdue Agric. Res. Programs. Back

2 Presented in part at the 88th Annu. Mtg. of Amer. Soc. of Anim. Sci., Rapid City, SD, Abstr. #480. This study was partially supported by IVY Laboratories, Overland Park, KS. Back

4 Dairyland Veterinary Associates, N 5404 Highway 151, Fond du Lac, WI 54937. Back

5 Frontier Genetics, 80756 Hickey Lane, Hermiston, OR 97838. Back

6 IVY Laboratories, Overland Park, KS 66221. Back

Received for publication January 3, 2002. Accepted for publication June 10, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


Berglund, L. A., D. C. Sharp, and W. Grubaugh. 1981. Effects of constant darkness on melatonin rhythms in pony mares. Biol. Reprod. 24(Suppl. 1):71A (Abstr.).

Bittman, E. L., R. J. Dempsey, and F. J. Karsch. 1983. Pineal melatonin secretion drives the reproductive response to day length in the ewe. Endocrinology 113:2276–2283.[Abstract/Free Full Text]

Bollinger, A. L., M. E. Wilson, A. E. Pusateri, M. L. Green, T. G. Martin, and M. A. Diekman. 1997. Lack of a nocturnal rise in serum concentrations of melatonin as gilts attain puberty. J. Anim. Sci. 75:1885–1892.[Abstract/Free Full Text]

Colquhoun, K. M., P. D. Eckersall, J. P. Renton, and T. A. Douglas. 1987. Control of breeding in the mare. Equine Vet. J. 19:138–142.[Medline]

Diekman, M. A., and T. A. Hoagland. 1983. Influence of supplemental lighting during periods of increasing or decreasing daylength on the onset of puberty in gilts. J. Anim. Sci. 57:1235–1242.[Abstract/Free Full Text]

Freedman, L. J., M. C. Garcia, and O. J. Ginther. 1979. Influence of ovaries and photoperiod on reproductive function in the mare. J. Reprod. Fertil. (Suppl.) 27:79–86.

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Hughes, J. P., G. H. Stabenfeldt and J. W. Evans. 1972a. Clinical and endocrine aspects of the estrous cycle of the mare. In: Proc. Annu. Conv. Am. Assoc. Equine Pract., San Francisco, CA.18:119–151.

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Kooistra, L. H., and O. J. Ginther. 1975. Effect of photoperiod on reproductive activity and hair in mares. Am. J. Vet. Res. 36:1413–1419.[Medline]

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