replications. Department of Animal Science, National and University Institute of Agriculture, {Received 29th April 1964)

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1 EFFECTS OF COHABITATION ON THE REPRODUCTIVE SYSTEM, KIDNEYS AND BODY COMPOSITION OF MALE RATS D. DRORI and Y. FOLMAN Department of Animal Science, National and University Institute of Agriculture, Rehovoth, Israel {Received 29th April 1964) Summary. Male rats were raised to different ages in cohabitation, i.e. with sexually receptive females, and unmated males were raised with spayed females or in all-male groups. The size of the testes, several accessory reproductive organs, kidneys and adrenals was compared. The body composition of the males was determined at 286 days of age. At all times after puberty, the males raised with receptive females had larger accessory reproductive organs and kidneys. At 286 days of age some of the accessory reproductive organs of the unmated males were 35 to 40% smaller, a degeneration of the testes and the coagulating glands had begun and their bodies contained 72% more fat and 6% less total ash. The findings suggest that in the male rat cohabitation is necessary for the proper maintenance of testicular androgen secretion. INTRODUCTION Steinach (1936) stated that male rats housed for several months in rooms con taining no females, and kept singly or in groups offour per cage, suffered atrophy of the testes and the accessory reproductive organs and consequent loss of libido; the atrophy of the reproductive organs was reversible upon prolonged contact with females or when the males were brought into the vicinity of females and exposed to their odours. Steinach kept no control males caged with or near females, and presented no data on organ weights or on the number of replications. Beach (1942) studied the copulatory behaviour of male rats raised singly, in all-male groups and in cohabitation. Copulatory activity was highest in the males raised singly, but higher in males raised in cohabitation than in those raised in all-male groups. Beach concluded that his results were at variance with Steinach's report of loss of sex drive in isolated or segregated males. The major purpose of this study was to compare the morphology of the reproductive organs of male rats kept with receptive females with that of un mated males kept with non-receptive females or in all-male groups. In addition, kidneys and adrenals were compared and gross body composition was determined at 286 days of age. 351

2 352 D. Drori and T. Folman MATERIALS AND METHODS Male rats weaned at about 25 days of age were used in all the experiments. They belonged to an albino strain of unknown origin. The animals were randomly bred in the authors' laboratory. The strain was rather tame: strange males could be introduced into cages containing males without noticeable conflict. The rats were fed ad lib. on a diet which has adequately supported growth and reproduction over 4 years. Under the conditions of management in the laboratory, sperm cells appeared in the epididymis when males reached body weights of 165 to 180 g or at the age of 45 to 55 days. Colony males over 180 g or older than 60 days of age im pregnated females. Hence, it may be said that males at 60 days of age were well past puberty. Conditions ofcohabitation were provided by housing the males with receptive females which were either intact or hysterotomized. Hysterotomy was performed by excising a length of about 2 mm from both horns of the uterus. The unmated males were kept either in all-male groups or with spayed females. Spayed females are invariably non-receptive (Barnett, 1963). After the males had been killed in chloroform vapour and bled, the following organs were removed and weighed, in one or more of the experiments : testes, penis muscles (i.e. the combined ischiocavernosus and bulbocavernosus), levator ani, seminal vesicles, coagulating glands, bulbourethral glands, penis, kidneys and adrenals. The organs were kept in physiological saline until weigh ing. The seminal vesicles were obtained intact and freed from the coagulating glands after immersion in Bouin's fixative for 10 min. Under these conditions it was possible to obtain the seminal vesicles without losing the secretion. This procedure did not affect their weight appreciably. In some instances the seminal vesicles were cleaned of secretion and the tissue was weighed. Body composition analyses were runonwhole rats. The ratswere starved for 24 hr before killing to empty the digestive tract. Water was determined by drying at 100 C to constant weight. Fat was determined by extraction in petroleum ether. The fat-free pelvis was cleaned and weighed and thereafter the whole fat-free rats were ground and ashed to constant weight at 600 C. Student's t test for paired observations was used where applicable. In Experi ment 3 the individual weights of organs and body constituents were expressed as percentages of body weight for statistical analysis and the t test for unpaired observations was used. RESULTS Preliminary trials Preliminary trials were conducted in an attempt to develop useful techniques ; 230 males were used in these trials. Fourteen males or more were used in each oftwelve comparisons. In cohabitation, males were raised with receptive females, which were intact or hysterotomized; an equal number of unmated males were raised with spayed females or in all-male groups. Two or three males were housed in one cage with one mature receptive female or with one spayed female ; in the all-male groups an additional male was used to bring the number of

