Sexually mature females were isolated from males for at least 3 weeks before 351. Sterility Research Institute, postimplantation losses.

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1 THE INFLUENCE OF THE AGEING OF RABBIT SPERMATOZOA IN VITRO ON FERTILIZING CAPACITY AND EMBRYONIC MORTALITY H. H. KOEFOED-JOHNSEN, A. PAVLOK and J. FULKA Sterility Research Institute, The Ryal Veterinary and Agriculture Cllege, Cpenhagen, Denmark, and Labratry f Animal Genetics, Libechv, Czechslvakia {Received 5th Octber 1970) Summary. Female rabbits were inseminated with spermatza which had either been freshly ejaculated r stred fr 24 r 48 hr at 5\s=deg\C. One viduct f each female was flushed 26 t 28 hr after inseminatin and hcg injectin. The va were examined and returned t the same viduct if cleavage had ccurred. This prcedure did nt affect the subsequent develpment f the va, as cmpared with thse in the undisturbed viduct. The number f implantatins was cunted at 8 days and the number f live fetuses at 20 days f pregnancy. Cmpared with fresh spermatza, the stred samples shwed significantly lwer fertilizing capacity and the fertilized va shwed significantly higher pre- and pstimplantatin lsses. INTRODUCTION Ageing f spermatza in vitr causes a decrease in their fertilizing capacity and an increase in embrynic mrtality. This has been amply demnstrated in relatin t artificial inseminatin in cattle by Salisbury & Flerchinger (1961, 1967). Dziuk & Hanshaw (1958) and First, Stratman & Casida (1963) fund increased embrynic mrtality after inseminatin with bar spermatza which had been stred fr 72 hr. Spermatza als gradually lse their fertility in the islated epididymis in viv, thugh after a much lnger time interval (Hammnd & Asdell, 1926; Tesh & Glver, 1966). Functinal changes evked by ageing ccur rapidly in spermatza in the female reprductive tract (rabbits -Hammnd & Asdell, 1926; Maurer, Whitener & Fte, 1969; Tesh, 1969; guinea-pigs Sderwall & Yung, 1940). The aim f the present wrk was t investigate the fertilizing capacity f rabbit spermatza aged in vitr by direct evaluatin f va, and the pre- and pstimplantatin lsses by examinatin f the uterine cntents. MATERIALS AND METHODS Sexually mature females were islated frm males fr at least 3 weeks befre 351 Dwnladed frm Biscientifica.cm at 12/24/ :44:05AM

2 352 H. H. Kefed-Jhnsen et al. inseminatin. Semen frm six males f knwn fertility was cllected by means f an artificial vagina and investigated micrscpically. Ejaculates f gd quality were diluted with a tris-egg ylk slutin (Kefed-Jhnsen, 1964) t a cncentratin f 75 t 100 IO6 spermatza/ml. An aliqut f the diluted semen was used immediately, and the remainder was cled t +5 C and stred fr 24 r 48 hr. The females were inseminated intravaginally with 20 t spermatza. An injectin f hcg (Physex-Le, Cpenhagen-10 i.u./kg bdy weight) was given immediately after inseminatin. In mst cases, tw t three females f each grup were inseminated with fresh semen, and the same number with semen which had been stred 24 and 48 hr, respectively. Hw ever, if ejaculates were finferir quality, the last grup f females (semen stred fr 48 hr) was reduced r the ejaculate was discarded. The females were subjected t lapartmy under ether anaesthesia 26 t 28 hr after inseminatin, the abdmen was pened by a flank incisin n the left side, r by an incisin in the linea alba. After checking the varies and deter mining vulatin pints n bth varies, a plyethylene catheter fitted with a glass tube was inserted frm the infundibular end int ne viduct t a depth f 1 t 2 cm. With a syringe and a thin needle (N. 20) intrduced