THE SURVIVAL OF THE SPERMATOZOON IN THE DOMESTICATED FOWL

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204 THE SURVIVAL OF THE SPERMATOZOON IN THE DOMESTICATED FOWL BY ARTHUR WALTON AND E. O. WHETHAM. (Institute of Animal Nutrition, University of Cambridge.) (Received iyth November, 1932.

1 204 THE SURVIVAL OF THE SPERMATOZOON IN THE DOMESTICATED FOWL BY ARTHUR WALTON AND E. O. WHETHAM. (Institute of Animal Nutrition, University of Cambridge.) (Received iyth November, 1932.) IN the large majority of animals which have internal fertilisation the effective length of life of the spermatozoon within the body of the female is extremely short. In no mammal is it known to extend from one oestrous period to another. A certain number of more exact determinations have been made. In rabbits, for example, fertilising capacity is retained for about 30 hours (Hammond and Asdell, 1926), and in sheep fertility diminishes presumably from death of the spermatozoa about 27hours after mating(mckenzie and Phillips, 1930). Data on the survival of motility, which it is to be supposed coincides approximately with fertilising capacity, has been collected by Hammond and Marshall (1925, chap. 6) and Yochem (1930), and the latter found 41 and 17 hours' survival in the uterus of the guinea pig and rat respectively. However, there are also many known instances of long survival, especially in the invertebrates. In the queen bee, for example, sperm may be stored 7 years and still retain its fertilising power (Bishop, 1920; Courrier, 1921), and in other Hymenoptera spermatozoa may be stored during hibernation or throughout the whole period of egg-laying. Many invertebrates have spermathecae in which spermatozoa are stored presumably for considerable periods. Among the vertebrates prolonged survival is much less common. Some teleosts which have internal fertilisation store spermatozoa for long periods (van Oordt, 1928). In some bats, which have their mating season in the autumn and in which the follicles do not ripen till the following spring, the spermatozoa remain alive within the female during the winter (Courrier, 1924; Redenz, 1929). In the male mammal spermatozoa may also be stored for a considerable time in the epididymus (Benoit, 1925, 1926; Hammond and Asdell, 1926; Redenz, 1924, 1925, 1926; Young, 1929, 1929 a, 1931). In all these cases of long survival the spermatozoa are stored under what are probably very favourable conditions, of which perhaps the most important is the dense concentration of the sperm-suspension. This is universally characteristic. It has long been known that spermatozoa survive better in concentrated suspension (Gemmill, 1900; Cohn, 1918). Gray (1928, 1928 a, 1931) provides the most likely explanation, having shown for the sea-urchin that dilution activates the spermatozoon and that the rate of senescence is an inverse function of the activity. In birds, although there is apparently no storage in concentrated mass, yet in many species insemination is sufficient to fertilise a large number of eggs laid over

2 The Survival of the Spermatozoon in the Domesticated Fowl 205 a long period. In the domesticated fowl the duration of fertility may be 3 weeks or occasionally longer, and as many as eleven fertile eggs may be laid after removal of the cock (refs. in Hammond and Asdell, 1926; Crew, 1926; Warren and Kilpatrick, 1929). It was the apparent anomaly of survival without evident concentration which particularly interested us. Many investigators have made microscopic preparations of the contents of the oviduct of a fertile hen in an effort to find spermatozoa (refs. in Ivanoff, 1924). Anderson (1922) could find no spermatozoa 15 hours after copulation. As far as we are aware only two papers exist in which the writers describe finding spermatozoa more than 24 hours after removal of the cock. According to both Payne (1914) and Warren and Kilpatrick (1929) the flagellum of the spermatozoon is actually destroyed during or soon after the first day in the oviduct, but sperm heads may be present which, they assume, are capable of fertilising the ova. If this be so it is difficult to see how the sperm heads are able to maintain their position against the ciliary current and are not carried down the oviduct, and the fertilisation of the ovum by a tailless spermatozoon is a somewhat novel concept in biology. Our own experience has been the same as that of the majority of workers; in spite of extensive examinations both of the fluid contents of the peritoneal cavity round the ovary and of smears taken from different parts of the oviduct, in no case have we been absolutely convinced of the presence of spermatozoa, although we have occasionally encountered a doubtful specimen. However, it must be admitted that failure to find spermatozoa is not an absolute criterion of their absence, for they may be very thinly scattered and obscured in the manifold crevices of the oviduct. When a virginal hen is first placed with the male, eggs which are passing down or are present in the oviduct are not fertilised. Presumably therefore fertilisation occurs not later than entry into the top of the oviduct. Harper (1904) found ova fertilised before they had entered the oviduct and suggested even that the spermatozoon might penetrate the follicular membrane of the ripe ovum when it was very thin and fertilise the egg before release. This assumes the constant presence of fertile spermatozoa in the neighbourhood of the ovary. In order to account for the failure to find spermatozoa in this situation Ivanoff (1924) postulated that almost immediately after insemination the sperm penetrated not only the follicular membranes of the ripe ova but also those of the immature, and in this way a whole clutch of eggs ripening on the ovary might be fertilised at once, superabundant spermatozoa being then eliminated. Since the publication of IvanofFs experiments, Crew (1926) found that the introduction of a second male reduced the period of the duration of fertility of the first cock by about 50 per cent. It is clear therefore that if Ivanoff's hypothesis is correct, replacement of spermatozoa within the immature ovum must take place. In order to test his hypothesis Ivanoff washed out with a disinfectant the oviduct and body cavity of a hen that had copulated some days previously. The hen continued to lay fertile eggs although it seemed likely that the treatment would have effectively rid the oviduct and body cavity of any spermatozoa present. His account is very short and lacking in detail but suggestive, and we thought the experiment might be interesting and valuable to repeat.