3 Effects of cohabitation on the male rat 353 animals per cage to three or four, respectively. The males were killed at 40 to 80 days of age. The following organs were compared : penis muscles, seminal vesicles, testes, kidneys and adrenal glands. No differences were found between the cohabitation males raised with intact females and those raised with hysterotomized ones. Likewise, there were no differences between the unmated males raised with spayed females and those in all-male groups. Between the males in cohabitation and the unmated males there was a consistent difference in penis muscles. The mean penis muscle weights are shown in Text-fig. 1. The penis muscles of the cohabitation males 1300 LT! E u> o ^ _li 60 Age (days) Text-fig. 1. Mean weights of penis muscles of male rats raised in cohabitation ( ) and unmated males (o). Fourteen or more males equally divided between both treatments were killed at different ages. Lines connect groups of males started simultaneously (preliminary trials). The points at 46, 51, 70 and 80 days represent means of seven ob servations, those at 67 days represent eighteen and forty-two observations, on cohabita tion and unmated control, respectively; each of the other points represents eight observations. were larger than those of the unmated ones. The differences tended to increase after puberty; the difference at 53 days and all the differences at 63 days or more were statistically significant. At the same time the mean body weights of these groups of males showed no consistent or significant differences. The seminal vesicle, testis and kidney weights of the males in cohabitation were not signifi cantly different from those of the unmated males, but tended to be slightly larger. There were no significant differences in adrenal weights. Experiment 1 The object of this experiment was to determine whether differences in addi tional organs would appear between the treatments if the males had been raised with two females rather than with one per cage. Twenty-seven pairs of male littermates were used. One member of each pair

4 354 D. Drori and Y. Folman was raised in cohabitation and its littermate was raised unmated. The cohabita tion males and the unmated ones were housed nine per cage. Two hysteroto mized females per cage were used in cohabitation and two spayed females were kept with the unmated males. Three males were removed from each cage and killed when they reached 140 g, three more when they reached 200 g and the remaining three when they reached 250 g. Killing weight had been allotted to littermate pairs at the time the experiment started ; thus, the members of each pair were killed at the same weight but not necessarily at the same exact age. In order to kill each male near his chosen weight, the males were weighed at least twice a week. The same organs were compared as in the preliminary trials ; in the males killed at 250 g the seminal vesicle tissue was also weighed. The results of this experiment are shown in Table 1. Like the penis muscles after puberty, the kidneys were significantly larger in the cohabitation males (P^O-05). As in some preliminary trials the seminal vesicles were smaller in the cohabitation males of the 62-day group. In the 81-day group the seminal vesicle Table 1 mean weights of reproductive organs, kidneys and adrenals of male rats raised in cohabitation and unmated males (experiment 1) Treatment Mean age (days) 45 ±1 45 ±1 Body wt(g) 139 ±3 140 Penis muscles Seminal vesicles Whole Tissue 31 ±4 37 ±7 Testes 1160 ± ±83 Kidneys 1530 ± ±36 Adrenal glands ±1 594* ± ± ± * ± ±57 34 ± ±5 251 ±5 1207** ± ± ± ** ± ± ± ± * ± ± The values are the means of nine males each, killed at predetermined body weights. Significant differences: *PsS0-05, **P<0-01. tissue was also weighed; whereas the difference in whole glands was only 9% in favour of the cohabitation males (and statistically not significant) the differ ence in net tissue weight was 37% and highly significant (P^0-01). This sug gested that in the preliminary trials and in the 62-day group of this experiment the differences in seminal vesicle tissue mass had been masked by a large and variable amount of secretion. Experiment 2 The purpose of this experiment was to extend the comparison of organs to the penis and the bulbourethral glands and to study the effect of reversing the treat ments on the comparative sizes of the different organs.

