Physiology & Behavior, Vol. 49, pp. 277-287. ©Pergamon Press plc, 1991. Printed in the U.S.A.

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The Effects of Short Day Exposure on Seasonal and Circadian Reproductive Rhythms in Male Golden Hamsters GERRY I. H O N R A D O , M A R Y B I R D A N D A L I S O N S. F L E M I N G 1 Department o f Psychology, Erindale College, University o f Toronto R e c e i v e d 21 M a y 1990

HONRADO, G. I., M. BIRD AND A. S. FLEMING. The effects of short day exposure on seasonal and circadian reproductive rhythms in male golden hamsters. PHYSIOL BEHAV 49(2) 277-287, 1991.--Male golden hamsters were exposed to long (LD 14: 10) or short (LD 10:14) photoperiods (Groups LP and SP, respectively) and tested 4 times over a 30-week period. At each test time, animals were tested twice, once in their subjective night and once in their subjective day. During each test animals were observed for approach and copulatory behaviors in response to hormonally primed receptive females. To determine gonadal condition, changes in testes size and sperm production over weeks in SP or LP condition were also measured. Results show that SP conditions induce gonadal regression (at 9 weeks) and recrudescence (at 17 weeks); gonadal function was fully restored by 21 weeks in SP. This pattern is mirrored by a decline in copulatory behaviors during regression, followed by a resumption of sexual behaviors during gonadal recrudescence. In contrast, approach measures showed an inverse pattern; males showed the highest level of approach behaviors at week 13, during gonadal quiescence. Short day conditions also induced changes in the circadian patterning of copulatory behaviors: whereas LP animals always showed more mounts, intromissions and ejaculations in the dark than in the light, between 1 to 13 weeks in short day conditions, SP animals show equal levels of copulatory behavior in the dark and in the light. At the time of gonadal recrudescence, SP animals start to show circadian patterns of sexual behaviors comparable to LP animals. These differences were not found for approach behaviors. The relationship between the different behavioral systems and physiological measures, and the effects on these of short day exposure are discussed. Golden hamster

Gonadal regression

Recrudescence

Short photoperiod

MUCH is known about the effects of seasonal change on the reproductive functions of animals in the wild (10, 17, 27, 60, 61, 64). In the laboratory, these seasonal reproductive rhythms can be induced by exposing animals to conditions which mimic the shortening days of fall and winter and the lengthening days of spring and summer. Long day breeders like the Syrian hamster undergo gonadal regression and loss of sexual behaviors when exposed to short photoperiods [less than 12.5 h of light per day; (17, 25, 45)] and gonadal and behavioral recrudescence when placed back into long photoperiods (greater than 12.5 h of light per day). Although under normal conditions recrudescence coincides with increasing day length, it will occur spontaneously in the absence of long day stimulation if the animals are maintained in short days for a period of 15 to 20 weeks, which corresponds to the usual length of winter (44,45). Considerable research has focused on the physiological [e.g., (7-9, 18, 19, 28, 30, 51, 54, 56-59)] and to a lesser extent, the behavioral (4, 20, 24, 32, 43) changes which occur during the regression phase of seasonal reproductive rhythms in rodents [cf. (10, 17, 64)]; however, only occasionally do these studies look at both behavioral and physiological changes simultaneously (12,

Long photoperiod

Circadian rhythms

19, 38). Moreover, the course of the recovery of sexual and gonadal functioning during recrudescence has not been adequately observed in the male [see (26, 33, 39, 50, 55)] or at all in the female (Honrado, Paclik and Fleming, submitted). Additionally, the sensitivity of these behavioral and physiological markers to circadian rhythm effects as a function of short photoperiod exposure has not been addressed. In the male Syrian hamster, lightdark rhythms in sexual behavior have been reported (21, 36-38). Are these circadian light-dark differences affected by exposure to short photoperiods? In this paper, we examine the effects of short photoperiod (SP) exposure on both physiological and behavioral measures in the male golden hamster. This study departs from previous work in 3 ways. First, we look at both the regression and recrudescence phases of seasonal reproduction; second, physiological and behavioral changes are assessed concurrently; and third, light-dark differences are monitored as a function of weeks in SP. METHOD

Animals and Housing Forty 12-week-old adult male hamsters (Mesocricetus aura-

1Requests for reprints should be addressed to Alison S. Fleming, Department of Psychology, Erindale College, University of Toronto, Mississauga, Ontario, Canada L5L 1C6.