3 206 ARTHUR WALTON ande. O. WHETHAM EXPERIMENTAL. Our first approach to the subject was to find if the number of eggs fertilised bore any relationship to the number ripening on the ovary at any one time. An ovum has two distinct periods of growth in a bird. During the first when growth is extremely slow the egg attains a diameter of 6 mm. The second, when growth is many times faster, is from 6 mm. up to the mature yolk of 30 mm. (Riddle, 1911; Romanoff, 1931). It was this second period which interested us. If Sudan III is fed to a laying fowl it is excreted in the yolks, and since yolk is laid down in concentric layers, a band of stained yolk will be present in all the ova ripening at the time. The position of the band in the egg after it is laid will depend on the stage of maturation of the particular egg at the time of the dose, the most mature yolk having a narrow band at the periphery while in the more immature yolks the colour is nearer the centre. A batch of six laying virginal hens were given 0-2 gm. Sudan III orally and then placed for 1 day only with fertile males. All eggs laid subsequently were incubated for 5 days and then examined for Sudan III and fertility. Unfortunately only three birds out of six were fertile and a second experiment, not here recorded, was a complete failure since none of the birds laid fertile eggs. However, the available data are sufficient to bring out the salient points. Table I records the result of examining the incubated eggs over a period of 5 days previous to the feeding of the Sudan III and for 22 days subsequently. It will be noted, first, that any eggs laid the day following the treatment were neither fertile nor stained with Sudan III. These eggs had already been liberated from the ovary and were on their way down the oviduct. Secondly, the length of fertility bears no exact relationship to the number of ova ripening on the ovary and stained with Sudan III. On the whole fertility extends a few days longer than the occurrence of the Sudan III, but traces of Sudan III are very difficult to identify in a fertile egg in which organic development and formation of blood has taken place. It was apparent that critical results could not be obtained by this method. In order to carry out the "Ivanoff" experiment efficiently we wished to make sure that the disinfectants we used were definitely spermicidal. We are indebted to Dr Baker and Dr Voge who very kindly suggested a number of substances. Of these we have used solutions of toluquinone, hexyl resorcinol and formalin all made up in normal saline, in concentration definitely toxic to mammalian sperm (Baker, 1931, 1932). Toluquinone proved definitely poisonous to the fowl. One bird did not survive the period of recovery from the anaesthetic and one bird lived but a few days. Hexyl resorcinol and formalin were tested for toxicity on cock spermatozoa in vitro. Spermatozoa from the vas deferens suspended in about equal quantities of egg white and normal saline served as a control, and in this solution the spermatozoa remained motile for nearly 24 hours. On mixing a drop of the control suspension with one or two drops of the spermicide it was found that the spermatozoa were immobilised immediately by the hexyl resorcinol and within 5 minutes by the formalin. After a trial period to ensure that the hens were laying fertile eggs the