5 Effects of cohabitation on the male rat 355 Sixteen pairs and eight quartets of male littermates were used, i.e. a total of sixty-four. One male of each pair and two of each quartet were raised in co habitation; their littermates were raised unmated. On both treatments eight rats were housed with two hysterotomized or two spayed females per cage, respectively. Of the sixteen pairs, eight were killed when they reached 160 g and 320r- (c) u Age ( days) Text-fig. 2. Mean weights of (a) penis muscles, (b) seminal vesicles, (c) penis, (d) bulbourethral glands, (e) testes and (f) kidneys of male rats raised in cohabitation (#) and unmated males (o). Solid lines represent the growth rates of 'through' groups; broken lines represent growth after reversal of treatments at a mean age of 67 days. Each point is the mean ofeight observations (Experiment 2). eight at 235 g. The eight quartets were used to observe the effect of treatment reversal. Two 'through' groups, each of eight males, one in cohabitation and one unmated, were raised to 100 days of age without any change in treatment. Their littermates formed two 'reversal' groups of equal number; these groups were initially 'cohabitation' and 'unmated', respectively, but their members were transferred from their initial treatment to the alternative one upon

6 356 D. Drori and T. Folman reaching 235 g and were killed at 103 days of age. One member of each quartet was used in each 'through' and 'reversal' group. All organ weights, except those of the seminal vesicle tissue and the adrenal glands, are presented in Text-fig. 2. Both the 'cohabitation' and the 'unmated' groups killed at 160 g had a mean age of 45 days and both groups killed at 235 g had a mean age of 67 days. The mean weights of the 'through' groups at 100 days were 287 and 278 g for the 'cohabitation' and the 'unmated' groups, re spectively. The unmated males switched to cohabitation weighed 272 g and the cohabitation males switched to the unmated treatment weighed 289 g. At 67 and 100 days (i.e. in the 'through' groups) all the organs were larger in the cohabitation groups (Text-fig. 2). Differences at both ages in penis and penis muscles were significant at the ==0-001 level, in bulbourethral glands at P^O-01 and in kidneys at P^O-05. Whole seminal vesicles were significantly different (P^0-01) at 100 days but not at 67 days; on the other hand the mean weight of the gland tissue at 67 days was 206 mg on cohabitation and 168 mg control on and the difference was significant at the P^0-001 level. The effect of reversal was plain. All the accessory reproductive organs (except the bulbourethral glands ofthe cohabitation-to-unmated group) and the kidneys changed their rate of growth. The seminal vesicles presented a notable excep tion in that their weight was larger in the 'reversal' group switched to and killed on the unmated treatment; however, the net tissue mass of the seminal vesicles in the latter group was 236 mg versus 259 mg for the 'reversal' group switched to and killed on cohabitation. Thus, the secretory fluid in the seminal vesicles again masked a trend which was evident in the tissue mass. Owing to an error, the seminal vesicle tissue of the 'through' groups was not obtained. The testes showed a slight trend similar to the other organs represented. Only in the 'through' groups (100 days) was the difference in testes weight significant {P^O-05). There were no significant differences in adrenal weights. At 45, 67, 100 and 103 days the mean weights of the adrenal glands were : 33 and 32, 42 and 42, 50 and 49, and 51 and 51 mg in the cohabitation and unmated males, respectively. Experiment 3 The purpose of this experiment was to study the long-term effect of both treatments. Thirteen pairs of males (nine littermate pairs and four pairs matched for age and weight at weaning) were divided between cohabitation and unmated treatment. From weaning to 200 days of age each group was housed in two cages, i.e. six or seven males per cage, and from 200 to 286 days of age in three cages, i.e. four or five males per cage. Two hysterotomized or two spayed females per cage were used in the respective treatments. The levator ani muscle and the coagulating glands were weighed in addition to the organs weighed in Experiment 2. Gross body composition was deter mined on all the males except one (unmated) which died of acute enteritis 3 days before the end of the experiment. This male was not available for body composition analysis, but its organs were weighed and included in the results. Mean organ weights are shown in Table 2 ; body composition and the weight of the pelvis are shown in Table 3.