near the utertubal junctin, the cntents f the viduct were flushed int a watch glass. Tw t three ml f medium TC 199, ph adjusted t 7-5 t 7-8 with sdium carbnate, were used fr flushing. The cntents f the viduct were investigated under a steremicrscpe at x20 magnificatin. All cleaved va were returned t the viducts by means f a thin glass capillary. The whle prcedure was carried ut under cnstant tem perature cnditins. Uncleaved va were evaluated under the micrscpe after fixatin with acetic acid: alchl and staining with lacmid (Chang, 1952). Only ne viduct frm each female was flushed, the ther served as a direct cntrl f the experimental prcedure. The females were again subjected t lapartmy 8 days after inseminatin in rder t check the number f im planted embrys and crpra lutea. Twenty days after inseminatin, the females were killed and their uterine cntents investigated. RESULTS The data summarized in Table 1 shw the prgressive decrease in the fertiliz ing capacity f stred spermatza and the increase in preimplantatin lsses. The decrease in fertilizing capacity after strage fr 24 hr reaches a statistically significant value. A further decrease, thugh nt statistically significant, was fund between 24 and 48 hr. Embrynic mrtality in the preimplantatin perid als increased with the time f strage in vitr. The experimental prcedures, i.e. flushing f viducts and return f va, appeared t have n adverse effect n the further develpment f the embrys, since the number fimplantatins expected in the cntrl uterine hrns did nt significantly exceed the actual implantatins in the treated nes. Table 2 shws the pstimplantatin lsses, including the number f live embrys after 20 days f pregnancy. The dependence n the age f the sperma tza was als evident here. The differences f pstimplantatin lsses between Dwnladed frm Biscientifica.cm at 12/24/ :44:05AM

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5 Functinal changes f stred rabbit spermatza 355 the grups inseminated with fresh spermatza and spermatza stred fr 48 hr were statistically significant, but they did nt reach the level f the pre implantatin lsses. DISCUSSION The changes which ccur during the strage f spermatza which are re spnsible fr their functinal inadequacy are as yet insufficiently knwn. The riginal findings f Salisbury, Birge, de la Trre & Ldge (1961) and mre recently f ther authrs cncerning the lss f spermatzal DNA during strage in vitr have nt been fully cnfirmed (Kefed-Jhnsen, Fulka & Kpecny, 1968; Miller & Blackshaw, 1968; Kpecny & Trebichavsky, 1969). Further detailed studies allwed Salisbury (1968) t assume that the changes evked by ageing affect the spermatzal DNA r DNP mlecule qualitatively rather than quantitatively and may cause damage t the genetic infrmatin transfer system. Spermatza stred in vitr can lse their fertilizing capacity t different degrees depending n the strage methd used. In ur experiments, in spite f the relatively high initial fertility, a decline was bserved, which was mre rapid than that reprted fr rabbit spermatza by Nrman (1964) wh used NJ 2 medium, r Miller & Blackshaw (1968) wh diluted ejaculates in IVT medium. Besides decreased fertility, high preimplantatin lsses, depending n the perid f strage, were bserved in the present study. These results are in accrdance with thse f Maurer et al. (1969), wh fund that spermatza ageing in the female reprductive tract were assciated with reduced fertility and cleavage rate, and a lwer prprtin f embrys develping int blast cysts. In cntrast, Tesh (1969) fund, fr the same type f spermatza, reduced fertility nly and t a lesser