THE INFLUENCE OF AGE AT CASTRATION ON THE SIZE OF VARIOUS ORGANS

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1 THE INFLUENCE OF AGE AT CASTRATION ON THE SIZE OF VARIOUS ORGANS BY JOHN R. BAKER. From the Department of Zoology and Comparative Anatomy, Oxford. (Received $th February 927.) (With Three Text-figures.) INTRODUCTION. IT has been known for a long time that the effects of castration are more marked if the operation is performed early than If It Is performed late. It was the object of the experiment described below to pet this fact upon a quantitative basis. I wish to thank Dr R. A. Fisher for most kindly explaining to me how to deal statistically with small samples, and Mr F. C. Sherlock for skilful practical assistance. Mr W. Hanson kindly arranged for his pigs to be castrated by a veterinary surgeon at the appropriate ages, and Messrs Marsh and Baxter cooperated In killing some of them and removing the necessary organs. METHOD. The experiment was carried out upon 7 male Large White pigs. Pigs were chosen because their large size enabled the small organs such as the pineal and pituitary to be weighed accurately. A pedigree Large White boar was the father of all the pigs Investigated. Their mothers were two Large White sows, not of pedigree stock. One of them was a daughter of the other, by the same boar that was the father of all the experimental animals. These facts are mentioned to show that the experimental animals were all closely related and unlikely to differ much In their Inheritance as regards the size of the organs studied. Six pigs were castrated at approximately 5 days, seven at days, and two at 2 days, while two were left uncastrated. In most farrows some pigs were castrated at-one age and some at another, so as to discount differences In size of organs due to environment rather than age at castration. All the pigs were killed at approximately days from birth, and the weight of each noted. The weight was obtained as follows (or In a very similar way). When the pig had been stuck with a knife, the escaping blood was caught In a bucket and weighed. This weight was added to the weight of the body, which was taken Immediately afterwards on a spring balance. From the total weight thus obtained was subtracted the weight of the large Intestine with Its contents. (It was not thought necessary to weigh the contents separately, since the weight of the large Intestine without the contents Is small.) There was little food In the small Intestine since the pigs were starved for a day before death, and for the same reason the contents of the stomach were usually small, but were weighed and

2 88 JOHN R. BAKER subtracted from the body-weight if not negligible. -5 kg. was subtracted for the weight of urine If the bladder was distended to the usual extent; otherwise the urine was weighed. The figure beyond the decimal point in the net weight expressed In kilograms Is not dependable. As soon as possible after death, the following organs were removed and placed in stoppered glass bottles: veslculae seminales, bulbo-urethrals (= Cowper's glands), adrenals, thyroid, pineal, and pituitary. These organs were then removed to the laboratory;, dissected free from fat and loose connective tissue, and weighed. A certain amount of error must necessarily have been caused by evaporation. The first four were weighed to the nearest 5 milligrams (or more accurately), the last two to the nearest milligram. For purposes of comparison the were multiplied by (to avoid decimals to some extent) and expressed as percentages of the net body-weight. The whole of the results obtained In this Investigation are given In the Appendix. The first column gives the serial number of each pig. The second column Indicates to which farrow each pig belongs. All the pigs designated by the same letter were born In the same farrow. The remaining columns are sufficiently explained by their headings. In order to present the figures given In the Appendix In a way In which they could be appreciated, the mean (x, as percentage of body-weight) of all the organs mentioned above of all the pigs castrated at each age were calculated for comparison with one another and with the mean of the same organs of the uncastrated pigs. The ordinary method of testing the significance of the differences between means, consisting In the calculation of the Probable Errors of the means, Is not really applicable to small samples such as those with which I had to deal. The following method was therefore employed to test the significance of the difference between two means. The Standard Error was calculated in each case from the following formula, the sums of the squares of the deviations from both means being lumped together: /7i i\ / S.E. = J - + T n J V \ a bj\ where a and h are the number of Individuals castrated at each age, d Is the deviation of each weight from the mean weight of Its group, and n = a + b 2. The difference between the means (or the difference between the mean of one group and the single observation constituting the other group) was then divided by the S.E., and a figure i obtained. To find whether t was sufficiently large to be significant, reference was made to the table of / given in Fisher's work on statistical methods (92s), where the lowest significant value of t for each value of n is given. In this table various grades of probability are tabulated; but only the P = -5 column was used, since this is accepted as a test of significance. The blanks in the Appendix are chiefly caused by the carelessness of butchers in failing to keep the organs of different pigs separate.