7 Effects of cohabitation on the male rat 357 All the accessory reproductive organs and the kidneys were considerably smaller in the unmated males. The coagulating glands of four unmated males had degenerated. Degenera tion was unilateral in three males and bilateral in one. Degenerated single glands weighed 10 to 12 mg versus 83 and 55 mg for normal single glands of the cohabitation and unmated groups respectively. Exclusion of the degenerated coagulating glands in calculating the mean of the control males did not change the level of significance of the difference (P^ 0-001). Table 2 mean weights of the reproductive organs, kidneys and adrenals of thirteen male rats raised in cohabitation and thirteen unmated males at 286 days of age (experiment 3) Treatment Body wt (g) ±13 Penis muscles 1930*** ± ±50 Levator ani 321*** ± Seminal vesicles tissue 359*** ± ±11 Coagu lating glands 166*** ±7 97 Bulbo urethral glands 101** ±3 85 ±5 Penis 422*** ±8 326 ±9 Testes 2810 ± ±95 Kidneys 2820** ± ±116 Adre nal glands Significant differences: **P^001, ***P^0001. Table 3 body composition of thirteen male rats raised in cohabitation and twelve unmated males at 286 days of age (experiment 3) Treatment Body wt Water Fat Fat-free organic matter Ash Fat-free pelvis ± ± ± ± *** ± ± ± !*** ± ± *** ± ±0-030 All values are in g. Body weights are dead weights ; a 24-hr starvation period with free access to water. Significant differences:*** P< the males were killed after Three unmated males exhibited unilateral degeneration of the testes ; two of these were among the four with degenerate coagulating glands (one bilateral and one unilateral). The degenerate testes of three males weighed 800, 816 and 1045 mg versus 1280, 1308 and 1380 mg for the respective normal contralateral testes. They were pale, of very low consistency and contained 9-4, 9-5 and 10-0% dry matter compared to a mean of 13-9% (and a range of 12-6 to 14-4%) in all the remaining testes. Of the five control males which had degenerate testes or coagulating glands both, or two were littermates and had two littermate counterparts in cohabitation; a third had one littermate and a fourth had three littermates on cohabitation.

8 358 D. Drori and T. Folman None of these six cohabitation males (nor any of the others) of degeneration in their reproductive organs. The adrenal glands significantly different. showed indications were not CORRELATION BETWEEN ADRENALS AND ACCESSORY REPRODUCTIVE ORGANS Although there were no significant differences in adrenal weight between the treatments, a negative correlation was observed between the weight of the accessory reproductive organs and the adrenal glands in both treatments. Thirty correlation coefficients were calculated between adrenal and penis muscle weight on groups of seven to thirteen males belonging to any one treat ment group. Of these correlation coefficients twenty-three were negative and seven were positive. Of the negative coefficients five were significant at P^ 0-05 and two at ^0 01, but none of the positive coefficients were significant. All the significant negative correlations occurred between 40 and 67 days of age. After 80 days of age the correlations tended to be low. DISCUSSION The gradual decrease in size of the accessory reproductive organs and the kidneys of the unmated males, their increased fat and decreased ash content and the considerable incidence of degenerated testes and coagulating glands among them, at 286 days of age, suggest that in the long run, in rats, cohabitation is necessary to maintain testicular androgen secretion at a normal level. It has been well established that the accessory reproductive organs develop and are maintained by testicular androgens. The seminal vesicles and the levator ani muscle are used as androgen bio-assay organs ; the penis is used as an external index ofandrogenic activity. The penis muscles in castrated rats respond to androgens (Wainman & Shipounoff, 1941) and the coagulating glands in crease in a nearly linear fashion under the influence of testosterone (Mann & Parsons, 1950). The kidney has also been shown to respond in weight to androgens (Korenchevsky & Dennison, 1934). The figures on body composition suggest the same explanation. The unmated males contained 72% more fat. Castrates of all species tend to deposit fat. A decrease in fat content in androgen-implanted male rats has been reported (Kochakian & Webster, 1958). The unmated males contained 6% less ash and the pelvis weight was 10% lower. In farm animals castration reduces the boneto-muscle ratio (Turton, 1962) as in our unmated males. Information on boneto-muscle ratio and on the effect of androgens on mineral content in rats is scanty (Gardner & Pfeiffer, 1943), but in so far as information is available, ossification in castrates appears to be favoured by moderate doses of androgen as in man. The negative correlation between the weight of the adrenal glands and that of the accessory reproductive organs suggests that at an early age the growth of the adrenals is checked by a hormone which stimulates the accessory reproductive organs. This hormone is probably testicular androgen. As to the opposite