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tus), weighing 100 to 135 g, were obtained from High Oaks Ranch Ltd., Greenwood, Ontario. The stimulus females used were also obtained from High Oaks Ranch. All animals were individually housed in plastic cages (48.3 x 26.7 × 15.6 cm) with food (Purina Rat Chow #5012 in pellet form) and water available ad lib. Upon arrival in the laboratory, the animals were placed into a room with a long day (LP) photoperiod (LD 14:10, lights on 2200 h). Two weeks later, 32 males were transferred from the long day room to one which had a short (SP) photoperiod (LD 10:14, lights on 0100 h); these males remained in this room for the remainder of the study. The rest of the males (N = 8) and all stimulus females were kept under LP conditions for the remainder of the study. During the light portion of each daily cycle, illumination was provided by fluorescent light bulbs. The light intensity for each room averaged 110 lux, as measured by a Gossen Profisix light meter. During both the dark and light portions of each cycle, a red light bulb (60 watts) provided the illumination needed to do behavioral tests. The temperature in each room was centrally regulated to be 22°C. Relative humidity was set at 55%. All males were screened for ability to copulate prior to the beginning of the experiment; males that did not show copulatory behavior towards receptive females were excluded from the study. The females were ovariectomized and then implanted with estradiol prior to the beginning of the experiment. Crystalline 17-beta-estradiol was packed in a 10-mm Dow Coming silastic tubing, measuring 3.2 mm o.d. and 1.6 mm i.d.; the ends of the capsules were closed with 4-ram lengths of wooden doming, and sealed with two applications of Medical Adhesive Silicon Type A (Dow Coming). The capsules were cleaned with alcohol and soaked in a 0.01 M saline buffer for two days prior to implantation under the skin of the female hamsters. Four hours before they were used for testing, these females were injected with 0.05 cc (500 txg/ 0.05 cc) progesterone (in sesame oil vehicle).

General Procedure SP (N = 32) and LP (N = 8) animals were tested at 4 different time periods. Time Period 1 tests occurred 1 to 5 weeks after the beginning of the experiment; Time Period 2 tests occurred 9 to 13 weeks after the beginning of the experiment; Time Period 3 occurred 17 to 21 weeks after the beginning of the experiment; and Time Period 4 occurred 25 to 29 weeks after the beginning of the experiment. Within each time period, both SP and LP animals were tested once in the dark and once in the light (circadian effect). To get a more detailed account of the changes that occur in short day conditions SP animals were divided into 2 groups (N = 16 each): Group 1 animals were tested 1, 9, 17 and 25 weeks after initial SP exposure and Group 2 males were tested 5, 13, 21 and 29 weeks after initial SP exposure. Two kinds of data were gathered: physiological measures and behavioral measures.

Physiological Measures Three physiological markers were measured: body weight, testes size (length and width) and sperm presence. Body weight was measured at 2-week intervals. The fight testis was measured using callipers (to the nearest 0.1 ram) after each behavioral test in the light (see below). This laparotomy was performed under ether anaesthesia. The males were not tested again for 8 weeks after this surgery; this interval gave animals enough time to recover. If a male ejaculated during testing, a vaginal smear was obtained from its female partner to assess the presence of sperm. Sperm presence was rated on a scale of 1 to 4: " 1 " meant that

HONRADO, BIRD AND FLEMING

no sperm was seen in the vaginal smear; " 2 " indicated a sparse sperm count (less than 20); " 3 " reflected a moderate sperm count (20 to 200); and " 4 " indicated a high sperm count (greater than 200) [adapted from (62)]. For statistical analysis " 1 " and "'~'" were combined into a low sperm category and " 3 ' and "'4'" were combined into a high sperm category.

