Relationship Between Fertility and the Nonprotein Sulfhydryl Concentration of Seminal Fluid in the Thoroughbred Stallion

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1 Relatinship Between Fertility and the Nnprtein Sulfhydryl Cncentratin f Seminal Fluid in the Thrughbred Stallin Frederick M. Haag, D.V.M., * and N. T. Werthessen, Ph.D. IN AN EARLIER REPORT! it was shwn that an inverse crrelatin existed between the nnprtein SH cncentratin f stallin seminal fluid and the ability f that fluid t maintain mtility in spermatza. Since mtility is ne f the prperties f spermatza which crrelates best with fertility, this was sme indicatin that ur findings might bear a valid relatinship t actual fertility. Hwever, it was recgnized that an actual field test wuld be the nly way t demnstrate cnclusively a relatinship between sulfhydryl cncentratin and fertility. A unique pprtunity t test ur hypthesis was given t us by the spnsrs f this research, and the fllwing reprt describes the results f an extensive field trial. Suthwest Fundatin fr Research and Educatin, San Antni, Texas, and the Department f Animal Pathlgy, niversity f Kentucky, Lexingtn, Ky. Guest f the Department f Animal Pathlgy, niversity f Kentucky, during the spring f Supprted in part by Greentree Stud, Inc., the Graysn Fundatin, and the Keeneland Fundatin. The authrs wish t express their appreciatin t Dr. Hagyard, Dr. W. R. McGee, Dr. F. Saeger, Dr. Hrace Davis, and Mr. F. McKenzie, whse kindness and cperatin made it pssible t cllect adequate specimens fr the study, and t Mr. Clarksn Beard, Mr. A. B. Hancck, Jr., Mr. Leslie Cmbs, and Mr. L. P. Dherty fr the truble these men tk in making available the material gathered frm the stud farms under their management. T Dr. F. E. Hull f the Department f Animal Pathlgy, niversity f Kentucky, their thanks fr his making available t Dr. Haag the labratry facilities f his department. Dr. Gldzieher's assistance in develping the methd used in this study was requisite fr its success. We wish t recrd ur appreciatin fr his assistance as well as fr his cgent advice as the study prgressed. 516

2 Vl. 7, N.6, 1956 SEMINAL SH AND FERTILITY 517 METHODS This study has taken advantage f the practices and requirements f Thrughbred hrse breeders; since these are quite rigid and specific, and cntribute t the study's significance, sme detailed descriptin is requisite. One f the bjectives in Thrughbred hrse breeding is the delivery f a fal as sn after January 1 as pssible, since hrses are entered in races accrding t their age. Irrespective f the date f birth, all Thrughbred hrses becme ne year f age the first January f their existence. Thus, as far as the rules are cncerned, a hrse brn n January 2 and anther brn n December 3 in any calendar year are bth 1 year ld n January 1 f the fllwing calendar year. Such a tremendus age difference hardly ever ccurs, but it is clear that under such rules a fal brn in June is liable t suffer a serius handicap cmpared t a future cmpetitr brn 4 r 5 mnths earlier. Other pints t be recgnized are that (1) artificial inseminatin is illegal. The stallin must cpulate with, r "cver," the mare. (2) Prir t a breeding seasn (which extends frm January t July) each stallin's wner arranges the stallin's breeding activities fr the cming seasn. Apprximately 2 t 3 mares are "bked" t a stallin, depending n the stallin's reputatin in regard t racing backgrund, fertility, and prved genetic capacity. (3) Stud fees are payable nly when the mare is prved pregnant r drps a living fal. (4) Offspring are valueless withut a pedigree. (5) Stud fees, in the area and fr the stallins n which this wrk was dne, are quite high, reaching $5 per impregnatin. These features have a number f cnsequences f imprtance in this study: 1. Chicanery, in the sense f hiding a pregnancy r having a mare cpulate with anther stallin, is valueless, as the mare's wner either btains n pedigree and has a cmpletely wrthless hrse, r pays an enrmus sum fr a genetic backgrund he culd have btained fr far less. 2. Each stallin has s many mares t cver that he is rarely capable f cpulating with a mare mre than nce per heat perid. "Duble cver" (tw matings per cycle) is s rare as t deserve special mentin when it ccurs, and such data are nt included in ur statistics. 3. A mare's wner may be paying as much as $5, per cpulatin; this, tgether with his extreme desire t induce pregnancy early in the seasn,