9 Effects of cohabitation on the male rat 359 action, Parkes (1945) states that the adrenals of rats exert noticeable androgenic activity only in castrates. The atrophy ofthe accessory reproductive organs and the limited but clear-cut degeneration of the testes and the coagulating glands at 286 days of age, under conditions which prevented heterosexual contact, confirm the statement of Steinach (1936) regarding the status of the reproductive system in segregated males. Steinach's claim that olfactory stimuli from females can maintain the reproductive system or restore it to normal has not been tested. Beach (1942) conducted copulation tests with males 100 to 110 days of age, which were raised in isolation, in cohabitation or in segregation (i.e. in an allmale group) and which weighed on the average 326, 264 and 251 g, respectively. Copulatory activity decreased in the same order. Beach states that his results differ from those of Steinach (1936) but suggests that the higher incidence of copulators among the isolated males was produced by greater excitability due to the novelty of contact with a second animal and to greater weight. This does not necessarily contradict the statement of Steinach that isolation of males for periods of 6 to 10 months brings about atrophy of the reproductive organs. Beach also suggests that the difference in incidence of copulators between the males raised in cohabitation and those segregated (which appear raised under more comparable conditions) had been the result of homosexual tendencies in the segregated males ; this difference can now be reinterpreted in to have been the light of the present findings, and explained by reduced androgen production in the segregated males as compared to males in cohabitation. REFERENCES Barnett, S. A. (1963) A study in behaviour, p Methuen, London. Beach, F. A. (1942) Comparison of copulatory behavior of male rats raised in isolation, cohabitation and segregation. J. genet. Psychol. 60,121. Gardner, W. U. & Pfeiffer, C. A. (1943) Influence ofestrogens and androgens on the skeletal system. Physiol. Rev. 23, 139. Kochakian, C. D. & Webster, J. A. (1958) Effect of testosterone propionate on the appetite, body weight and composition of the normal rat. Endocrinology, 63, 737. Korenchevsky, V. & Dennison, M. (1934) Effect on male rats of the simultaneous administration of male and female sexual hormones and the relation to the assay of the hormones. Biochem. J. 28, Mann, T. & Parsons, U. (1950) Studies on the metabolism of semen. 6. Role of hormones. Effect of castration, hypophysectomy and diabetes. Relation between blood glucose and seminal fructose. Biochem. J. 46, 440. Parkes, A. S. (1945) The adrenal-gonad relationship. Physiol. Rev. 25, 203. Steinach, E. (1936) Zur Geschichte des maennlichen Sexualhormon und seiner Wirkung beim Säuge tiere und beim Menschen. Wien. klin. Wschr. 49, 161. Turton, J. D. (1962) The effect of castration on meat production and quality in cattle, sheep and pigs. Anim. Breed. Abstr. 30, 447. Wainman, P. & Shipounoff, G. C. (1941) The effect of castration and testosterone propionate on the striated permeai musculature in the rat. Endocrinology, 29, 975.