>HE VITALITY OF THE SPERMATOZOA IN THE MALE AND FEMALE REPRODUCTIVE TRACTS

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1 >HE VITALITY OF THE SPERMATOZOA IN THE MALE AND FEMALE REPRODUCTIVE TRACTS BY JOHN HAMMOND, M.A. AND S. A. ASDELL, M.A., PH.D., Institute f Animal Nutritin, Schl f Agriculture, Cambridge. (With Tw Diagrams in text.) {Received ith June 96.) CONTENTS.. Intrductin..... Vitality in the male tract.. Vitality in the female tract. Discussin and cnclusins 5. Summary PAGE INTRODUCTION. THE fertility f a mammal depends first, n the number f va which are shed at each heat perid, secndly n the number f these which becme fertilised, and thirdly n the number f fertilised va which develp t birth. The secnd f these factrs is (except fr pathlgical cnditins, such as cclusin f the Fallpian tubes, etc.) mainly ne f time relatins between the life f the va and f the sperms. The duratin f fertility f the va has already been dealt with(i), it being shwn that they are nly capable f being fertilised fr abut - hurs after they leave the fllicle. The present paper is cnfined t an accunt f the life f the sperm. A determinatin f the length f fertile life f the sperms, bth in the male and female tracts f the rabbit, has been made. It must be stated at nce that the main pint treated is that f length f fertility rather than that f mtility. Wrk with Mr A. Waltn n the length f life f the sperm utside the bdy has shwn that althugh sperms under the cnditins f these experiments may still be mtile, yet their fertility has been lst, a pint which was indicated by Wlf's () results, and accrdingly a distinctin has been made fr the purpses f this paper. This distinctin appears t be justified, fr further evidence t be described indicates that under certain circumstances the sperms drawn frm the epididymis f a buck, althugh mtile, are infertile. While much wrk has been dne n the length f time f mtility the practical aspect f fertility appears t have been neglected. A study f the length f fertility f the sperms in the reprductive passages is a necessary preliminary in any cnsideratin f the cnditins under which artificial inseminatin may be practised. The length f life in the male tract gives an indicatin f the rder f lngevity which must be aimed at under the best cnditins utside the bdy; while, as vulatin in mst species ccurs at a definite in the reprductive cycle, an imprtant factr in success must be the capability sperms t live until vulatin takes place. In ther wrds, we require t knw the length f time in which successful inseminatin may ccur; thus Ander-

2 56 JOHN HAMMOND and S. A. ASDELL sn () states that wing t the shrt life f the sperms the time relatins between inseminatin and vulatin is imprtant in mares, and Ivanffu) finds that ^ ceptin as a result f a mare being inseminated utside "heat" is exceptinal. mst species cpulatin is allwed nly at the time f estrus, immediately befre the ccurrence f vulatin. In thse cases in which there is a prlnged estrus, r in which, as in man, cpulatin ccurs at any time in the cycle, fertility as measured by the percentage f cpulatins which are successful is lw. This may be explained n the assumptin that, if the sperms live in the female tract fr a limited perid nly, cpulatins which ccur earlier than this with reference t vulatin are sterile. The rabbit has been chsen as the experimental animal in this investigatin fr several reasns. The mst imprtant is that the de remains n heat fr lng perids and usually vulates nly n citus, vulatin succeeding citus by an average f hurs (Heape(s)); va may cnsequently be prduced at any definite time as required. Als as the prcess f reprductin in the rabbit has been wrked ut in detail () results culd be btained with greater certainty than if a fresh species were used. In additin, the rabbit has the advantage that it breeds rapidly, mates under bservatin and is cmparatively cheap t maintain.. VITALITY IN THE MALE TRACT. Methds. In rder t determine fr hw lng the spermatza remain capable f fertilisatin in the male tract, ligatures and divisin f the middle part f the epididymis between the upper and lwer ples were made (see Diagram I) n each side thrugh an abdminal incisin alng the linea alba. In a few f the earlier experiments n special precautin was taken t withdraw the testes t the scrtum frm the abdminal cavity after the peratin and in mst cases they returned naturally, but as ne case ccurred (F 5) where the testis was retained in the abdmen by adhesin t the fatty tissue surrunding the bladder, and especially as in this particular case the buck was sterile when mated, the testes were replaced in the scrtum after the peratin in all subsequent experiments. Care was taken nt t injure r ligature any bld vessels supplying the testes r epididymis during the peratin, fr damage t these sn causes marked degeneratin f these rgans. By this peratin the supply f newly-frmed sperms frm the testes was cut ff and the length f time that thse already frmed at the date f peratin, and stred in the lwer ple f the epididymis, culd remain capable f fertilisatin was tested by mating such bucks at definite intervals after the peratin with des n heat. The des used fr this purpse were carefully selected and fr the mst part were thse which had prduced a litter a few days previusly and the yung f which had been remved as sn as they were brn, r thse which were at the end (after the 6th day) f a pseud-pregnant perid. Occasinal cases f sterility frm adherent Fallpian tubes d, hwever, smetimes ccur, but des s suspefl have been mated t nrmal bucks afterwards and where they failed t prc a litter a pst-mrtem was made, the animal being marked as prbably sterile

3 Vitality f Spermatza in Male and Female Reprductive Tracts 57 t the time f the last knwn litter. N animal already under suspicin f ity was used in any f the experiments. The tatting f numbers in the ears f all rabbits and the systematic keeping f all breeding recrds enabled the writers t trace the histry f all animals, in mst cases since birth. The bucks used were tested fr fertility by mating with des befre they were perated n. The majrity were bucks which had been reared by the writers and were abut 9- mnths ld, well ver the age f puberty, which ranges frm abut (strain F) t 6 (strain E) mnths. In additin at the critical perids, lder bucks which had been successfully used as stud animals were als perated n. On the whle the duratin f life f the sperm in these lder bucks was rather less than in the yunger nes. In rder t ensure a gd supply f sperms in the epididymis the bucks were usually nt allwed t serve any des fr days t a week befre the peratin, while during this perid and between the time f peratin and mating they were especially well fed and kept in gd cnditin t lwer the chances f absrptin f sperm ccurring. In mst cases the buck was mated nce with the de n the first ccasin n which he was used after the peratin, n the secnd ccasin twice and n the third ccasin thrice. The purpse f the tw cpulatins n the secnd ccasin was t ensure if pssible that the des shuld nt be sterile thrugh lack f sufficient semen, the ttal amunt available in these bucks being limited by the supply stred up in the lwer ples f the epididymis befre the peratin. In this way an attempt was made as far as pssible t equalise the amunts f semen ejaculated in each f the first three des with which the buck was mated. In a few cases (E 7, a) where truble was experienced in getting the buck t mate at the appinted time it was killed and des were inseminated frm the fluid cntained in the lwer ples f the epididymes diluted with Ringer's r Waltn's fluid (buffered istnic salt slutin mixed with sugar). Table I shws the detailed results btained frm a number f such ^ ^ s. When the first results f the spermatza living as lng as 7 days after the peratin were btained it was at first dubted whether the cnnectin between the epididymis and testis had been effectively cut ff (see bucks C -c Diagram I. Shwing methd f peratin n bucks. L, L, ligatures; C, cut; T, testis; EH, head f epididymis; ET, tail f epididymis ; V, vas deferens.

