Culture conditions required to induce capacitation and the acrosome reaction of canine sperm in vitro

100F AS a prerequisite to testing the functional capacity of caninle 8 sperm usiIng anI oocyte pcieetration assay, it is first necessary AR to establish that speriml can survive and undergo capacitationi E 60 and the acrosome reaction in vitro. Mahi and Yanagimachi Q 40 (1978) developed a culture medium named caninie capacitacn20 tion medium ((t(tm) which was shown to be useful for sUpporting spontaneous capacitation and the acrosomreactionI TALP TALP TALP CCm CC of caniine sperm in vitro, and when used at pH 7 8, these (pH74) (pH78) Hepes (pH74) (pH78) events were further enhaniced. In a previous study, the present Meditum authors have shown that the dual fluorescent labelling techFIG 1: Chlortetracycline fluorescent staining patterns of nique described by Ward and Storey (1984), which incorpocanine sperm incubated in five different media for eight rates chlortetracycline and Hoechst 33258, is useful for hours at: a) 20°C, b) 39°C. (Means of two replicates and rates chlortetracvclliie and Hoechst 33258, IS uIsetul for I~~~~~~~~~~~~~~ standard deviations) moniltoring chainges in the functional status and viability oft canine sperm (Hewitt and England 1998). The present study used this technique in an attempt to establish optimal culture conditions for caniine sperm by comparing the use of ccm. no significant effect of culture maediumn or temperature upoIn wsvith a tyrodes medium supplemented with albumin, lactate the percentage of spermii capacitated after eight hours and pyruvate (lIAI.I') (BavisterandYanagimachi 1977, Bavister (P=0 7909), but there was a significaint effect of temperature and others 1983). UpOIn the percentage of AR sperimi after eight hours (P=0 0293). Fluorescenit labels and other solutions were prepared as The proportions of the three staininlg patterins after eight hours described by Hewitt and England (1998). The sperm-rich at 20 and 39°C are shown in Fig Ia aild b. fraction of the ejaculate was collected by digital manipulatioin By modifying the TALP, Mahi anid Yanagimachi (1978) from four beagles of unknown fertility and was pooled. The showed that CCm allowed caniine sperm to spontaneously sample was divided into five equal volumes. Each was diluted capacitate and acrosome react in vitro. They showed that with one of the followving: TALI (pH 7-4), TALP (pH 7-8), capacitation, the acrosome reactioni and the maintenance of TALP/HEPES, C(CM (pH 7.4) or CCNI (pH 7.8) at a dilution of 3:1 motility was greatest in this mediumil at pH 7.8, and that after and washed by centrifLgation. The concentration of the fincal seven hours culture at 37°C, 80 to 90 per cent of sperm wvere sample was between 5 and 10 x 106/ml. Each of the five samstill motile and, of these, 46 per cenit were acrosome-reacted. ples was divided into 10 aliquots and incubated at 20 or 39°C Their observations were nmade usnllg unstained sperm under under 5 per cent carbon dioxide in air for zero, two, four, six phase contrast microscopy. or eight hours. Following incubation, samples were stained In this study, culture in all media at all pHs and temperafor viability and acrosomal status and fluorescence tures allowed the proportion of sperill that were capacitated microscopy was used for the assessment of staining patterns and acrosome-reacted to increase, but the rate and extent of (Hewitt and England 1998). For each treatment, only live these changes varied considerably with each treatmeint. In sperm, identified by the absence of Hoechst 33258 staining general, the effect of temperature was marked, with changes were examined. The staining patterns were recorded as an F from F to B and AR patterns OCCUIrring more rapidly at 39°C. pattern (uncapacitated and acrosome intact), B pattern The percentage of AR cells was significantly greater in all treat(capacitated aind acrosome intact) and an AR pattern (capacments at 39°C. For sperm incubated at 390C, the percentage itated and acrosoimie-reacted sperm) (Ward and Storey 1984). of AR cells after eight hours in culture was increased in both Data were analysed statistically in terms of the percentage Ar.AIP and Ccm by the raised pH. Although these data indicated of capacitated sperm (B pattern plus AR pattern cells) and the that both media at the higher pH w%rere better at supporting percentage of acrosome-reacted sperm (AR pattern cells) after capacitation, the difference was not significant. In addition, eight hours of culture. Normality of the data was assessed both media at the higher pH caused a higher occurrence of Vc'tcrinary Rccord (1999) using the Kolmogorov-Smirnov test and data were then acrosome reactions to be observed. This difference again was 144, 22-23 analysed using a two way analysis of variance. not significant. In all culture media at both temperatures, the proportion In attempting to find the optimal conditions that accuD. A. Hewitt, HJSc, of F pattern cells decreased as the proportion of B and ARt cells rately mimic those found inl the female reproductive tract, it G. C. w. England, increased. The increase inl percentage AR sperm occurred more may be that a compromise is required because, ideally, the in lOVetMedl>, PhIa), t)VR1, CertV'A, rapidly and was shown to be significantly greater in all treatvitro culture conditions selected should encourage capacita1)VRepct, I)ipAC,'l ments at the higher temperature. For sperm incubated in TALP tion, but limit the occurrence of the spontaneous acrosome Departmenlt of Falrm medium1 at 39°C, the percentage of AR sperm after eight hours reaction, thus increasing the ability of sperm to fertilise Anlimall anld Fquline in culture was increased from 15 to 24 per cent by the raised oocytes in the oviduct. However, trends shown in this experM\edicinle anld Sulrgery, pH. A similar pattern was seen in CClM# (AR sperm increasing iment and evidence from other studies indicate that further Royall Vterinairyr College, from 13 to 37 per cent). Overall, the CCMS at pH 7 8 and 39°C investigations should be undertaken incorporating the two Unliversity of Lonldonl, caused a higher rate of acrosome reactions, although the effect media used in this study at a pH of 7 8, and a temperature of HawNkshead Lanle, North of TAI.P at pH 7 8 and 39°C was similar. Despite this, there was 39°C to establish ideal conditions. MvmmIlIls, Haltfield AL9 OTA