4 The Survival of the Spermatozoon in the Domesticated Fowl 207 fowls were separated from the males for 2 days before operation. The operation was carried out under anaesthesia obtained by intramuscular injection of 0-75 c.c. per kg. of " Roche-Numal," a derivative of barbituric acid, kindly supplied by the Hoffman-La Roche Co., Ltd. This usually gave deep anaesthesia but occasionally a little ether or chloroform was used in addition. The incision was made between the last two ribs on the left side. In some birds free access to the body cavity surrounding the ovary was blocked by one or more large yolks and these we had to remove by suction from a filter pump. (The number of yolks removed bore no relation to the subsequent results.) The end of the oviduct was then secured and spermicide run into it from the fimbriated end until considerably distended. It was then ligatured and left until the end of the operation, in order that the disinfection of the fimbriated end should be as complete as possible and in order that a considerable volume of fluid should pass down the oviduct. In several cases the fluid passed completely down the oviduct and out at the cloaca, being found in the cage an hour or so later. We then washed out the body cavity thoroughly, including the outside of the fimbriated end of the oviduct and round the ovary, removing the fluid as far as possible through the suction tube. If this was not done we found that the fluid got into the lungs through the air sacs and obstructed the bird's breathing. Respiratory movements of the bird aided the thorough distribution of the fluid in the body cavity. About 200 c.c. of fluid usually passed into and out of the bird during the operation, which occupied about 1 hour. The wound was then closed with catgut ligatures. Recovery from the anaesthetic was complete in about 4 hours, and on the following day, with the exception of the two birds treated with toluquinone, the birds appeared practically normal and could be returned to the laying pens. The results are shown in Table II. The results of incubating the egg for 5 days previous to removal of the bird from the mating pen are recorded. It will be noted that on the day following removal, egg-laying was generally inhibited. On the day of the operation (column B) the egg was generally laid while the bird was going under the anaesthetic before the operation. A period of inhibition of egg laying followed the operation. The period is variable but to some extent depends upon the strength and apparent toxicity of solution used. Thus with 1/16 per cent, hexyl resorcinol birds Nos. 7, 8, 9 and 10 did not lay until 32, 25, 18 and 28 days respectively after the operation. With 1/32 per cent, hexyl resorcinol No. 11 laid a soft shelled egg on the 9th day and a cracked egg on the 13th. The third egg laid on the 15th was, however, fertile, but subsequent eggs laid with regularity proved infertile. With 1/16 per cent, formaldehyde, one bird, No. 12, laid fertile eggs on the nth, 13th and 14th days, and infertile eggs with normal periodicity subsequently. No. 13 laid a broken egg on the 18th and an infertile egg on the following day, but did not continue to lay regularly. The third bird, No. 14, laid fertile eggs on the 12th and 13th days and infertile eggs subsequently. Of the controls with NaCl hen No. 15 laid a fertile egg 3 days after the operation, an egg laid on the following day was unfortunately lost, and eggs laid subsequently were infertile. The last three birds, Nos. 17,18 and 19, did not lay until the 13th, 14th and 12th days respectively after the operation and all eggs were infertile. It will be seen from the data available that

The Survival of the Spermatozoon in the Domesticated Fowl 209 the fertility of the eggs laid is not significantly affected by the operation either with regard to the percentage fertility or the time

6 The Survival of the Spermatozoon in the Domesticated Fowl 209 the fertility of the eggs laid is not significantly affected by the operation either with regard to the percentage fertility or the time of survival after removal of the cock. These results tend to confirm Ivanoff's hypothesis, but on this hypothesis the number of eggs fertilised is determined by the number ripening on the ovary at the time of insemination and survival is determined by the time of liberation of these eggs. Now from the table it will be seen that the maximal survival is unaffected in time while the number of fertile eggs laid is considerably reduced. Since this might be due to changes in the ovary a number of laying birds were Sudan III fed and either 1 or 2 days later operated on as before and killed at specified times after the operation. The ovary and peritoneal cavity were examined. The results are shown in Table III. Two birds were killed a week after operation. Fowl No. 20, which had been treated with 1/16 per cent, hexyl resorcinol, showed obvious pathological changes. Degenerate yolks were present on the ovary, there was considerable exudate and detritus in the peritoneal cavity and many connective tissue adhesions. Fowl No. 21 was treated with more dilute spermicide (1/32 per cent, hexyl resorcinol). This bird laid an egg immediately after operation which contained an outer ring of Sudan III. Killed a week later it showed an almost normal appearance and was about to lay, an egg being found in the oviduct. A trace of Sudan III was found in the centre of this egg, showing that it must have been quite immature at the time of operation. Presumably yolks intermediate in development between that laid just after the operation and this one had been absorbed. Large normal yolks without a trace of Sudan III were present on the ovary. Fowl No. 22 was treated with the same concentration of hexyl resorcinol (1/32 per cent.) but killed only 2 days later. The peritoneal cavity and internal organs were normal except for the ovary. This showed two large degenerate yolks, both with surfaces much crenated. Both contained irregular circles of Sudan III. A small yolk was apparently normal and contained a little Sudan III near the periphery, showing that little or no deposition of yolk had occurred subsequently to the operation. Similar results were obtained with treatment with formaldehyde and with normal saline. It may be mentioned that this last series of experiments was carried out during the summer when birds may spontaneously stop laying. Great care was, however, taken to select only birds in full laying condition and without any signs of moulting. CONCLUSIONS. The experiments were designed primarily to test the hypothesis of Ivanoff that in the domesticated fowl immature ova are fertilised on the ovary and that survival of fertility after removal of the male is not necessarily a criterion of survival of the spermatozoon in the female tract. We have shown that there is a rough correspondence between the number of eggs in the stage of maturation in which yolk (and incidentally Sudan III) is laid down and the number of fertilised eggs which are laid subsequently. The correspondence is, however, not absolute and may be coincidental. A repetition of IvanofFs own experiment (irrigation of the genital tract with spermicide) has confirmed his results that fertile eggs may be laid subsequently. The time of survival has not been significantly altered and it has been