4

5 Vitality f Spermatza in Male and Female Reprductive Tracts 59 s... p.... m V ).... C;.... 'u V ) ' g.... s.... I... * In V) $.... m b " " b.... b.n *... z.... 'u... PI Y.... PI b.... n.... PI N.... PI -... PI R O O.. - w *** * O ", m C w.t: ", S m

6 i6 JOHN HAMMOND and S. A. ASDELL and F ); the buck rabbits were therefre again bred t nrmal des t test pint and having prduced n litters were killed and examined by inspecting fluids in the vas and lwer ple f the epididymis fr spermatza. There culd be n dubt, hwever, that the peratin was successful, fr while there were n sperms in the lwer ple f the epididymis (at and 6 days after peratin) the upper ple was packed with mtile sperm. Subsequently when the mtility f the sperm was being tested (see belw) this fact was repeatedly cnfirmed. The cases (bucks C and C 7) where the buck was nt fertile at.the first r secnd mating but was s subsequently are pssibly t be explained by lack f vulatin by the de (which may ccasinally ccur) r t ejaculatin f semen mainly cmpsed f vesicular fluid, which may happen in an easy ejaculatin taking place after a prlnged sexual rest where a lt f this secretin has accumulated (see F 75, Table III). In the summary f results given in Table II these cases have been classed as ptentially fertile at the first mating. Table II. Life f sperm in male tract. Summary f Table I. Cnsideratin f individual bucks nly t last fertile mating r, if sterile, t first mating. Number f bucks perated Number f bucks fertile Percentage f bucks fertile I-IO Days after peratin - S.5 - s 6 - ( + I») O Fertile nly when time in female tract reduced, excluded frm percentage. t Excluding ne sterile in which testes were retained in abdmen after peratin. The summary shws that nrmal fertility is maintained up t abut the th day after the peratin, 9 per cent, f the bucks (excluding ne in which the testes remained in the abdminal cavity) tested being fertile up t the th day. After this time the fertility begins t fall ff s that between and days nly 6 per cent, f the bucks tested were fertile, while between and days nly per cent, gave litters. The detailed accunt given in Table I shws that there is cnsiderable individual variatin between bucks as t the length f time the sperms remain fertile, the causes fr this, hwever, have nt been specially investigated, althugh suggestins have been made in the discussin belw. Since, as will be shwn belw, the life f the sperms in the female tract is very shrt and the sperms after their life in the male tract have t exist in that f the female fr hurs (until vulatin ccurs), it was thught that it might be pssible t btain litters frm these perated bucks beynd the time nrmally pssible by reducing the time the sperms have t exist in the female tract (see Diagram This was tried in three cases (E 55, X 5 and E 559, see Table I) by mati^^ des with a sterile buck 8 r 6 hurs befre they were mated with the perated buck, i.e. the sperms were inserted r -^ hurs befre vulatin, the latter nr-

7 Vitality f Spermatza in Male and Female Reprductive Tracts 6 ccurring in the rabbit hurs after citus. In the first tw cases, at hurs vulatin, n result was btained, but pssibly the interval was t shrt fr such attenuated sperms t reach the va in the time. In the third case, hwever, at J hurs befre vulatin the attempt was successful and a litter f five was btained days after the buck had been perated n, althugh he was sterile when mated nrmally t des bth befre and after this time. Such a single result wants cnfirmatin but it suggests that attenuated sperms have mre chance f fertilising va' when the interval between inseminatin and vulatin is shrt, prvided sufficient time is given fr them t reach the upper ends f the tubes. Mtility. In the case f sme f the bucks which were killed in rder t inseminate des (see Table I) frm the fluids in the lwer ples f the epididymis mtile sperms were fund (E 7 at the 5th day and B at the 9th day) althugh the des s inseminated failed t prduce yung, the des f curse having been mated t a sterile buck just befre inseminatin s as t cause vulatin. After this fact was ascertained the mtility f the sperms was watched by cllecting a drp f fluid frm the vulva f the de after each mating made by the bucks and afterwards by carefully examining the fluids f the lwer epididymis when the bucks were killed. Table III gives the results btained, the size f the litter prduced (when mated) being shwn by the figure in brackets. The last bservatin in the case f each buck was btained frm examinatin f the lwer epididymis at death while the thers represent bservatins n drps btained frm the vulva f the des with which he was mated befre this time. The facts given in this table shw that the sterility f the bucks which begins t appear after the th day fllwing the peratin was nt due t exhaustin f the supply f sperms in the lwer ple f the epididymis, as in every case up t 6 days sme sperms were fund in this psitin when the buck was killed. In a few cases sperms culd nt be bserved in the drp btained frm the vulva f sme f the des after service (bucks F 75, E, E 8, see Table III) and it is pssible that in these cases the vesicular fluid frmed the large bulk f the ejaculatin; in these same bucks, hwever, sperms were seen in ther ejaculatins and were present in the lwer epididymis when the bucks were killed. The fact that the mtility f the sperms persists lng after they have ceased t be capable f fertilising va is als shwn cnclusively by Table III. It has been shwn abve that fertility diminishes greatly frm t days after the peratin, the mtility f the sperms, hwever, des nt begin t decline perceptibly until after abut days, as will be seen frm the summarised accunt shwn in Table IV; after this time ( days) the prprtin f the bucks cntaining nly dead (immtile) sperms rises cnsiderably, althugh in ne case mtility persisted up t 6 days. In bucks which had cpulated frequently the sperms had all disappeared frm the lwer epididymis frm 9 t 57 days after the peratin. e writers are indebted t Mr A. Waltn fr many bservatins n the hisf the sperms during this perid which will be published by him, but it may be said here that many were fund in the bent frm befre their mtility was lst cmpletely, the tail being bent ver and lying almst parallel t the head s

8 6 JOHN HAMMOND and S. A. ASDELL Table III. Observatins n mtility f sperm in male tract. In drp frm vulva f de when mated, r frm lwer ple f epididymis when killed (last bservatin in each buck). + -Mtile sperm seen. $ -Dead sperm nly seen. - N sperm seen. ()» (), (5) = Size f litter prduced if mated r des inseminated (last bservatin n each buck). Days after peratin Buck <r *() E 7 ().. +(). () B +() E g.' + E7 'M) E +().. +() () () P +(). +(). () F75 (). +(). +(). +(). +() E8 + E +(). +()* +() () C 5 >. +(). +(). -t(). <f>() B *() () () E 8 + (). (). (). C6 +()» *(). «(). E ss +(). +(). +(<>). () () X5 +()*. *() +() E 55Q +().. +() C) (S)t F +() E86 ' ' E69 E59 C 7 C Fi B 8 Days after peratin S SO a E7.... B I.... E9.... E7 E *. P.!(O).... (O) F75 *! & : : : : : : < > : : : : : : : : : : : : : : C5 * Bj.. E8 * C6. «... E55 +<»)... () X5 «().... *().. E 559 () +().... +()... F... E86. *... E69 E59 *.. C.. C7 O. C. F B8... Sme f the sperms bserved t be bent, with kink in tail. t By reducing length f time in female.

