EFFECTS OF INTERRUPTED PHOTOPERIODS ON THE INDUCTION OF OVULATION IN ANESTROUS MARES 1

EFFECTS OF INTERRUPTED PHOTOPERIODS ON THE INDUCTION OF OVULATION IN ANESTROUS MARES 1 K. Malinowski, A. L. Johnson and C. G. Scanes Rutgers the State University 2, New Brunswick, NJ 09803 ABSTRACT The ability of interrupted photoperiods to induce early estrus and ovulation was examined. Horse mares were exposed to long (16 b light) or short (10 h light), noninterrupted photoperiods, ambient light, or various interrupted photoperiod treatments from December 1 to April i5 (135 d). Follicular development was assessed by rectal palpation and estrous behavior was determined by teasing with a stallion. Serum concentrations of progesterone were used as an indicator of corpus luteum function. Differences among the light treatment groups were compared for the following behavioral and ovarian characteristics: days to first detectable 3-cm follicle, days to first estrous behavior, days to first ovulation, the number of mares ovulating within the treatment period, and the number of ovulations within the treatment period per mare. Compared with the ambient and IOL:14D (L = h of light and D = h of darkness) photoperiod treatments, ovulation was advanced to the greatest extent by a photoperiod of 16L:SD and the interrupted photoperiod IOL:SD:2L:4D. These two stimulatory photoperiod treatments were characterized by the presence of light 8 to 10 h after dusk. Therefore, the present data are consistent with an external coincidence model for the induction of seasonal breeding in horses, with the photoinducible phase occurring within the period 8 to 10 h after dusk. (Key Words: Photoperiod, Intermittent Light, Ovulation, Estrus, Anestrus, Mares.) Introduction Most mares are seasonally polyestrus and undergo periods of sexual quiescence associated with the winter months (Ginther, 1979). Increasing the daily photoperiod with artificial lighting during the winter months can induce the early resumption of ovarian activity (Burkhardt, 1947). A 16-h daylength has been used to induce estrus in anestrous mares (Loy, 1967; Sharp and Ginther, 1975; Sharp et al., 1975; Oxender et al., 1977; Freedman et al., 1979). However, the photoperiod stimulation of reproduction in the mare has yet to be critically studied in terms of identifying a photoinducible phase as has been done for other vertebrate species (e.g., quail: Follett, 1973; Follett and Milette, 1982; hamster: Reiter, 1973; ram: 1Supported by the New Jersey Agr. Exp. Sta. (publication D-06414-O1-85). 2Dept. of Anim. Sci. We acknowledge the assistance of Susan Becker, Stephen P. Dey, Jr., Linda Kravitz, Wendy A. Lifters and Ellen Smiga in obtaining blood samples. 3 Agway, Bordentown, NJ. Received December 13, 1984. Accepted April 19, 1985. 951 Ravault and Ortavant, 1977; Lincoln et al., 1981; ewe: Legan and Karsch, 1980). In the present study we have examined whether photoperiodism in the mare can be explained according to the external coincidence model (Bunning, 1960). Photoscan experiments were designed to determine the time within the scotophase during which a pulse of light coincides with the mare's photoinducible phase and results in the stimulation of cyclic ovarian activity. Materials and Methods Eighteen cycling, nonpregnant, Standardbred, horse mares between the ages of 3 and 17 yr were obtained in the fall of 1981, and 21 and 23 mares of a similar status and age range were obtained in the fall of 1982 and 1983, respectively. Throughout the duration of the 3-yr study all mares were fed a standardized diet (adjusted to maintain body weight) of a commercial sweet-grain mixture 3, mixed hay and supplemental seasonal pasture. Water and trace mineral salt 3 were available ad libitum. All mares received 10 h of natural daylight daily while outside in exercise lots (from 0700 to 1700 h Eastern Standard Time). Before the JOURNAL OF ANIMAL SCIENCE, Vol. 61, No. 4, 1985