3 518 HAAG & WERTH ESSEN Fertility & Sterility has given rise t the practice f placing all available semen int the mare at each cpulatin. This is accmplished primarily by btaining and using the "tail end" sample. A stallin's penis when remved frm the mare after cpulatin is nt cmpletely flaccid and may still retain frm 2 t 15 cc. f semen which did nt enter the mare. Breeding practice tday calls fr the squeezing f this fluid frm the penis (the "tail-end sample"), and injecting it int the uterus f the mare. Thus if an aliqut f the tail-end sample culd be btained and if this were representative f the whle ejaculate, measurements culd be made n a semen sample actually used fr impregnatin. Examinatin f Tail-end Samples T test this pint, a number f ejaculates and the crrespnding tail-end samples were btained frm the labratry's stallins by use f the artificial vagina and subsequent "stripping" f the penis in the usual fashin. Ampermetric titratin 2 demnstrated a sufficiently clse and cnstant crrelatin between the tw fractins t permit us t prceed with a field trial based n the tail-end sample. Further preparatins fr the field trial invlved a study f the effect f freezing in dry ice f an uncentrifuged sample, and the lability in strage f the small samples (1 t 5 cc.) that wuld be available. N significant alteratins were fund. The technic f cllectin as finally develped was as fllws: 1. T each breeding farm (r shed) a daily supply f clean plastic centrifuge tubes bearing the stallin's name, labratry cde number, and the serial number f the sample f the particular hrse was prvided. 2. The dry ice cntainer at each statin was replenished daily. 3. Samples were picked up daily frm each farm, maintained ver dry ice at all times, and brught back t the labratry fr strage ver dry ice until assayed. 4. At the time f assay the samples were allwed t melt and were then centrifuged; the supernatant fluid was remved, and tw aliquts f 1 cc. each used fr determinatin f the SH cncentratin whenever the vlume permitted. One cubic centimeter f seminal fluid was pipetted directly int 9. cc. f the electrlyte used in the titratin. Prtein precipitatin tk place and went t cmpletin while the specimen remained in the dry-ice bx vernight. The slutin was centrifuged and the supernatant used fr titratin. An additinal 2 cc. f electrlyte was added and titratin carried ut. The ampermetric technic f Gldzieher et al. was used. 2

4 Vl. 7, N.6, 1956 SEMINAL SH AND FERTILITY 519 RESLTS Stallins and mares f all ranges f fertility prvided the material fr this study. The "raw" data uncrrected fr mare sterility are presented in Fig. l. The number f samples in each grup is given in brackets and the per cent f pregnacies is pltted as the rdinate. Fr the sake f simplicity the nnprtein SH cncentratin is pltted n the abscissa in terms f tenths f mls. f.1n AgN3 used in the determinatin. This is dne in view f the fact that "blank" titratins with p-chlrmercuribenzate usually culd nt be 6 C/) 5 W 4 «z (!) 3 W a::.. ~ 2 W a:: - ~ 1.-- raw data: 132 bservatins.---- (441) (353) (248) (133) (77) (34) (15) (8) (II) SH CONCENTRATION Fig. 1. Fertility f single inseminatins vs. seminal fluid SH level, raw data. dne; therefre, the bserved values are actually slightly higher than the true SH cncentratin. The values f AgN3 pltted n the abscissa may be cnverted easily int micrgrams f nnprtein sulfhydryl per cubic centimeter f seminal fluid by multiplying by 3.37 (.1 cc. f O.OOlN AgN3 is equivalent t 3.37 flg. sulfhydryl). In Fig. 2, matings t mares that returned fur times t the stallin as well as thse that ccurred within their first heat perid after faling have been