7 210 ARTHUR WALTON ande. 0. WHETHAM shown that the fact that fewer fertile eggs are laid subsequently to treatment can be explained by degeneration and absorption of the larger ova. Nevertheless we are by no means convinced that the hypothesis is correct. The apparent absence of spermatozoa from the oviduct may be explained by their wide distribution among the folds and crevices of the oviduct, a position which would also render them practically inaccessible to the spermicidal solution, and while in general the dispersed spermatozoon survives but a limited period in the uterus of the mammal it is not inconceivable that the conditions within the oviduct of the fowl may be peculiarly favourable to maintenance and survival of fertilising capacity. For example, while in mammals the sperms are produced from testes in the scotum at a temperature below that of the body cavity and uterus, in birds the testes are located internally and sperms are produced at the same temperature as that of the oviduct. The penetration by the spermatozoon of a relatively thick membrane such as covers the surface of the immature ovum and its subsequent fertilisation, although not necessarily impossible, is on general biological principles governing fertilisation unlikely and will require more conclusive experimentation before acceptance. Experiments to elucidate the problem further are contemplated. SUMMARY. 1. Fertility in the hen persists after removal of the cock for about 15 days. Normal spermatozoa are not found in the oviduct throughout this period. 2. Confirming Ivanoff we have shown that fertility persists after irrigation of the peritoneal cavity and oviduct with spermicidal solution. 3. Survival of fertility after treatment persists as long as in the unoperated bird, but the number of fertile eggs laid is reduced by degeneration of the ova. 4. These results conform to Ivanoff's hypothesis that spermatozoa may penetrate the immature ova previous to ovulation, but reasons are advanced for regarding this assumption as unwarranted without further evidence. We are indebted to Mr E. T. Hainan and Mr M. Pease of the Poultry Institute for much help and advice. REFERENCES. ANDERSON, W. S. (1922). Kentucky Agric. Expt. Sta. Bull BAKER, J. R. (1931). Journ. Hyg. 31, 189. (1932). Journ. Hyg. 32, 171. BENOIT, J. (1925). Rtcherches sut les votes excritrices du testicule chez let mammiferes. Strassburg. (1926). Arch. ccanat. cthutol. et d'embryol. 6, 175. BISHOP, G. H. (1920). Journ. Exp. Zool. 31, 267. COHN, E. J. (1918). Biol. Bull. 34, 167. COURRIER, R. (1921). C. R. Soc. Biol. 85, 941. (1924). Arch, de Biol. 34, 369. CREW, F. A. E. (1926). Proc. Roy. Soc. Edin. 46, 230.

8 The Survival of the Spermatozoon in the Domesticated Fowl 211 GEMMIIX, J. F. (1900). Journ. Anat. and Physiol. 34, 163. GRAY, J. (1928). Brit. Journ. Exp. Biol. 5, 337. (1928 a). Brit. Journ. Exp. Biol. 5, 345. (1931). Journ. Exp. Biol. 8, 202. HAMMOND, J. and ASDELL, S. A. (1926). Brit. Journ. Exp. Biol. 4, 155. HAMMOND, J. and MARSHALL, F. H. A. (1925). Reproduction in the Rabbit. Edinburgh. HARPER, E. H. (1904). Amer. Journ. Anat. 3, 349. IVANOFF, E. I. (1924). C. R. Soc. Biol. 41, 54. MCKENZIE, F. F. and PHILLIPS, R. W. (1930). Proc. Amer. Soc. Animal Production, p. 3. VAN OORDT, G. J. (1928). Tijdtchrift der Ned. Dierkundige Vereemging, jde ser. 1, 1. PAYKE, L. F. (1914). Oklahoma Sta. Circ. 30. REDENZ, E. (1924). Arch.}. Entwickelungsmech. 103, 593. (1925). Arch.f. Enttcickelungsmech. 106, 290. (1926). Wurzburger Abdhandlign. aus d. Geiantgebeit d. Medizen. 24 (5), 107. (1929). Zeit.f. Zellforch. u. Micros. Anat. 9, 734. RIDDLE, O. (191 I). Journ. Morph. 22, 455. ROMANOFF, A. L. (1931). Biochem. Journ. 25, 994. WARREN, D. C. and KILPATRICK, C. (1929). Poultry Sci. 8, 237. YOCHEM, D. E. (1930). Biol. Bull. 56, 274. YOUNG, W. C. (1929). Journ. Morph. and Physiol. 47, 479. (1929 "). Journ. Morph. and Physiol. 48, 475. (1931). Journ. Exp. Biol. 8, 151.

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