952 MALINOWSK! ET AL. start of the experiment on December 1 of each year, mares were randomly assigned to light treatments as indicated in figure 1. Mares were housed two per pen. Light-tight stalls were artificially lighted with full spectrum, cool-white fluorescent light panels to provide approximately 1,076 lux of light intensity at head level. The temperature in the animal facility was not controlled, but followed the normal seasonal pattern for New Brunswick, New Jersey throughout the duration of the experiment. Mares in the ambient group were exposed to naturally decreasing and increasing photoperiods for New Brunswick (40~ during the time period December 1 to April 15. All mares were bled (10 ml blood) thrice weekly throughout the study via jugular venipuncture. Serum was collected and frozen at -20 C until assayed for progesterone. Mares were observed for signs of true behavioral estrus by teasing at least thrice weekly with a stallion. True behavioral estrus (vs occasional signs of estrous behavior that were sporadically noted in anestrous mares) was indicated when a mare displayed signs of winking, squatting and urination after exposure to the stallion for a continuous period of 3 to 5 d. To assess ovarian activity and follicular development, mares were rectally palpated once each week during anestrus and at least twice weekly during estrus. Serum concentrations of progesterone were determined by radioimmunoassay (RIA). The progesterone RIA was essentially as described by Johnson and van Tienhoven (1980) and was validated for mare serum. The sensitivity of the assay was.1 ng/ml and the within- and between-assay coefficients of variation, were 12 and 15%, respectively. Experiments for all 3 yr ended on April 15 (d 135). Ten mares (over a 3-yr period) were eliminated early in the treatment period due to their failure to enter anestrus. Differences among the light treatment groups were compared for the following behavioral and ovarian criteria: time to first detectable 3-cm follicle (d), time to first estrous behavior (d), time to first ovulation (d; determined by rectal palpation, and the initiation of corpus luteum function as indicated by serum concentrations of progesterone), the number of mares ovulating within the treatment period and the number of ovulations within the treatment period per mare. Data on time to first 3- cm follicle, estrous behavior and ovulation were? I~.: IlO: 2L:4D ~ - - - 05 Or C~ II IS B 17 m 9 23 ~ o5 os Or Figure 1. Description of noninterrupted and interrupted photoperiod treatments. Mares in group 1 were exposed to ambient light throughout the experiment. Mares in groups 2 through 7 were exposed to 10 h ambient light from 0700 to 1700 h, and any supplemental light was full-spectrum, cool-white fluorescent light. Open portion of bar represents light, stippled portion represents darkness. analyzed by a general linear model of a randomized block design with the year effect removed; differences among treatment means for each variable were tested by the Newman- Keuls test. Differences among treatments for number of mares ovulating within the treatment period were calculated by chi-square analysis. R esu Its Data on the effect of various photoperiods on follicular development, estrous behavior and the induction of ovulation are shown in table 1. The mean number of days until a 3-cm follicle was first detected and days to the first true seasonal estrous behavior among treatments ranged from 56.6 to 130.0 d, and 70.5 to 120.2 d, respectively. A noninterrupted photoperiod of 16 h light (16L:SD) and an interrupted photoperiod of 10L:8D:2L:4D stimulated early follicular development compared with all other photoperiod treatments (P<.05). Although there was a tendency for the number of days to first estrus to be related to days to first 3-cm follicle, there were no significant differences among treatments in time to the first true seasonal estrous behavior. Also displayed in table 1 are the results of time to first ovulation, number of mares ovulating within the treatment period and number of ovulations within the treatment period per mare. The mean time until the first ovulation ranged from 63.4 d to greater than 135.0 d. The mean number of mares ovulating within the treatment period ranged from 0 to 8.0 and the mean number of ovulations within the

