Proceedings of the 5th International Symposium on EQUINE ...
Proceedings of the 5th International Symposium on EQUINE ...
Proceedings of the 5th International Symposium on EQUINE ...
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Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 3TABLE 2: Pregnancy rate resulting from inseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> fresh/frozen n<strong>on</strong>-sorted/sorted spermTreatment Mares Pregnant Pregnancy Pregnant/inseminatedinseminated (16 d) rate (%) E Fresh n<strong>on</strong>-sorted 10 4 40Fresh sex-sorted 16 6 382/84/8Frozen/thawed n<strong>on</strong>-sorted 16 6 38Frozen/thawed sex-sorted 15 2 13 0/7 2/8technique when n<strong>on</strong>-sorted sperm wereinseminated. Pregnancy rates were notsignificantly lower when hysteroscopicinseminati<strong>on</strong> was used for sorted vs n<strong>on</strong>-sortedspermatozoa.The lack <str<strong>on</strong>g>of</str<strong>on</strong>g> pregnancies in Experiment 1following deep intrauterine inseminati<strong>on</strong> wasunexpected. One cause for <str<strong>on</strong>g>the</str<strong>on</strong>g>se poor results couldbe <str<strong>on</strong>g>the</str<strong>on</strong>g> small inseminati<strong>on</strong> volume used (100 µl).Buchanan et al. (2000) was able to achieve 35%pregnancy rates with a similar method, while usinga larger volume for inseminati<strong>on</strong>. Ano<str<strong>on</strong>g>the</str<strong>on</strong>g>rsignificant difference between <str<strong>on</strong>g>the</str<strong>on</strong>g> 2 studies lies in<str<strong>on</strong>g>the</str<strong>on</strong>g> treatment <str<strong>on</strong>g>of</str<strong>on</strong>g> sperm prior to inseminati<strong>on</strong>. In <str<strong>on</strong>g>the</str<strong>on</strong>g>previous study, n<strong>on</strong>-treated sperm wereinseminated within 1 h <str<strong>on</strong>g>of</str<strong>on</strong>g> collecti<strong>on</strong>. In <str<strong>on</strong>g>the</str<strong>on</strong>g> presentexperiment, sperm were inseminated 8 h or morefollowing collecti<strong>on</strong>, and were subjected t<strong>on</strong>umerous treatments, including 2 centrifugati<strong>on</strong>steps. Fur<str<strong>on</strong>g>the</str<strong>on</strong>g>r studies are needed to determine <str<strong>on</strong>g>the</str<strong>on</strong>g>applicability <str<strong>on</strong>g>of</str<strong>on</strong>g> this method <str<strong>on</strong>g>of</str<strong>on</strong>g> horn inseminati<strong>on</strong>.Based <strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g> results <str<strong>on</strong>g>of</str<strong>on</strong>g> Experiment 1, deep uterineinseminati<strong>on</strong> with <str<strong>on</strong>g>the</str<strong>on</strong>g> aid <str<strong>on</strong>g>of</str<strong>on</strong>g> a video-endoscope is<str<strong>on</strong>g>the</str<strong>on</strong>g> preferred method for low-dose inseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>n<strong>on</strong>-sorted, as well as sex-sorted spermatozoa.Experiment 2 was c<strong>on</strong>ducted to compareeffects <str<strong>on</strong>g>of</str<strong>on</strong>g> sexing and freezing stalli<strong>on</strong> sperm <strong>on</strong>fertility using a 2 x 2 factorial design: sexed orunsexed vs fresh or frozen. Oestrus wassynchr<strong>on</strong>ised in 41 mares as presented in <str<strong>on</strong>g>the</str<strong>on</strong>g> firstexperiment. Mares were administered 3,000 iuhCG iv ei<str<strong>on</strong>g>the</str<strong>on</strong>g>r 6 h (fresh sperm) or 30 h(frozen/thawed sperm) prior to inseminati<strong>on</strong>.Hysteroscopic inseminati<strong>on</strong> was performed <strong>on</strong> allmares with 5 x 10 6 motile sperm (230 µl). Mareswere randomly assigned to <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> 4 treatmentgroups: Group 1 (n=10): inseminated with fresh,n<strong>on</strong>-sorted spermatozoa; Group 2 (n=16):inseminated with fresh sex-sorted spermatozoa.Group 3 (n=16): inseminated with frozen n<strong>on</strong>sortedspermatozoa. Group 4 (n=15): inseminatedwith frozen, sex-sorted spermatozoa.C<strong>on</strong>centrati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> sperm were adjusted beforefreezing, based <strong>on</strong> predetermined average postthaw motilities, so that each dose c<strong>on</strong>tainedapproximately 5 x 10 6 motile sperm post-thaw.Mares in Groups 1 and 2 were inseminatedapproximately 8 h after collecti<strong>on</strong>, based <strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g>time needed to process and sort <strong>on</strong>e inseminati<strong>on</strong>dose. From collecti<strong>on</strong> to inseminati<strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g>spermatozoa were protected from light and held atroom temperature in an adapted HEPES-bufferedtyrodes soluti<strong>on</strong> (Parrish et al. 1988).No difference was found (P>0.1) in pregnancyrate for mares inseminated with fresh n<strong>on</strong>-sorted,fresh sex-sorted or frozen n<strong>on</strong>-sorted spermatozoa(Table 2). Fewer mares became pregnant followinginseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> frozen, flow-sorted spermatozoacompared to <str<strong>on</strong>g>the</str<strong>on</strong>g> o<str<strong>on</strong>g>the</str<strong>on</strong>g>r treatments, but thisdifference was not significant, likely due to lowgroup numbers.These studies dem<strong>on</strong>strated that hysteroscopicinseminati<strong>on</strong> can be used to obtain pregnancieswith low numbers <str<strong>on</strong>g>of</str<strong>on</strong>g> fresh and frozen equinespermatozoa, as well as low numbers <str<strong>on</strong>g>of</str<strong>on</strong>g> sex-sortedspermatozoa. Fur<str<strong>on</strong>g>the</str<strong>on</strong>g>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<str<strong>on</strong>g>of</str<strong>on</strong>g>f, L.A. and Squires, E.L (2000)Inseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> mares with low numbers <str<strong>on</strong>g>of</str<strong>on</strong>g> ei<str<strong>on</strong>g>the</str<strong>on</strong>g>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 Robins<strong>on</strong>,J.J. (1997) Producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> lambs by low doseintrauterine inseminati<strong>on</strong> with flow cytometricallysorted and unsorted semen. Theriogenol. 47, 267(Abstract).Fugger, E.F. (1999) Clinical experience with flowcytometric separati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> human X- and Y-chromosome bearing sperm. Theriogenol. 52, 1435-1440.Johns<strong>on</strong>, L.A. (1991) Sex preselecti<strong>on</strong> in swine: Alteredsex ratios in <str<strong>on</strong>g>of</str<strong>on</strong>g>fspring following surgicalinseminsperm. RJohns<strong>on</strong>, L.preselecspermReprod.Morris, L.HHysterosperma7