9 Vitality f Spermatza in Male and Female Reprductive Tracts 6 that they tend t wander rund in circles instead f prgressing in a straight As Table III shws, there is great variatin between different bucks as t the time at which the mtility f the sperms is lst, just as there is with fertility. It wuld appear that the sperms prduced by sme bucks are deficient in vigur as cmpared with thse f thers and that in extreme cases this may lead t sterility in nrmal matings, althugh mtile sperms may have been bserved in the semen f such animals. Table IV. Mtility in male tract. Summary f Table III. Days after peratin Number f cases Number with mtile sperm Number with dead sperm nly N sperm seen * 9 7 * 6 5t All in drp frm vulva f de after mating. t All frm lwer ple f epididymis f buck n pst-mrtem. Size f litter. A pint f interest in cnnectin with the life f the sperms, as it passes frm full fertility t sterility, is whether n the brder-line between these tw phases a cnditin exists in which small litters are prduced by des mated with these bucks and whether fetal degeneratin may be caused in this way. Owing t the way in which the experiments were planned it is necessary first t cnsider the effect f the rder in which the des were mated with the buck; as already mentined the de first mated usually cpulated nce, the secnd twice, and the third thrice, t ensure as far as pssible equal numbers f sperms being injected int each de. As in the first few cases (see Table I) investigated the bucks did nt prduce yung when mated subsequently t the first six matings, few attempts were made t cntinue mating beynd this and six matings t three des made in this way were arbitrarily taken as a standard number in each case. The last clumn in Table V shws that there is n reductin in size f litter with the first three des s mated. As the des t which the bucks were mated belnged t several different strains, which varied in fertility, each strain has been averaged separately. Having shwn that the methd f mating adpted did nt prduce small litters, the riginal prblem may nw be cnsidered. Frm the facts given in Table V it wuld appear that there is sme indicatin that small litters are prduced n the brder-line between full fertility and sterility, due t the attenuatin f the sperm as a result f age. The number f litters btained at this time, hwever, are frm the nature f the case few, but the results are similar in the case f each strain, a^nigh naturally mre evident in the mre fertile strains. That the small litters which ccur at this time are nt due t lack f va prduced by the des is shwn by the fllwing tw cases in which the de was killed during pregnancy:

10 Strain f de F-Plish X-Plish x Belgian E-Belgian ::: I ::: " '5 ' ' ' ' Average I; ' IE 8.5 7'9 7' 7' * By reducing length f time in female.

11 Vitality f Spermatza in Male and Female Reprductive Tracts 65 Buck E 9 was mated n the 6th day after the peratin with de E 5; ^ e, killed during pregnancy, cntained tw nrmal fetuses and ten crpra lutea f pregnancy, shwing that althugh ten fllicles had ruptured, nly tw va had develped. (b) Buck E 9 was mated n the 8th day after the peratin with de E 6; this de, killed during pregnancy, cntained six nrmal fetuses and eleven crpra lutea f pregnancy. In the rabbit in nrmal matings there are ften mre crpra lutea than fetuses during pregnancy, but the difference is rarely s wide as this, the average f missing va being under per cent. (6). The pssible causes f small litters are referred t belw. Sex rati. The sex rati f the yung prduced as the result f matings made after the sperm had existed fr different lengths f time in the epididymis is given in Table VI. After the th day, when the fertility was falling ff, the percentage f females was marked, but the prprtin f males brn in any ne litter is ntably very variable, s that data based n a small number f litters cannt be very reliable. Table VI. Life f sperm in male tract. Sex f yung prduced. Day after peratin mated Males Females Percentage males N. f litters Ttal - 6., 5.. S IS 7 8 Ttal ,. 8 S8-i Ttal - 5,,, 8. i, Ttal Ttal

12 66 JOHN HAMMOND and S. A. ASDELL. VITALITY IN THE FEMALE TRACT. Methds. It has been shwn in a previus publicatin ( that spermatza in the rabbit d nt retain their fertilising pwer fr days in the female passages. This was determined by making a mating befre the 6th day f pseud-pregnancy (fr the rabbit will, if well nurished, ften cpulate during this time) when large crpra lutea are present in the vary which prevent vulatin ccurring after citus; a secnd mating was then made with a (sterile) vasectmised buck at the th day, at which time the crpra lutea have atrphied sufficiently t allw f vulatin ccurring. Owing t variatins in the exact day (between 5 and 9) n which the crpus luteum atrphied in different individuals (just as the duratin f the estrus cycle varies in ther species) it was nt pssible by this methd t btain reliable data fr the life f the sperm in the female passages fr perids f under days, the reasn being that vulatin might either ccur at the first citus with a fertile buck r at the secnd citus with a sterile ne. A further attempt t slve the prblem was therefre made by the use f rabbits shwing dminant and recessive Mendelian clur differences. Des f a recessive (albin) clur were used; these were made pseud-pregnant by cpulatin with vasectmised bucks and at varius times frm the th t 6th day f pseudpregnancy were mated with hmzygus bucks f a dminant clur (aguti) and again n the th day f pseud-pregnancy with bucks f the recessive (albin) clur (see Table VII). Shuld vulatin ccur at the first citus the yung wuld all be aguti, but shuld it nt ccur until the secnd citus, then, if the sperms frm the first mating lived, there wuld be a chance f getting yung f bth clurs in the litter, r if they did nt live, then nly albin yung wuld be prduced. Cnsideratin f the duratin f pregnancy furnished a further check n the date f vulatin, nrmal des f this strain ging - days frm the time f mating (see Cntrls, Table VII). In the first series f animals (see Table VII) the time f the secnd citus was kept cnstant at days and the first citus was varied frm t 6 days, but n litters f mixed clurs were prduced. In the secnd series the time f the first citus was kept cnstant at 5 days and the time f the secnd citus was varied frm 6 t 7 days; here again n litters f mixed clurs were prduced. A number f cntrl animals in which n secnd mating was made gave data as t the average duratin f pregnancy in this strain and als shwed that vulatin might ccur as early as the th day r farl t ccur as late as the 7th day. This variatin at which the time f vulatin might ccur therefre gave a lwer limit f days, belw which it was impssible t determine the duratin f fertility f the sperm in the female tract, de N. D at days' interval between the cpulatins failing t give a litter f mixed clur. It was evident frm these experiments, hwever, that the length f life f the sperms in the female tract was t be ^ in hurs rather than in days. The methd f artificially inseminating des n heat was finally adpted and fr

13 Vitality f Spermatza in Male and Female Reprductive Tracts I 67 X X X X X X X X X X X = E.... x x x X... P w. S X X x. x x x x!. X X X """". X x... 'L)