PHOTOPERIOD-INDUCED OVULATION IN MARES 95; 3 TABLE 1. EFFECTS OF INTERRUPTED PHOTOPERIOD ON FOLLICULAR DEVELOPMENT, ESTROUS BEHAVIOR AND THE INDUCTION OF OVULATION IN THE ANESTROUS MARE (MEAN -+ SE)a, b First detectable First estrous First Treatment n 3-cm follicle, d behavior, d ovulation, d Ambient 7 126.6 8.9 d 114.8 +- 10.6 d 130.7 -+ 4.6 d 10L:14D 6 130.0-+ 3.5 d 120.2+ 16.5 d 135.0-+.0 d 16L:8D 5 56.6 -+ 2.1 e 72.0-+ 5.7 d 63.4 *- 3.4 e 10L:2D:2L:10D 8 111.4 8.9 d 117.2 -+ 13.4 d 119.9 -+ 7.7 d IOL:4D:2L:SD 9 112.7 + 13.3 d 103.8 + 13.4 d 121.9 12.2 d IOL:6D:2L:6D 8 108.9 9.3 d 114.0 11.7 d 122.3 7.9 d IOL:8D:2L:4D 8 70.0 + 7.5 e 70.5 -+ 8.7 d 78.0 7.8 e atreatment period is from December 1 to April 15 (135 d). bsome mares were eliminated before the termination of the experiment. CSome mares did not ovulate within the treatment period. d'evalues in the same column with different superscripts differ (P<.05). Number of mares ovulating within treatment period, % 1 (14.3) e 0 (0.0)e 5 (100.0) d 4 (50.0) e 3 (33.3) e 3 (37.5) e 8 (100.0) d Number of ovulations within treatment period/ mare c.1.2 d.0 +.0 e 3.2 -+.2 d.9 +-.5 e.7 -+.5 e.6.3 e 2.6 +-.4 d treatment period per mare ranged from 0 to 3.2. Exposure of mares to 16L:8D or 10L:8D: 2L:4D resulted in a significant reduction in the number of days until first ovulation, and increased both the incidence of mares within the treatment group that ovulated and the total number of ovulations within the treatment period. It should be noted that, irrespective of treatment, once ovulations were initiated, mares displayed normal ovarian cycles for the remainder of the treatment period, as indicated by serum progesterone concentrations (data not shown). Discussion According to the external coincidence model for photoperiodism, stimulation of ovarian development and the induction of ovulation should occur when light extends into the photosensitive phase. Our results suggest that the mare conforms to the external coincidence model for photoperiodism. A photoperiod treatment of 10L:8D:2L:4D when initiated in early December, can stimulate follicular development and advance the time to ovulation in anestrous mares to a treatment interval comparable with the conventional 16L:8D photoperiod (Oxender et al., 1977; Palmer et al., 1982). By contrast, interrupted photoperiods of 10L:2D:2L:10D, 10L:4D:2L:8D and 10L: 6D:2L:6D failed to decrease significantly the time to first ovulation when compared with the ambient light or a 10L:14D photoperiod treatment. In contrast to previous reports in other species where the suspected photoinducible phase occurs early in the subjective night from about 12 to 16 h after dawn (reproductive function, quail: Follett, 1973; hamster: Reiter, 1973; serum prolactin levels, sheep: Ravault and Ortavant, 1977), our data agree with Palmer et al. (1982) in that the putative photoinducible phase appears to be fixed to the time of dusk, and it occurs 8 to 10 h after the onset of darkness. By contrast if the photoinducible phase were fixed relative to dawn, the interrupted photoperiod 10L:4D:2L:SD would be expected to have been comparable with 10L: 8D:2L:4D in its ability to stimulate ovarian activity. This was not observed from results of our experiments. Traditionally, an asymmetric interrupted photoperiod experiment is designed so that the second light pulse in the various photoperiod treatments scans the entire scotophase, thus simulating photoperiods of increasing duration. There are two possible types of phase shifts. Firstly, when the second pulse falls early in the night, phase delays occur that are similar to the