Behavioral Measures Males were tested for sociosexual behaviors at 2 times in their circadian day: in the dark (15 h after light onset) and, one day later, in the light (9 h after light onset). For the SP animals this corresponded to a dark test at 1600 h and a light test at 1000 h. The LP animals were tested once in the dark at 1400 h and again, one day later, in the light at 0800 h. Presumably, the dark tests coincided with all the animals' active period (around CT 13 to CT 14) while the light tests coincided with the animals' rest period [around CT 6 to CT 8: cf. (16)]. At the beginning of each test, males were acclimatized to a test arena (36 x 48 x 20 cm) for 2 rain, after which a stimulus female in heat was introduced. For 15 min, the behaviors of both animals were recorded using an S & K Computer-based Event Recorder (S & K--Norpark Computer Design, 761 Euclid Ave., Toronto) unless the male initiated a fight, in which case the pairs were only allowed to interact for 2 minutes. If a pair fought, the male was given a second stimulus female with which to interact. For each trial, frequency and total duration of a number of behaviors were recorded. These behaviors were divided into 2 functional categories. Approach behaviors of" the male [cL (32, 34.47-49)]. These approach behaviors are similar to measures of proceptive behavior usually observed in female hamsters. The term proceptive refers to "appetitive" nonconsummatory sexual behaviors. Male approach: a movement towards the female while she is stationary. Male follow: any movement towards the female while she is in motion. Male sniff: any sniffing motion directed at any part of the female's anatomy. These 3 behaviors were statistically correlated to each other throughout the experiment. Thus, in analyzing these data, the 3 behaviors were combined into a single approach category. Sexual behaviors of the male [cf. (11, 49, 50)]. Mount: the male is on top of the female, usually mounting from behind while his 2 front paws grab the female's lateral torso. Intromit: the male mounts the female and then inserts his penis into the vagina of the female; the male usually thrusts a few times at a constant rate before withdrawing his penis. Ejaculation: the male intromits accompanied by an increase in thrusting rate just prior to ejaculation; the duration of penile insertion is also longer than usual. The proceptive behaviors of the female were also recorded as was lordosis behavior [cf. (13, 29, 48, 49)]. However, these data were not analyzed because the same females were used repeatedly.

Data Analysis The design of the study was such that 4 variables were manipulated, The first variable was Photoperiod: the males were either exposed to LP or SP conditions. The second variable was Time Period: all males were tested at Time Periods 1, 2, 3 and 4. Comparing across Time Period shows when SP animals undergo regression and recrudescence and determines whether LP animals show any change in behavioral and

PHOTOPERIOD AND REPRODUCTION IN MALE HAMSTERS

physiological measures across Time Period. A Photoperiod x Time Period interaction indicates at which Time Period differences between SP and LP animals occur. The third variable was Group: within each time period, the short day animals were divided into 2 groups. Group 1 SP animals were tested during the first half of each time period, whereas Group 2 animals were tested during the second half of each time period. Comparisons of these SP groups allows us to determine more precisely when regression and recrudescence occur. The fourth variable was Circadian time of testing: all males were tested in the dark and in the light. Measurements in the dark versus the light allows us to determine whether the phasing of behavioral and physiological circadian rhythms differ between SP and LP animals (Photoperiod × Circadian interaction), and if so, at what time period these differences occur (Circadian × Photoperiod interaction). The SPSS/PC + V2 was used for all analyses.

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Changes in Physical and Physiological Measures as a Function of Time Period or Weeks in Short or Long Photoperiod Conditions In the first set of analyses, the physiological characteristics of LP animals were compared to those of the SP animals (Photoperiod effect) across the 4 time periods (Time Period). When the body weights of animals were analyzed as a function of Time Period, no significant differences were obtained between the SP and LP animals. All animals showed an expected increase in weight as a function of age [significant Time Period effect: F(3,108) = 14.75,p

The effects of short day exposure on seasonal and circadian reproductive rhythms in male golden hamsters.

Male golden hamsters were exposed to long (LD 14:10) or short (LD 10:14) photoperiods (Groups LP and SP, respectively) and tested 4 times over a 30-we...
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