14 68 JOHN HAMMOND and S. A. ASDELL Table VIII. Life f sperm in female tract. Size f litters prduced by at different intervals (N) f time befre mating with a sterile buck. (N + hurs = life in female tract.) Each lt represents a sample f semen tested. Inseminatins were made frm the vagina f ne de t that f anther unless therwise stated. Cntrls. Des inseminated but did nt cpulate Interval in hurs between inseminatin l de and cpulatin with a sterile buck Date f in-, * ^,. *-. seminatin Nt tried Wuldnt -- i viii. *...V.* ix. * ix s.x. O O 6 II.X O.. O 8. X. O O 5. O 8.x... xi xi.. 7 il 5. xi.. xii. 6. xii.... xii. H il. xii.. 9. xii. 8 -" >-5tll. 8 8.i. 5t. nil 9 8. i. 5 U. K 8. i i 8. i i ii ii ii ii ij iii. 5. %. iii

15 Vitality f Spermatza in Male and Female Reprductive Tracts 69 Table VIII {cat.). Cntrls. Des inseminated but did nt cpulate Interval in hurs between inseminatin f de and cpulatin with a sterile buck K Date f in-,-» ^_. * N seminatin Nt tried Wuldnt - - i iii iii iii. 5% Z5- i". 5. iv. 5t il. iv iv iv iv v v vii. 5.. i i 8. Vll viii viii. li 5II 7- viii. 8 il 5. ix. 5 6 il il Oil Oil H 9X ' 5 ^ 8 6. x. 5.. f II il 8. x. 5 * Inseminated int uterus. f Semen btained frm the epididymis f a buck. X Semen btained by spnge frm vagina and diluted with Waltn's fluid. Semen diluted with Waltn's fluid. II Semen diluted with Ringer's fluid. ^ De prbably sterile, i.e. had adherent tubes when killed sme time afterwards; ** De sterile, i.e. had adherent tubes when killed shrtly afterwards. BJEB IV

16 7 JOHN HAMMOND and S. A. ASDELL this purpse des which had recently prduced a litter r which had cmple pseud-pregnant perid were selected. The time f inseminatin was nted des were then mated t sterile (vasectmised) bucks at varius intervals after this. Since vulatin wuld ccur hurs after the mating the length f life f the sperm culd be calculated as the time between inseminatin and cpulatin + hurs. At first (Table VIII, dates. viii.,. viii. and 6. ix. ) in rder t avid any stimulatin f the vaginal passages by the inseminatin, which might pssibly cause vulatin, a few des were inseminated int the uterus direct via an abdminal incisin. Previus investigatin (), hwever, had shwn that vulatin after citus ccurs as a cnsequence f the rgasm attendant n it rather than as a result f the mechanical intrmissin f the penis, s that direct inseminatin int the vagina, being a much simpler and easier methd, was adpted in all subsequent experiments. Hwever, since an rgasm might be prduced n inseminatin if the de was nt t frightened by the methd f cntrl adpted, it was cnsidered advisable t have a number f cntrls which were nt mated after the inseminatin. Incidentally als a number f des which were inseminated refused t mate when tried with the buck, sme prbably because they were nt n heat (althugh every effrt was made t select des which were s) and thers because they were ften tried under rather unusual circumstances, i.e. at night with the aid f a flash lamp, which had t be dne, hwever, in rder t get the required interval f time between inseminatin and cpulatin; thers again were f a nervus temperament and difficult t mate. Examinatin f the literature als shwed that there is evidence that vulatin is but rarely caused by artificial inseminatin in the rabbit. Heape(7) inseminated des and fund that sperms reached the tubes but n vulatin ccurred. Yamane and Egashira(8) btained three litters frm 6 des (8* per cent.) artificially inseminated withut citus, whereas ver 6 per cent, were fertile when artificially inseminated in assciatin with a sterile citus, figures which are nt very different frm the results given belw. Lush (9), frm inseminatin f des, after cpulatin with vasectmised bucks, nly btained a - per cent, fertility, but since his nrmal matings nly gave a 9-7 per cent, fertility the lw figure is prbably due t matings made when the des were nt n heat and t the frequence f sterility amng his des. The semen used fr the purpse f inseminatin was derived frm the vagina f a de which had just cpulated with tw r three bucks. Mre than ne buck was used in almst every case, in rder t increase the bulk f the semen btained and als t avid, as far as pssible, testing samples which might be deficient in vitality due t lack f vigur in any particular buck. The cllecting de was killed immediately after cpulatin by breaking its neck and was hung up by the hind legs. A clip was put n the vulva immediately, s that peristaltic cntractins did nt drive the semen ut; the abdmen was cut wen and the bladder nicked with a sharp pair f scissrs s that the urine fell ^^n the flr; if this was nt dne the bladder cntracted when it was tuched and drve the urine int the vagina and spilt the sample f semen. After nicking the bladder

17 Vitality f Spermatza in Male and Female Reprductive Tracts 7 jher clip was placed at its base t prevent the semen running back int the ier when the vagina was tuched; a small nick was made in the vaginal wall belw this pint and the end f the inseminatra glass tube abut J inches lng with a rubber teat at the end (like a funtain-pen filler)was inserted and the semen was sucked up int it. When this was remved anther clip was placed ver the nick in the vagina t prevent the remaining semen running ut. The des t be inseminated were at hand and were held by ne f the writers, sitting dwn, with the head and fre legs f the de held between the knees and the hind legs held uppermst, ne in each hand; the ther then inserted the inseminatr int the vulva, taking care t pass it ver the pelvic brim befre squeezing the bulb and ejecting the cntents. By using a glass tube it was pssible t see hw much semen had been cllected and hw much was given t the de; the tube tapered frm the pint, with a diameter f abut \ inch, t the base with a diameter f abut \ inch and care was taken t have the pint well runded s as t avid danger f damaging the vaginal walls. The sterilised instrument was kept befre use within a biling tube which was immersed in a water bath at bdy temperature. In this way it was thught that the transference f the semen frm the cllecting de t thse being inseminated was as near as pssible t natural cnditins. The amunt f semen btained frm the cllecting de varied and cnsequently the number f des which culd be inseminated frm ne animal als varied; in Table VIII the lts inseminated frm each cllecting de (and cnsequently bucks) have been shwn separately; this has been dne s that the variatins with different samples can be seen. During the curse f the investigatin several ther methds f cllecting the semen were tried but all shwn in Table VIII were cllected in this way except where therwise stated. The disadvantage f the abve methd is that a de has t be killed each time a set f rabbits is t be inseminated and in times f shrtage f des (. i. 5 and 8. i. 5) bucks were killed and the semen frm the epididymis diluted with Ringer's fluid was used; the results did nt differ materially frm thse in which the semen had been btained frm the vagina f the de, but the des injected with the semen s btained were nt mated at critical perids. Anther methd used t cllect the semen frm a de was t insert a small cmpressed net-spnge int the vagina with a pair f frceps befre the de was mated, leaving the string r thread hanging frm the vulva. It was, hwever, a. tedius business getting the de t cpulate under these circumstances as the thread was cntinually getting in the way f the penis and withut the thread the spnge culd nt be easily recvered. After cpulatin the spnge was squeezed ut in Ringer's r Waltn's fluid and des were inseminated with this; the methd was used n tw ccasins (5. iii. 5 and. iv. 5). In several ther cases where there was difficulty in btaining sufficient semen fr all the des available it was slightly dited r the vagina f the cllecting de was rinsed ut with Ringer's r Waltn's i^pl, either fr the whle set r fr ne r tw des in a set. The results btained in this way (see Table VIII) did nt differ materially frm thse in which the semen had nt been s diluted.