3 Influence of Age at Castration on the Size of Various Organs 89 RESULTS. The results are shown in full in the Appendix and graphically in Figs, and 2. Table I is an abridgment of the Appendix. In the graphs the mean weight of each organ at days (expressed as a proportion of the body-weight) is plotted against age at castration. In Fig. the ordinate is on a much smaller scale than in Fig. 2, so as to include the relatively huge of the vesicuiae seminales and bulbo-urethrals in uncastrated pigs. The of the organs of the uncastrated pigs are plotted as though these pigs had been castrated on the day of death (approximately days). 4r Vesicuiae seminales (xrooo) / I o Si 2 5 Thyroid (x ) Bulbo-urethrals (x ) a o 2 «* m es 5* I Bulbourethrals 2 Not castrated Age at castration (days) (equivalent to. castration at days Fig.. =age at death) JS CO g Table I. TOO 2 Age at castration (days) Fig. 2. Vesicuiae semimks (xiooo) /. Adrenals (x IOOO) Pineal (xloojooo) Pituitary (xio,ooo) Age at castration (days) So 2 Not castrated Mean body (kg.) at days Mean at days ( x, as percentage of body-weight) Vesicuiae seminales IO'I 56 Bulbourethrals 2* I4-4 2 Adrenals Thyroid 4- H'9 8-9 Pineal Pituitary -5 Q Taken by themselves, these graphs are not very helpful, for they show only the means and not the range of variation in each case. For instance* it is not possible to determine by examination of Fig. 2 whether pigs which have been castrated at days have significantly smaller thyroids than those which have

4 9 JOHN R. BAKER been castrated at 5 or 2 days. To remedy this I have written the word "significant" between the comparable figures in the table, to indicate those cases in which it was age at castration, and not chance alone, that determined the differences. The significance of the differences between means was calculated as explained under the heading "Method." Each organ will now be considered separately. Vesiculae seminales. It is well known that the vesiculae are small in castrated mammals. Figs. and 2 show graphically the effect of castration at different ages. It will be noted that it makes little, or possibly no, difference whether the pig be castrated at 5 or days, the difference between the means not being statistically significant; but when castration is delayed to 2 days the difference in size is very great s for the glands are about four and a half times as heavy. In uncastrated pigs the vesiculae are more than two hundred times as large as in those which have been castrated at 5 or days. We must conclude that the influence of the testes upon the vesiculae is felt chiefly, or perhaps entirely, after the age of days; and that the effect is very much greater between 2 and days than between and 2 days. Bulbo-urethrals. Nagel (quoted by Marshall (922)) states that these glands are of normal dimensions in castrated men, but Schneidemiihl (also quoted by Marshall) records atrophy after castration in other mammals. In pigs I find that castration has a very marked effect. If castration is deferred.to 2 days the bulbourethral glands grow much larger than they do in pigs which have been castrated at 5 or days. In uncastrated pigs they are very much larger, though the difference is not so great as in the case of the vesiculae seminales (Figs. and 2). Adrenals. It has been stated by Paton (9) that the adrenal cortex tends to enlarge after castration. My experiments (which deal only with the gland as a whole) show on the contrary that pigs castrated at days have smaller adrenals than those castrated at 2 days. When the mean for all pigs castrated at days is compared with the figure for the single individual castrated at 2 days, the value of t is found to be 2-55, significance being reached at The difference between pigs castrated at 5 and days is not significant, but might become so with larger samples (Fig. 2). Thyroid. The figures for the thyroid indicate that it makes little or no difference when the animal is castrated, though this organ varies in size far more than the others. A most interesting fact however emerges, namely that all the male pigs born in the same farrow tend to have thyroids of about the same size, while the different farrows differ markedly from one another in this respect. This is clearly shown in Fig., in which pigs born in the same farrow are bracketed together irrespective of age at castration. Environmental influences are evidently important in determining the size of this organ. Pineal. From my experiments it appears that in pigs time of castration has no effect upon the size to which the pineal grows, though Biach and Hulles (quoted by Marshall (922)) record atrophy of the pineal after castration in kittens. An interesting point with regard to the pineal is that it tends to grow to a certain size