5 52 HAAG & WERTHESSEN Fertility & Sterility eliminated. This bviusly increases the ver-all fertility rate and further sharpens the significance f the crrelatin t sulfhydryl. It is evident that the fertility f ejaculates with a nnprtein sulfhydryl cncentratin f abut 3 units (1 p.g.j cc.) r higher is greatly impaired; when the level reaches 6 units (2 p.g. / cc. ), fertility is virtually nil. Figure 3 presents infrmatin n tw selected stallins. Stallin CAB CJ) L&.J (3 z 6 5 <t 4 (!) L&.J a: a.. 3 ~ L&.J 2 a: L&.J a.. 1 r-- r-- r bservatins: adjusted r-- fr female infertility factr r- r--- r--- ( 351) (284) (21) (11) (64) (28) ( 1) ( ) (7) Fig LO-I SH CONCENTRATION Fertility f single inseminatins vs. seminal fluid SH level, adjusted fr female infertility factr. maintained a cnsistently lw level f sulfhydryl; his fertility was high, and bth the failures in a ttal f 12 cpulatins ccurred when he was mated with mares wh had faled 9 days previusly. Stallin CLH, n the ther hand, shwed great variability in his sulfhydryl cncentratin. Eleven fluids shwed a level f 3 units r higher (1 p.g.j cc.) ut f a ttal f 3. In the 19 matings where the tail-end sample shwed a level lwer than 3 units, there were 13 pregnancies; in the eleven samples with a sulfhydryl f 3 r higher, there were nly 3 pregnancies. This is statistically significant at the 2 per cent level.

6 Vl. 7, N.6, 1956 SEMINAL SH AND FERTILITY 521 Cnclusin. A lgical cnsequence f this bservatin is that the failure f a particular cpulatin can be predicted with reasnable certainty, simply by measurement f the sulfhydryl cncentratin f the tail-end sample f seminal fluid <t a::: _... - LLJ 2 :I: en Fig. 3. a M < : cab 83% IC IC - - -j-- 8 IC, '" c.~ CLH 37% 6% KEY: ~ = pregnancy x = fai lure (difference between 37% 8 6 % significant at the 2 % level) Fertility vs. seminal fluid SH level in cnsecutive samples frm individual stallins. DISCSSION These data seem t demnstrate clearly (at least in the Thrughbred stallin) that the presence f an unknwn but measurable factr in the seminal fluid cntributes markedly t the fertility f the ttal ejaculate. When in additin it is recgnized that ur results are entirely uncrrected fr the intrinsic fertility ptential f the spermatza themselves, as they may vary frm sample t sample and stallin t stallin, they appear t gain added significance. Were it pssible t measure in additin a fertility index f the spermatza, an ver-all measure f even greater reliability culd result.

7 522 HAAG & WERTH ESSEN Fertility & Sterility The significance f these data as they apply t ther species is nt knwn. Few animals prvide a suitable quantity f seminal fluid fr even the mst sensitive technics, nr d the practices cncerned with their breeding permit s clear a study as that described. This may well be the reasn why bservatins such as urs have nt been made heretfre. Studies in ther species, particularly the human, are currently in prgress, and the chemical identity f the active factrs is under investigative attack. Results f these studies will be reprted subsequently. SMMARY AND CONCLSIONS It has been demnstrated that a relatinship exists in the stallin between fertility and the nnprtein sulfhydryl cncentratin f seminal fluid. The relatinship is inverse; a sharp decline in fertility ccurs with cncentratins exceeding 1 P,g. sulfhydryl! cc., and infertility f the specimen is virtually certain abve 2 P,g.j cc. Fluids cntaining less than lo P,g.j cc. have a 55 t 6 per cent pssibility f causing pregnancy in the grup f Thrughbreds studied. REFERENCES 1. MARDEN, W., and WERTHESSEN, N. T. Influence f seminal fluid n sperm mtility. Fertil. & Steril. 7:58, GLDIEHER, J. W., RAWLS, W. B., and GOLDIEHER, M. A. The uptake f sulfhydryl cmpunds by rat adrenal, liver, and muscle as measured by an imprved ampermetric technique. ]. Bil. Chern. 23:519, 1953.

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