18 7 JOHN HAMMOND and S. A. ASDELL The methd f inserting the inseminatr int the vagina after cpulatin and withdrawing the semen is nt very safe and has nt been used in ^Be experiments, fr the de frequently urinates when this is being dne and that this ccurs cannt always be detected easily unless'the amunt f urine is large. Urinatin is als liable t ccur in the spnge methd, but here it takes place as the spnge is being inserted befre cpulatin and s can be detected and anther spnge used after the bladder has been emptied. Table VIII gives a picture f the way in which the experiments were cnducted first a few des were inseminated at each cnsecutive time interval t find the apprximate length f life, then larger numbers at certain mderately wide intervals t put these results n a statistical basis, this being fllwed by filling in the gaps between critical perids and btaining larger numbers at critical times. The incidence f sterility in the des as shwn in this table appears larger than it really is wing t the fact that the same des were used ver and ver again in the curse f the experiments. The bulk f the sterile animals, which had adherent Fallpian tubes, culd be traced t ne r tw lts which had been inseminated with a rather lng and pinted tube and it is pssible that this penetrated the vaginal wall and set up peritnitis, the ciliated ends f the tubes naturally cllecting the dibris frm the abdminal cavity, this leading t adhesins. Where a de had nt prduced a litter fr abut mnths she was tested by a nrmal mating and if she prved sterile the results btained during the previus mnths had t be questined back t the time the last litter was prduced. The advantage btained by using the des ver and ver again was that f nting the fertility and size f the litter prduced at the different times in the same de; unfrtunately, wing t lack f space it has been fund impssible t incrprate the numbers f the des in Table VIII. Fertility. Table VIII shws the results btained in each animal inseminated; a few f these des were suspected f sterility and have been s marked in the table; these results, hwever, are nt included in the summary f results shwn in Table IX. Of the 8 cntrl animals which were inseminated but nt tried with the buck nly r -6 per cent, prved fertile, shwing that where des are kept in separate cages, as these were, there is very little chance f spntaneus vulatin r f vulatin as a result f inserting a tube int the vagina. Of the des which were inseminated and tried with the buck afterwards but which refused t cpulate r - per cent, prduced litters, the chances f vulatin being increased, presumably by the increased liability t an rgasm where the de was jumped by the buck, althugh nt actually mated. The frmer are the true cntrls, fr citus was nt attempted in the experimental animals until the interval required after inseminatin had elapsed. In cntrast t the lw degree f fertility where n mating ccurred after inseminatin were the cases in which the des were mated with vasectmised bucks frm t + 8 hurs after inseminatin; f the des which were mated at^k time (r 9 per cent.) prduced a litter, a degree f fertility nly slightly belw that btained in nrmal matings extending thrughut the jiear. After this time (8 hurs

19 Vitality f Spermatza in Male and Female Reprductive Tracts 7 r inseminatin, which means a life f 8 hurs in the female passages befre atin) the chances f fertility began t decrease, thus at - hurs (r - hurs in the female tract) f des which were mated with vasectmised bucks (6 per cent.) were fertile, at 6 hurs ut f 8 (55 per cent.), and at 8 hurs 8 ut f ( per cent.) were fertile. After sterile citus at the th hur after inseminatin, r a life in the female tract f hurs, nly ut f ( per cent.) were fertile, while cmplete sterility was btained in the case f 9 des mated at and hurs and after. Thus it may be cncluded that the fertility f the sperms begins t fall ff after abut hurs in the female tract t hurs when cmplete sterility is reached. While this may be taken as a general statement, the results shwn in Table VIII indicate that there may be cnsiderable variatin in different samples, in all prbability due t differences in the vigur f sperms btained frm different bucks and frm the same bucks under different cnditins f nutritin. Table IX. Life f sperm in female tract. Summary f all experiments excluding cases f des prbably sterile. Cntrls. Inseminated but did nt cpulate Number f hurs between inseminatin and sterile citus Nt tried Wuld nt - i Ig umber f des inseminated [umber f des fertile (i.e. prduced litters) 8 I II 8 g O O Percentage fertile -6 g '5 * 9 O Size f litter. Table X gives the size f the litter prduced frm sterile matings made at different times after inseminatin. The stck f rabbits cnsisted f three pure strains (and sme crsses between these) which are being inbred t prduce hmzygsity fr determining physilgical differences as regards fertility and grwth; cnsequently the nrmal average size f the litter in the different strains varied cnsiderably, s that in judging the effect f the length f the survival f the sperms n the size f litter it is necessary t cnsider each f these strains separately. Fr this purpse the average litter sizes f each strain btained frm nrmal matings have been given fr cmparisn. It will be seen that litters belw nrmal size begin t be prduced at and after the th hur (r hurs after inseminatin), the time at which the decrease in fertility f the des, r lss in vitality f the sperms begins t shw itself, while at 6- hurs (r 6- hurs after inseminatin) this decline is very marked. Tw litters f 7 and 8 at hurs are exceptinal and may pssibly be explained earlier vulatin than usual; statistically they lie within the range f pssithat vulatin was induced in these cases at the time f inseminatin. By the abve-mentined means it has thus been fund pssible t prduce experimentally small litters frm des which nrmally give large litters.

20 Table X. Lije f sperm in female tract. Size f litter prduced. Strain f de F A~~~~~~,f nrmal natings* Cntrls. Inseminated but did nt cpulate - Nt tried Wuld nt Number f hurs between inseminatin and sterile citus D E I I C I I X F D E C X