5 Influence of Age at Castration on the Size of Various Organs 9 (the mean of all individuals studied is -62 gm.) irrespectively of the size to which the body grows. Thus a small pig has a relatively large pineal while a large pig has a relatively small one. This can be expressed mathematically by finding (a) the standard deviation from the mean of the actual and (b) the standard deviation from the mean of the expressed as percentages of the body-weight. The figures for (a) and (b) are 2 and 2 per cent, respectively. One would of course have supposed that (a) would have been much larger than (b). Pituitary. Tandler and Gross, Fichera, Cimorini, and Sehonberg and Sakaguchi (quoted by Marshall (922) and Lipschiitz (924)) record increase in size of the pituitary after castration in rabbits, cattle, buffaloes, dogs and men, while Livingston (quoted by Marshall) finds no effect in rabbits. My findings indicate that in pigs castration has little or no effect upon the growth of the pituitary. 25 4> u o ft m es os O a X Fig.. Body-weight. There is considerable variation in body-weight, and, not unnaturally, a tendency for all pigs born in the same farrow to approximate more closely to one another in weight than to pigs born in other farrows. Since in most farrows some pigs were castrated at one age and some at another, this fact does not render it impossible to note the effect of early and late castration on weight. From the figures given in Table I it will be noted that the heaviest pigs are those which were castrated at 5 days; but the means are not statistically different. CONDITIONS IN THE ADULT UNCASTRATED MALE. An uncastrated male pig (the father of all the experimental animals) was killed at the age of several years for purposes of comparison. His body-weight was 24 kg. The relative of his organs are shown in Table II, which should be compared with Table I. It will be observed that the vesiculae seminales and bulbo-urethrals are smaller

6 92 JOHN R. BAKER in this boar, relatively to the body, than those of an uncasttated pig days old. In other words, the body grows faster than these organs after days. The same seems to apply to the adrenals. The head and neck had unfortunately to be sold, so there are no figures for the thyroid, pineal, and pituitary. Table II. (x, as percentage of body-weight) Vesiculae seminales 97 Bulbourethrals 85 + Adrenals 5-6 DISCUSSION. The experiment described above shows that the testes exert a very marked influence on the vesiculae seminales and bulbo-urethrals between the ages of and 2 days, and have also a significant effect upon the adrenals during this period* Between 5 and days, on the contrary, the effect is slight if there is any at all. (The differences between the means are not significant statistically, but since they are all in the same direction as the differences between the -day and 2-day means, they might well become significant with larger samples.) There are two conceivable explanations of this fact: after days, either the testes pour out a larger amount of hormone into the blood-stream or the glands respond more readily to the hormone. From histological considerations I regard the second alternative as the more probable. If the gonad hormone suddenly began to be poured out at a certain time during development, one would expect marked differences between the condition of the interstitial cells of the testes before and after that time; for there is a great deal of evidence that the hormone is secreted by these cells. That there are no such differences was shown by Ancel and Bourn in their well-known researches (9) on the interstitial cells in the testes of the pig. These authors write: "Les cellules interstitielles du verrat offrent les memes caracteres cytologiques que celles de 'animal jeune." This fact is clear from their figures. Figs. 5 and 7 in their paper, showing the structure of the interstitial cell in the suckling or slightly older pig, should be compared with Fig. 7, showing the adult condition. In both cases the interstitial cells are loaded with secretory products. Further, these cells are abundant at all stages. These considerations show that Steinach's term "puberty gland," as a synonym for the interstitial gland of the testis, is rather misleading. They give some support to Crew's hypothesis (92) of intersexuality in goats and pigs. Crew supposes that there is a more or less definite time in the development of each accessory organ, during which it is capable of being stimulated to develop by the testicular or ovarian hormone, while before or after this time no amount of gonad hormone will affect it. The investigations described in this paper, taken in con-