954 MALINOWSKI El" AL. light around dusk in a complete photoperiod. In our experiment the interrupted photoperiod 10L:2D:2L:10D would be expected to be interrupted as a constant 14L:10D. Secondly, when the second pulse occurs later in the night (as seen in 10L:8D:2L:4D), pulse advances may occur that cause a reorganization in the positions of the circadian rhythms such that 10L: 8D:2L:4D is not read as 20L:4D but as 16L:SD (Follett, 1981). Summarized in figure 2 is a model for the photoperiodic stimulation in the horse mare. The greatest stimulation of follicular development and early ovulation was seen in two of the light treatment groups: a noninterrupted photoperiod of 16 h of light (16L:8D) and the interrupted photoperiod 10L:8D:2L:4D. A common feature of these treatments is the presence of light 8 to 10 h after dusk. By contrast, the photoferiod treatments 10L:2D:2L:10L, 10L:4D:2L:8D and 10L:6D:2L:6D were nonstimulatory when compared with the ambient and 10L:14D photoperiod treatments. A simplified interpretation of results seen in asymmetric interrupted photoperiod experiments would be that in such treatments where the second pulse occurs early in the night, the main photoperiod acts as dawn and the second pulse as dusk. In treatments where the second pulse occurs late in the night, the shorter pulse acts as dawn and the main photoperiod as dusk (Wada, 1979). The possibility of a phase shift in our experiments is unlikely based upon the finding that there were no differences in peak or nadir concentrations of daily rhythms of... E=oo=: : Heun horn Cu~t ~ O~rkr, ne Figure 2. Interpretation of results according to the hypothesis that the photosensitive phase occurs 8 to 10 h after dusk. The degree of ovarian stimulation is based on the number of days from the beginning of the treatment period (December 1) to flrst ovulation. Open portion of bar represents light, stippled portion represents darkness; + indicates stimulation within the treatment period, 0 indicates no stimulation. cortisol, or time of peak or nadir concentrations of daily rhythms of cortisol, or time of peak or nadir concentrations in any of the noninterrupted or interrupted photoperiod treatments (Johnson and Malinowski, 1983). Secondly, if phase-shifting had occurred, then the interrupted photoperiod treatments IOL: 4D:2L:8D and 10L:6D:2L:6D would be expected to have stimulated ovarian activity to an extent similar to the 16L:8D and IOL:8D: 2L:4D photoperiod treatments. That the IOL: 8D:2L:4D photoperiod treatment resulted in early follicular development and ovulation comparable with 16L:8D, suggests that the photoinducible phase in the mare is fixed to dusk, not dawn and that phase-shifting did not occur. Finally, it is known that the pineal gland participates in the measurement of daylength through the changes in the secretory pattern of the indoleamine, melatonin, in seasonally reproductive species (Reiter, 1977). It remains to be determined what effect(s) interrupted photoperiods have on daily rhythmic melatonin secretion in mares. The effects of various interrupted photoperiods on the daily rhythm of melatonin secretion are presently under investigation in our laboratory. A possible explanation for the lack of a significant difference in time to first estrous behavior is the considerable degree of variation in behavioral response to teasing among mares. Moreover, the decision of whether a mare is in estrus is usually made on a subjective basis (Ginther, 1979). There is little doubt that both of these aspects contributed to the variability in the mean time to first estrous behavior, as reflected in large standard errors (table 1). In summary, these studies have shown that the mare can be utilized as a model for studies concerning the mechanism of photoperiodic time measurement. Our results suggest that an interrupted photoperiod treatment of 10L:8D :2 L:4D, when initiated early in December, can advance the time of follicular development and ovulation in anestrous mares to a treatment interval comparable with a conventional, noninterrupted 16L:8D photoperiod. Further research is needed to determine the exact mechanism by which night-interrupted schedules work in the mare and to give further insight into the neuroendocrine pathway by which light is perceived and how it is translated into a message that results in gonadal stimulation.

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