Proceedings of the 5th International Symposium on EQUINE ...

Havemeyer Foundation Monograph Series No. 3TABLE 2: Pregnancy rate resulting from insemination <strong>of</strong> fresh/frozen non-sorted/sorted spermTreatment Mares Pregnant Pregnancy Pregnant/inseminatedinseminated (16 d) rate (%) E Fresh non-sorted 10 4 40Fresh sex-sorted 16 6 382/84/8Frozen/thawed non-sorted 16 6 38Frozen/thawed sex-sorted 15 2 13 0/7 2/8technique when non-sorted sperm wereinseminated. Pregnancy rates were notsignificantly lower when hysteroscopicinsemination was used for sorted vs non-sortedspermatozoa.The lack <strong>of</strong> pregnancies in Experiment 1following deep intrauterine insemination wasunexpected. One cause for <strong>the</strong>se poor results couldbe <strong>the</strong> small insemination volume used (100 µl).Buchanan et al. (2000) was able to achieve 35%pregnancy rates with a similar method, while usinga larger volume for insemination. Ano<strong>the</strong>rsignificant difference between <strong>the</strong> 2 studies lies in<strong>the</strong> treatment <strong>of</strong> sperm prior to insemination. In <strong>the</strong>previous study, non-treated sperm wereinseminated within 1 h <strong>of</strong> collection. In <strong>the</strong> presentexperiment, sperm were inseminated 8 h or morefollowing collection, and were subjected tonumerous treatments, including 2 centrifugationsteps. Fur<strong>the</strong>r studies are needed to determine <strong>the</strong>applicability <strong>of</strong> this method <strong>of</strong> horn insemination.Based on <strong>the</strong> results <strong>of</strong> Experiment 1, deep uterineinsemination with <strong>the</strong> aid <strong>of</strong> a video-endoscope is<strong>the</strong> preferred method for low-dose insemination <strong>of</strong>non-sorted, as well as sex-sorted spermatozoa.Experiment 2 was conducted to compareeffects <strong>of</strong> sexing and freezing stallion sperm onfertility using a 2 x 2 factorial design: sexed orunsexed vs fresh or frozen. Oestrus wassynchronised in 41 mares as presented in <strong>the</strong> firstexperiment. Mares were administered 3,000 iuhCG iv ei<strong>the</strong>r 6 h (fresh sperm) or 30 h(frozen/thawed sperm) prior to insemination.Hysteroscopic insemination was performed on allmares with 5 x 10 6 motile sperm (230 µl). Mareswere randomly assigned to one <strong>of</strong> 4 treatmentgroups: Group 1 (n=10): inseminated with fresh,non-sorted spermatozoa; Group 2 (n=16):inseminated with fresh sex-sorted spermatozoa.Group 3 (n=16): inseminated with frozen nonsortedspermatozoa. Group 4 (n=15): inseminatedwith frozen, sex-sorted spermatozoa.Concentrations <strong>of</strong> sperm were adjusted beforefreezing, based on predetermined average postthaw motilities, so that each dose containedapproximately 5 x 10 6 motile sperm post-thaw.Mares in Groups 1 and 2 were inseminatedapproximately 8 h after collection, based on <strong>the</strong>time needed to process and sort one inseminationdose. From collection to insemination <strong>the</strong>spermatozoa were protected from light and held atroom temperature in an adapted HEPES-bufferedtyrodes solution (Parrish et al. 1988).No difference was found (P>0.1) in pregnancyrate for mares inseminated with fresh non-sorted,fresh sex-sorted or frozen non-sorted spermatozoa(Table 2). Fewer mares became pregnant followinginsemination <strong>of</strong> frozen, flow-sorted spermatozoacompared to <strong>the</strong> o<strong>the</strong>r treatments, but thisdifference was not significant, likely due to lowgroup numbers.These studies demonstrated that hysteroscopicinsemination can be used to obtain pregnancieswith low numbers <strong>of</strong> fresh and frozen equinespermatozoa, as well as low numbers <strong>of</strong> sex-sortedspermatozoa. Fur<strong>the</strong>r studies are needed toimprove pregnancy rate with sex-sorted, frozenequine spermatozoa.REFERENCESBuchanan, B.R., Seidel, G.E. Jr, McCue, P.M., Schenk,J.L., Herickh<strong>of</strong>f, L.A. and Squires, E.L (2000)Insemination <strong>of</strong> mares with low numbers <strong>of</strong> ei<strong>the</strong>runsexed or sexed spermatozoa. Theriogenol. 53,1333-1344.Cran, D.G., McKelvey, W.A.C., King, M.E., Dolman,D.F., McEvoy, T.G., Broadbent, P.J. and Robinson,J.J. (1997) Production <strong>of</strong> lambs by low doseintrauterine insemination with flow cytometricallysorted and unsorted semen. Theriogenol. 47, 267(Abstract).Fugger, E.F. (1999) Clinical experience with flowcytometric separation <strong>of</strong> human X- and Y-chromosome bearing sperm. Theriogenol. 52, 1435-1440.Johnson, L.A. (1991) Sex preselection in swine: Alteredsex ratios in <strong>of</strong>fspring following surgicalinseminsperm. RJohnson, L.preselecspermReprod.Morris, L.HHysterosperma7