21 Vitality f Spermatxa in Male and Female Reprductive Tracts mmw.. C( H C( I..I.+,,MI WC(MNNm....

22 76 JOHN HAMMOND and S. A. ASDELL In rder t shw that these small litters were nt chance results, such as ccur in any strain, Table XI has been drawn up. This shws the distributi^ litter size in nrmal matings f the different strains as well as thse prduced after inseminatins where the sperm had remained fr extended perids in the female tract befre vulatin ccurred. Frm this table it will be seen that the distributin in litter size is nearly similar t that f nrmal matings where the sterile citus has ccurred frm t + 8 hurs after inseminatin, but that the distributin gets prgressively shifted t a smaller size f litter as the interval at which the sterile citus was made is raised t - hurs and 6- hurs. There can be little dubt, therefre, that as the end pint f the vitality f the sperms in the female tract is being reached small litters instead f large nes are prduced. In rder t make quite certain that the small size f litter btained at the ith-oth hur was nt due t a smaller number f va being shed, des which had been inseminated at intervals f - hurs befre a sterile citus were killed n r after the th day after citus and the number f crpra lutea and fetuses were cunted. Table XII, which gives the results f this examinatin, shws quite definitely that the small-sized litters and the sterility which cmmences frm the th hur is due t lack f fertilisatin, per cent, f the va shed being " missing " at the th hur (r after the sperm had remained hurs in the female tract befre vulatin), 5 per cent, at the 6th hur and per cent, at the 8th, th and th hur. In the case f de N. C, where the sperms had remained 6 hurs in the female tract befre vulatin, nly ne fetus was present, althugh 9 va had been shed (as shwn by the number f crpra lutea present). It will be nted als that the percentage f atrphic fetuses fund (- per cent.) is belw nrmal, which suggests that attenuatin f the sperms results in nn-fertilisatin r failure f the vum t develp rather than in lack f vigur in develpment. Diagram II has been prepared in rder t ffer an explanatin f the causes fr the gradual decline in the size f litter frm inseminatins ccurring frm t hurs befre vulatin. It is suggested that the number f sperms available (in the upper ends f the Fallpian tubes) t fertilise va varies at different times after inseminatin in the frm f the curve X; the first sperms prbably reach the upper tube - hurs after inseminatin and the number increases rapidly t a maximum, this being fllwed by a slw decline as the sperms die ff due t the cnditins fund in the female tract. When this end pint f the life f the sperms therefre just verlaps the variability curve f the time f vulatin (K) a small litter will result while the litter will be increased in size as the time f inseminatin begins t apprach that f vulatin. In a nrmal mating at B the sperm curve Y rises well abve the vulatin curve and persists well ver it s that there is very little chance f a small litter bejj prduced in a nrmal mating thrugh lack f sperms, except in the rare case which the frm f the sperm curve differs cnsiderably frm that given in Diagram II, fr example, in cases f deficient vigur f the sperms frm a buck in the

23 Vitality f Spermatza in Male and Female Reprductive Tracts 77 incipient stages f sterility r experimentally thrugh lwered vitality due t the l^«i f time the sperms remained in the male tract (see abve). The shape f the curve f the available va (K) is due firstly t individual variatin frm ne de t anther as t the exact time f vulatin as well as t variatin in the time at which the different fllicles f the same de rupture, f the latter evidence is available frm experiments in cnjunctin with Mr A. Waltn (t be published shrtly) in which the prcess f vulatin has been watched under the micrscpe in the living animal. Secndly, the shape f this curve is due t the shrt length f time during which the va remain capable f fertilisatin(o; ther experiments nw in prgress (which will be published in detail later) shw that a fertile mating (C) made frm 9 t hurs after a sterile ne at B als results in a small-sized litter, this being due t the variability in the vulatin curve cmbined with the shape f the sperm curve r time taken by the sperm t reach the 5 5 I I I TimeinJ Hurs -» T T (D Inseminafin Sterile Citus OvuiaHn Diagram II. Illustrating the pssible causes f small litters due t lengths f vitality f the sperm and va. K = Curve f available (upper tube) va, due t variability in time f vulatin and t their shrt life. X, Y and Z = Curves f available (upper tube) sperm, due t the time taken t reach the upper tubes and t the shrt life in the female tract. X = Frm inseminatin (A) at 8 hurs befre sterile citus (B) and 8 hurs befre the mean time f vulatinsmall litter prduced. y = Frm a nrmal fertile citus at fi hurs befre the mean time f vulatinnrmal large litter prduced. Z = Frm a fertile citus at C made hurs after a sterile citus at Bsmall litter prduced. upper ends f the tubes, nly the last-shed va being fertilised by the first sperms t arrive in the upper parts f the tubes. Incidentally the prductin f small litters in either f the abve ways is nw being used by ne f the writers t btain evidence n fetal nutritin as affected by the size f the litter; each yung f the litters f ne r tw frm des f high fertility brn under these circumstances weighs up t 9 gm. r mre at birth as cmpared with abut 55 gm. fr a litter f 7 r abut gm. fr a litter f. They are als specially well develped at birth and mstly have lng hair like a rabbit f a week r days ld. The exact shapes f the curves shwn in Diagram II require further investigatin, experiments nw in prgress will prbably enable a mre accurate estimate t"be made. As drawn they represent average curves, but individual variability, whether due t breed r feeding cnditins, will affect, them. There is evidence fr

24 78 JOHN HAMMOND and S. A. ASDELL this in the data presented in Table VIII, the pint f death f the sperr varying in different samples f semen pssibly accrding t the vigur f the frm which it was btained. In the same way the vulatin curve varies when a large r small number f va are shed by the de, fr (as is partly shwn in Table X) it is easier t btain small litters frm des nrmally prducing a large number f va than frm thse shedding a smaller number; in the latter case there is a greater tendency t give a full litter r nne at all. Table XII. Life f sperm in female tract. Pst-mrtems n des killed after inseminatin at different times befre sterile citus. Majrity killed at th day after citus. Hurs between inseminatin and mating with sterile buck 6 8 N. f de Crpra lutea K7 Nrmal fetuses Atrphic fetuses Missing va N. f de Crpra lutea Nrmal fetuses Atrphic fetuses Missing va N. f de Crpra lutea Nrmal fetuses Atrphic fetuses Missing va Pregnant Nt pregnant Average As % f eggs shed C6 F c < C C77 Fz i J- fzn C76 C8 C E55 F Hurs between inseminatin and mating with sterile buck N. f de Crpra lutea Nrmal fetuses Atrphic fetuses Missing va N. f de Crpra lutea Nrmal fetuses Atrpliic fetuses Missing va Pregnant Nt pregnant Average As % f eggs shed E E68 E 9 7 O O - ' O O O ' E< 55 «i 5 ) 7 5 c < c> c> i ) 7 5 IC Sex rati. The sex rati f the yung prduced frm litters brn as a result f sterile matings made at different perids after inseminatin are shwn in Table XIII. There is a slight excess f males in sme f the litters prduced frm the matings made near the end pint f the vitality f the sperm in the female tract, but in view f the great variability fund in sex ratis where small numbers are being dealt with it is cncluded, since n very striking result is shwn, that little r n differ^fc in the sex rati is caused by the length f time the sperm remains in the female mrct befre vulatin ccurs.