extent an increase f pstembrynic lsses. He did nt bserve any effect n preimplantatin develpment. Thibault (1967) described an increased incidence f digyny after fertilizatin with ageing spermatza. In females with a cnsiderably reduced fertilizatin rate, especially thse which were inseminated with spermatza which had been stred fr 48 hr, the 'undercrwding' effect (Hafez, 1968) may have been ne f the causes f higher embrynic mrtality. ACKNOWLEDGMENTS The authrs are grateful t Dr A. McLaren fr reading the manuscript and fr her criticism. One f us (J.F.) wuld like t thank Prfessr J. Mustgaard, Ryal Veterinary and Agricultural Cllege, Cpenhagen, fr financial supprt. REFERENCES Chan, M. C. (1952) Fertilizability f rabbit va and the effect f temperature in vitr n their sub sequent fertilizatin and activatin in viv. J. exp. Zl. 121, 351. Dwnladed frm Biscientifica.cm at 12/24/ :44:05AM

6 356 H. H. Kefed-Jhnsen et al. Dziuk, P. J. & Hanshaw, G. (1958) Fertility f bar semen artificially inseminated fllwing in vitr strage. J. Anim. Sci. 17, 554. First, N. L., Stratman, F. W. & Casida, L. E. (1963) Effect f sperm age n embry survival in swine. J. Anim. Sci. 22, 135. Hafez, E. S. E. (1968) Fetal survival in undercrwded and vercrwded unilaterally pregnant uteri in the rabbit. Prc. Vlth Cngr. Anim. Reprd., Paris, 1, 575. Hammnd, J. & Asdell, S. A. (1926) The vitality f the spermatza in the male and female repr ductive tracts. Br. J. exp. Bil. 4, 155. Kefed-Jhnsen, H. H. (1964) Kunstig saedverfring hs kaniner. Aarsberetn. Inst. Sterilitetfrsk. 7, 33. Kefed-Jhnsen, H. H., Fulka, J. & Kpecny, V. (1968) Stability f thymine in spermatzal DNA during strage in vitr. Prc. Vlth Cngr. Anim. Reprd., Paris, 2, Kpecny, V. & Trebichavsky, I. (1969) Resistance f tritium activity in adenine-2,8-t labelled rabbit sperm. Annls Bil. anim. Bichim. Biphys. 9, 463. Maurer, R. R., Whitener, R. H. & Fte, R. H. (1969) Relatinship f in viv gamete aging and exgenus hrmnes t early embry develpment in rabbits. Prc. Sc. exp. Bil. Med. 131, 882. Miller, O. C. & Blackshaw, A. W. ( 1968) The DNA f rabbit spermatza aged in vitr and its relatin t fertilizatin and embry survival. Prc. Vlth Cngr. Anim. Reprd., Paris, 2, Nrman, C. (1964) Further studies n the preservatin fmammalian sperm at variable temperatures. Prc. Vth Cngr. Anim. Reprd., Trent, 4, 296. Salisbury, G. W. (1968) Fertilizing ability and bilgical aspects f sperm strage in vitr. Prc. Vlth Cngr. Anim. Reprd., Paris, 2, Salisbury, G. W., Birge, W. J., de la Trre, L. & Ldge, J. R. (1961) Decrease in Feulgen-psitive material (DNA) in in vitr strage f bvine spermatza. J. biphys. bichem. Cytl. 10, 353. Salisbury, G. W. & Flerchinger, F. H. (1961) In vitr aging f spermatza and evidence fr embry nic r early fetal mrtality in cattle. Prc. Vlth Cngr. Anim. Reprd., The Hague, 3, 601. Salisbury, G. W. & Flerchinger, F. H. (1967) Aging phenmena in spermatza. I. Fertility and pre natal lsses with use f liquid semen. J. Dairy Sci. 50, Sderwall, A. L. & Yung, W. C. (1940) The effect f ageing in the female genital tract n the fertilizing capacity f guinea pig spermatza. Anat. Ree. 78, 19. Tesh, J. M. (1969) Effect f the ageing f rabbit spermatza in uter n fertilizatin and prenatal develpment. J. Reprd. Fert. 20, 299. Tesh, J. M. & Glver, T. D. (1966) The influence f ageing f rabbit spermatza n fertilizatin and prenatal develpment. J. Reprd. Fert. 12, 414. Thibault, C. (1967) Analyse cmparée de la fécndatin et de ses anmalies chez la brebis, la vache et la lapine. Annls Bil. anim. Bichim. Biphys. 7, 5. Dwnladed frm Biscientifica.cm at 12/24/ :44:05AM