7 Influence of Age at Castration on the Size of Various Organs 9 junction with Ancel and Benin's histological results, seem to show that there is at any rate a time before which the hormone (though present) has little or no effect, and that the accessory sexual organs become progressively more and more capable of responding to it. It is apparently at about the age of days that the vesicuke seminales and bulbo-urethrals begin to respond in the pig, and between 2 and days their response is very much greater than it was between and 2 days. Careful examination of the figures given in the Appendix reveals the fact that in each castration-age-group, the pigs which have large vesicuke seminales tend to have large bulbo-urethrals and those with small vesicuke tend to have small bulbo-urethrals. Previous considerations rule out the possibility that the correlated size of these organs is to be accounted for by early or late outpouring of the testishormone. The amount of testicular hormone poured out (before castration) can hardly be the cause, for it is well known from experiments that a portion of testicular substance only one-fiftieth the size of two normal testes suffices in mammals for full development of the accessory sexual organs. It is not a case of heterogony, for it is not simply the large pigs that have these organs highly developed. The case is similar to what everyone knows to be the case in men; namely that some have secondary sexual characters more developed than others. It appears to me possible that the potency of the sex-chromosomes may vary and affect the size to which the accessory organs grow, just as we have good reason to suppose that the sex-chromosomes of insects vary in potency and affect the degree of development of the sexual organs; though there is 5 of course, this difference, that in mammals the accessory organs cannot develop save in the presence of the testicular hormone. (This explanation would of course not cover "eunuchoid" individuals, for here the small size of the accessory sexual organs is undoubtedly caused by the very small size of the testes.) This hypothesis would be rendered the more likely, if it could be shown that the size of these organs is not determined by general metabolic conditions. SUMMARY. Male pigs were castrated at the ages of 5, and 2 days. Others were not castrated. All were killed at days. The of various sexual and endocrine organs at days were determined, and a comparison instituted between pigs castrated at each age. The following results were obtained: () The vesicuke seminales, bulbo-urethrals, and adrenals are smaller when castration is performed at days than when it is performed at 2 days. There is much less difference (possibly none) according to whether castration is performed at 5 or days. In uncastrated pigs the vesicuke seminales and bulbourethrals are enormously larger even than those of pigs castrated as late as 2 days. (2) It is probable that some change occurs in these organs at about the age of days, rendering them progressively more and more sensitive to the gonad hormone. BjEB*viii f

8 94 JOHN R. BAKER () The thyroid,, pineal, and pituitary are not significantly affected by time of castration. Body-weight also is not significantly affected. A general discussion is given on the causes governing the size of the accessory sexual characters in mammals. REFERENCES. ANCEL, P. and BOUIN, P. (9). Arch, de Zool. Exp. Ser., 4, 47. CREW, F. A. E. (92). Proc. Roy. Soc. B, 95, 9. FISHER, R. A. (925). Statistical Methods for Research Workers. (Oliver and Boyd, Edinburgh.) LIPSCHUTZ, A. (924). The Internal Secretions of the Sex Glands. (Heffer, Cambridge.) MARSHALL, F. H. A. (922). The Physiology of Reproduction. (Longmans, Green and Co., London.) PATON, N. (9). Regulators of Metabolism. (Macmiiian, London.)

9 APPENDIX. at h s) 2 a 2 2 Bodyweight at death (kg.) s 89 Vesiculae seminales I-8I -48 I-I i-55 i' i body i-68 i-4-4 I-IO i i' * Bulbo-urethrals 2-7 ' IO 2- S i body ' Adrenals.,_ S-i s. 6s _ body 5* ' ' _. Thyroid i body i io _ Pineal * "* body _ -8.~ Pit o