25 Vitality f Spermatza in Male and Female Reprductive Tracts 79. DISCUSSION AND CONCLUSIONS. much f the literature n this subject has been referred t in a previus publicatin () it is nt prpsed t qute it here in any detail. The first pint t cnsider is hw far the shrt life f the sperms in the female tract is peculiar t the rabbit and whether this species is typical f mammals in general. Table XIII. Life f sperm in female tract. Sex rati f yung brn. ( ) sexed at weaning, remainder f litter died befre this time. ltrls: Nt tried with buck Tried, but wuld nt cpulate i r - CO - i Is nsei ci n ii rile D O w'* 6 8 IO 6 8 O Males i, Females,,()..5,7.7. 5>(< J), 6, 5,,,7, 5,6,,,,(),,,i,,5,().,5,,, (). 5,,, () 7,,,, 8,,,,,, I,,,,, i,,,,,, i,.,, 6,, 5, Males Females Ttals Males 9 ] J ) 6 J Females 5 5 Percentage f males It is well knwn that in the case f insects (bees) the sperm may live fr a year r mre in the female tract. Amng warm-blded animals, the sperm f the bat, which mates in the autumn, lives thrughut the winter in the uterus and is said t fertilise the va which are shed in the spring (Currierd)), althugh mating has been seen t ccur again in the spring; the bat is peculiar, hwever, in that it hibernates during the winter, and in that very large quantities f semen are stred in the uterus during this perid. In the turkey, ne mating is sufficient t fertilise the whle clutch f eggs laid up t, perhaps, days afterwards. In the fwl, numerus investigatins have been made, mst authrs agreeing that the fertility f the eggs laid gradually falls ff after the cck is remved frm the pen until abslute sterility btains abut the 6th day (Gilbert (n), Frateurdz), Laurie (), Chappellierd), Payne (5), Elfrdd6), Gray (7), Philips (8) and Kauppd9)), althugh as in rabbits sme variatin in the actual time f abslute sterility was fund n different ccasins. In the duck Chappellier(i) fund abslute sterility frm the 7th t nth day. In the case f birds, hwever, the testis is abdminal and the natural temperature at which the sperms are frmed is similar t that f the viduct, v^^t\ is nt the case in mst mammals where the scrtal temperature is lwer than th^bdminal (see Table XIV). Amng the dmestic animals there is n evidence, frm frequency distributins 9O

26 8 JOHN HAMMOND and S. A. ASDELL f pregnancy duratin recrds, that the sperms may live fr weeks (the nrmal interval between heats) in the female tract and the experiments in arti^m inseminatin that have been made have shwn while it is rarely pssible t fertilise an animal utside the heat perid. Thse cases where the finding f mtile sperms in the female tract have been reprted several days after mating als are nt evidence f their fertility, as has been shwn in the experiments described abve. Sbtta () states that in the muse the sperms live in the uterus nly fr a few hurs and are then absrbed, while Hehne and Behne(ai) fund that the sperms lived nly a shrt time in the uterus f the guinea-pig. Rbinsn () fund in the ferret that the shrtest time after citus that vulatin ccurred was J hurs, while in ne case it had nt ccurred after 9J hurs, which wuld indicate that the sperm may live lnger in this species. Benit(), wh studied the mtility f the sperms in the male tract, fund in the guinea-pig and muse that after unilateral castratin the sperms in the epididymis frm the side frm which the testis was remved were mtile up t mnth and 5 days and mnths and days respectively, times which crrespnd very clsely with the maximum length f survival in the rabbit. With regard t man vulatin is usually held t ccur abut days after the beginning f the menstrual flw, and Siegel () has fund, frm the recrds f men n leave during the war, that the result f a single mating made at different times during the cycle is attended by very different degrees f fertility, accrding t the perid f the cycle at which it was made. Such a finding brings man int line with the majrity f ther species in which cnceptin is nly pssible fr a shrt perid (estrus) during the cycle. By his statistical methds, cmpiled frm the averages f different individuals, Siegel fund that the percentage f fertility increases frm the beginning f menstruatin, reaches the high pint 6 days later and remains abut the same height t the th day and then declines t the nd day, after which time there is abslute sterility. It shuld be pinted ut, hwever, that while this may express the average prbability in the mass, it is nt necessarily true f the individual, fr example, Giles (5) fund that the length f the cycle may vary frm t 5 days and s the time f vulatin, if all were gruped n the 8-day cycle, wuld vary frm 7 t + 7 days frm the nrmal, thereby causing a variatin in degree f fertility at different times in the cycle such as fund by Siegel fr wman. It wuld, therefre, seem pssible that the time in the cycle during which any individual wman is capable f cnceptin may be mre limited than is usually suppsed, and the case f wman cannt, n the present evidence, be cited as evidence fr a lengthy survival f the fertility f the sperm in the female tract. This thery culd, hwever, be tested by experiment n the mnkey n the lines f a single mating made at the mst unlikely time fr cnceptin first, viz. the 6th day f the cycle, and shuld this fail t be fertile, wrking back by -day perids t the th, nd day, and s n, until the perid was fund at which^bceptin ccurred; afterwards the ther directins culd be prceeded with, 8th, nd, th, etc. until the ther pint in the cycle at which fertility ccurs is fund.

27 Vitality f Spermatza in Male and Female Reprductive Tracts 8 In such a way it might be fund pssible t btain fr mnkeys much real infrmatins t the curse f the cycle and the time f estrus f which s little is knwn as cmpared with that f the lwer species f animals, since it is bscured by the fact that citus may be allwed at any time during the cycle r during pregnancy. The rabbit als allws citus during pregnancy. With regard t the time differences bserved between the lss f mtility f the sperms and the lss f their pwer t fertilise va, the writers are nt inclined t think that there is any abstruse mechanism invlved in these differences whereby the fertilising pwer is destryed withut affecting the mtility. The results given abve suggest rather that difficult cnditins which exist fr the sperm in the female passages, which allw f a life f hurs instead f days, are sufficient t kill ff sperms f lw vitality under hurs, the time at which vulatin nrmally ccurs after citus in the rabbit. Althugh the sperms frm the perated bucks may appear mtile when examined under the micrscpe befre inseminatin, yet they may nt be able t exist in a fertile state fr anther hurs under the cnditins they meet in the female tract, even thugh they might be able t d s under the mre favurable cnditins fund in the male tract. An indicatin f this was btained (see abve, p. 6) when the time in the female tract was reduced and a litter was prduced days after the buck had been perated n. Benitt), wh fund that sperms taken frm the testes were immtile while their mtility increased as they passed dwn the epididymis, suggests that the secretins f the epididymal epithelium are necessary fr the acquisitin f mtility by the sperms; the writers are inclined t believe, hwever, that the increase in their mtility is mre prbably a part f their life-cycle, just as in the failing mtility in ld age, rather than any specific functin f the secretin, fr sme mtile sperms have been seen in the testis itself. The main fact which stands ut in this investigatin is the difference in length f time that the sperms can retain their fertility in the male tract (8 days) as cmpared with that in the female tract ( hurs). At present ne can nly speculate as t the cause fr this; in all prbability, hwever, it is dependent nt n ne factr alne but n a number f cntributry causes f which sme may be mentined. Crew (6) first suggested, and Mre (7) has published experimental evidence, t shw that high temperatures cause the seminiferus tubules t becme atrphic. Since the female rgans are abdminal while the male are scrtal the pssibility presents itself that the difference in length f life f the sperm in the tw tracts is due t the temperature differences s prduced. The actual temperature differences between the abdmen and the scrtum in a number f bucks were taken during the curse f the peratins described abve and were fund t be n the average abut F. (see Table XIV). The actual scrtal temperature f different individuals, hwever, varied as much as this withut any apparent ill-effects n the sperm, althugh in all cases it was belw the abdminal temperature. ^wither pssible cause f the shrt fertility f the sperm in the female tract is the presence f leuccytes in these rgans. In semen btained frm des a few days after parturitin ne f the writers has, in cnjunctin with Mr A. Waltn, bserved

28 8 JOHN HAMMOND and S. A. ASDELL the sperms clustering rund leuccytes and apparently attempting t fertilise the in this way the majrity f the sperms wuld becme flcculated after ren fr sme time in the female tract. There is, as is well knwn, difficulty in breeding frm females which have an excessive discharge f leuccytes frm the tract. Table XIV. Temperatures ( F.) f scrtum and f abdmen in the rabbit. N. f buck P E B E 8 C 7 E X S E 86 E8 E 59 A F 8 C E 69 C 5 E 8 C6 E 9 E C8 F E5S F75 E559 Av. with testes in scrtum 5 Av. with testes ut f scrtum 9 Average (all) Abdminal temperature IOI-I IOI-I IOII S IOI-O i-6 Scrtal temperature Testes in scrtum ' '7 Testes nt in scrtum 'S Benit () fund that althugh the sperms in the epididymis may remain mtile fr mnths in the perated side f a unilaterally castrated muse r guinea-pig, yet after bilateral castratin they became immtile abut the 8th-ith day after the peratin, as had als been fund by Marshall and Jlly(8) in the rabbit. Benit bserved that under these latter cnditins the secretin f the epithelium f the epididymis ceased, whereas in the unilaterally castrated animals it cntinued. This suggests that the sperms are nurished as well..as stred by the epididymis and it might be taken that the lack f prper nurishment was a cause fr their early lss f fertility in the female tract. The results given abve suggest that in spite f the large numbers f spermatza usually prduced in the semen f nrmal males it is quite pssible that smallsized litters may be prduced by males whse sperms are deficient in ^ althugh still numerus and mtile. Scientific pinin has hithert been against the idea f a male being a gd r

29 Vitality f Spermatza in Male and Female Reprductive Tracts 8 bad "getter" f yung, it being held that since the number f sperms is usually s l^b it is a questin f all r nne f the va being fertilised. The results given abve rather supprt the pinin ften held amng farmers that a bar may influence the size f a litter prduced by a sw, r that a cw will nt "hld" t ne bull (which may be fertile with ther cws) whereas she may if mated t anther bull, especially if the latter is a hardy rugh scrub r mngrel animal, which ften has mre vigur than highly-bred pedigree stck. Rbertsn (9) states that in Kansas it is the practice t put the mare t a hrse a few minutes befre she is mated with the ass stallin and that in the usual way mules nly are prduced, which suggests that the spermatza f the ass have sme advantage ver that f the hrse stallin. Marshall () describes an experiment in which he inseminated a Dandie Dinmnt bitch with mixed semen frm a Dandie Dinmnt and a mngrel dg. The resulting yung were all mngrels. The sperms f the unrelated type were superir in fertilising pwer t thse f the related type. Crew () has just recently shwn that in duble matings where the sperm f ne individual is lw in vigur, caused by the length f time it had remained in the female tract r by the age and lack f vigur f the cck frm which it came, the newer r mre vigrus sperm had a greater chance f fertilising the eggs f the hen. Lewis () has als btained evidence that the sperm f sme bars will live lnger utside the bdy than that f thers. In many f the results frm the life f the sperm in the female tract, where the semen frm dminant and recessive clured bucks was mixed, and recessive clured des inseminated, the yung prduced were ften f ne clur nly, suggesting a mre vigrus cnditin f the sperm frm sme males than frm thers. In the literature f sterility amng dmestic animals many cases are quted in which n pathlgical r anatmical defect culd be fund t accunt fr the fact; these cases are mre numerus where intensive systems f agriculture are practised and where matings are made "by hand," i.e. the male nly being allwed access t the female nce during the heat perid fr service. Under natural cnditins the female wuld cntinue t be served by the male until she went ff heat and s, even thugh the sperms frm the first service might nt live until the time f vulatin, thse frm the last service wuld prbably d s. The results given abve suggest that many f these cases may be due t delayed vulatin in the female, alne r tgether with a lw vigur f the sperms. It seems pssible that the very lw fertility f travelling stallins (nly 5-6 per cent, f the mares served each year prduce fals()), where the mare is served nly nce in a heat perid lasting abut -7 days, may be due t this causethe shrt time during which the sperms remain fertile in the female tract. N such lw percentage f fertility exists in any ther class f farm stck, in all f which the length f the heat perid is much shrter; nr des it ccur in the wild hrse r amng pnies where the stallin is run with the mares. With dgs it is the practice f sme breeders t mate the bitch at days after the nset f prestrum and then again days later if the bitch is still n heat in ^ t make certain f catching the vulatin. wuld appear that the determinatin f the time f vulatin in relatin t the beginning and end f heat and the natural causes fr variatin in this are f the

30 8 JOHN HAMMOND and S. A. ASDELL utmst imprtance t breeders f dmestic animals. It has been fund that in cw vulatin nrmally ccurs abut hurs after the beginning, r hu^ the end f heat () and that in the sw vulatin ccurs abut 6 hurs after the beginning f heat (); but little r nthing is knwn f the causes which affect its variability. This knwledge is essential fr the successful emplyment f artificial inseminatin, fr where this methd is being used, particularly under cnditins in which the vigur f the sperms is likely t suffer (such as being kept fr sme hurs utside the bdy), it wuld appear that the nearer the time f vulatin (allwing due time fr ascent t the tubes) it is perfrmed the greater will be the chances f success. The methds described abve fr determining the life f the sperms in the male and female tracts appear t ffer an experimental means f determining the cnditins which affect the vigur f the bdy cell and f the sperm in particular, i.e. hw far this can be influenced by feeding cnditins (ver-fatness, lack f vitamines, etc.), breeding cnditins (hybrid vigur r inbreeding), r by management, Wdward (5) stating that the mtility and lngevity f bull sperms culd be increased by daily exercise f the animal n a treadmill. These methds wuld appear t ffer a mre certain and refined test that that f mating the male directly with a number f females r f bserving the length f life f the sperms utside the bdymethds which, fr example, were used by Llyd-Jnes and Hays (6) when determining the effect f excessive sexual activity by the male, as a result f which they fund a slight decrease in the size f the litter frm the th service made by the buck within hurs. 5. SUMMARY. () Methds and experiments are described fr testing the length f fertility f the sperms in the male and female tracts f rabbits. () The sperms may retain their fertility in the male tract up t 8 days, but in the female tract nly up t hurs. () In cases where the sperms had remained in the female tract fr - hurs befre vulatin small litters were frequently prduced; it is suggested that this is due t the end pint f the vitality f the sperms falling within the limits f time during which the prcess f vulatin ccurs. () The bearing f these results n prblems f fertility and sterility is discussed. The experiments were perfrmed at the Field Labratries, Miltn Rad, Cambridge, in cnnectin with the Institute f Animal Nutritin, and the expenses were largely defrayed ut f a grant made t the Institute by the Ministry f Agriculture and Fisheries. T Mr S. Tadman and Mr V. Swann the writers' thanks are due fr the care and feeding f the animals during the curse f these experiments.