Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 310080n=12% c<strong>on</strong>ceived6040n=14n=34n=820n=25025 milli<strong>on</strong> 25 milli<strong>on</strong> 5 milli<strong>on</strong> 5 milli<strong>on</strong> 5 milli<strong>on</strong> 300 milli<strong>on</strong> 5 milli<strong>on</strong>0.5 ml 0.5 ml 0.1 ml 0.1 ml 0.1 ml 0.5 ml 0.1 mlC<strong>on</strong>venti<strong>on</strong>al AI Ipsilateral UTJ Ipsilateral UTJ Cranial to cervix C<strong>on</strong>tralateral UTJ Epididymal UTJ Epididymal UTJn=12n=19Fig 1: C<strong>on</strong>cepti<strong>on</strong> rates achieved in mares inseminated hysteroscopically and c<strong>on</strong>venti<strong>on</strong>ally with low numbers <str<strong>on</strong>g>of</str<strong>on</strong>g>frozen-thawed ejaculated and epididymal spermatozoa.inseminated <strong>on</strong>ce with frozen-thawed ejaculatedspermatozoa from fertile stalli<strong>on</strong>s at a fixed timeafter an ovulati<strong>on</strong>-inducing dose <str<strong>on</strong>g>of</str<strong>on</strong>g> g<strong>on</strong>adotropin.Not <strong>on</strong>ly were high c<strong>on</strong>cepti<strong>on</strong> rates obtained usingboth c<strong>on</strong>venti<strong>on</strong>al uterine body and hysteroscopicuterotubal inseminati<strong>on</strong> techniques, <str<strong>on</strong>g>the</str<strong>on</strong>g> number <str<strong>on</strong>g>of</str<strong>on</strong>g>frozen-thawed spermatozoa inseminated was <strong>on</strong>ly5–10% <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> minimum number <str<strong>on</strong>g>of</str<strong>on</strong>g> frozen thawedspermatozoa used c<strong>on</strong>venti<strong>on</strong>ally in commercialinseminati<strong>on</strong> programmes.When <str<strong>on</strong>g>the</str<strong>on</strong>g> inseminati<strong>on</strong> dose was 25 milli<strong>on</strong>motile spermatozoa, <str<strong>on</strong>g>the</str<strong>on</strong>g> hysteroscopic uterotubalmethod <str<strong>on</strong>g>of</str<strong>on</strong>g> inseminati<strong>on</strong> held no advantage over<str<strong>on</strong>g>the</str<strong>on</strong>g> c<strong>on</strong>venti<strong>on</strong>al uterine body inseminati<strong>on</strong>technique. When <str<strong>on</strong>g>the</str<strong>on</strong>g> inseminati<strong>on</strong> dose wasreduced to <strong>on</strong>ly 5 milli<strong>on</strong> spermatozoa in 0.1 mlextender, however, <str<strong>on</strong>g>the</str<strong>on</strong>g> hysteroscopic uterotubalmethod showed a definite advantage overc<strong>on</strong>venti<strong>on</strong>al depositi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> inseminate justcranial to <str<strong>on</strong>g>the</str<strong>on</strong>g> cervix.Not surprisingly, <strong>on</strong>ly <strong>on</strong>e mare c<strong>on</strong>ceivedafter depositing <str<strong>on</strong>g>the</str<strong>on</strong>g> low dose <str<strong>on</strong>g>of</str<strong>on</strong>g> frozen-thawedspermatozoa <strong>on</strong>to <str<strong>on</strong>g>the</str<strong>on</strong>g> uterotubal papillac<strong>on</strong>tralateral to <str<strong>on</strong>g>the</str<strong>on</strong>g> side <str<strong>on</strong>g>of</str<strong>on</strong>g> ovulati<strong>on</strong>. This reflects<str<strong>on</strong>g>the</str<strong>on</strong>g> fragility <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> frozen-thawed cells and <str<strong>on</strong>g>the</str<strong>on</strong>g>irpoor ability to migrate around <str<strong>on</strong>g>the</str<strong>on</strong>g> uterus to achievea satisfactory sperm reservoir at <str<strong>on</strong>g>the</str<strong>on</strong>g> site <str<strong>on</strong>g>of</str<strong>on</strong>g>fertilisati<strong>on</strong>.The low number <str<strong>on</strong>g>of</str<strong>on</strong>g> pregnancies obtained inmares inseminated with frozen-thawed epididymalspermatozoa was disappointing. The epididymalspermatozoa showed an appreciably lower motilitythan <str<strong>on</strong>g>the</str<strong>on</strong>g> ejaculated spermatozoa, both before andafter freezing, and <str<strong>on</strong>g>the</str<strong>on</strong>g>y exhibited a higherproporti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> acrosome-reacted spermatozoa afterfreezing. Thus, it appears that <str<strong>on</strong>g>the</str<strong>on</strong>g> l<strong>on</strong>gevity,viability and fertilising potential <str<strong>on</strong>g>of</str<strong>on</strong>g> epididymalspermatozoa is much less than ejaculatedspermatozoa after freezing and thawing, and thisdeficiency cannot be overcome by hysteroscopicdepositi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> sample close to <str<strong>on</strong>g>the</str<strong>on</strong>g> site <str<strong>on</strong>g>of</str<strong>on</strong>g>fertilisati<strong>on</strong>.The most important outcome <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> study wasthat high c<strong>on</strong>cepti<strong>on</strong> rates can be achieved inmares inseminated with low numbers <str<strong>on</strong>g>of</str<strong>on</strong>g> frozenthawedejaculated spermatozoa at a fixed timeafter <str<strong>on</strong>g>the</str<strong>on</strong>g> administrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> an ovulati<strong>on</strong> inducingagent.REFERENCESKloppe, L.H., Varner. D.D., Elmore. R.G., Bretzlaff,K.N. and Shull. J.W. (l988) Effect <str<strong>on</strong>g>of</str<strong>on</strong>g> inseminati<strong>on</strong>timing <strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g> fertilizing capacity if frozen/thawedequine spermatozoa. Theriogenol. 29, 429-439.Loomis, P.R., Amann, R.P., Squires, E.L. and Pickett,B.W. (l983) Fertility <str<strong>on</strong>g>of</str<strong>on</strong>g> unfrozen and frozen stalli<strong>on</strong>spermatand pacMorris, L.HHysterospermapreovulPace, M.M.insemincomp<strong>on</strong>5
Equine Embryo TransferHYSTEROSCOPIC INSEMINATION OF NON FROZENAND FROZEN UNSEXED AND SEXED <strong>EQUINE</strong>SPERMATOZOAA. C. Lindsey, L. H. A. Morris*, W. R. Allen*, J. L. Schenk † , J. K. Graham,J. E. Bruemmer and E. L. SquiresAnimal Reproducti<strong>on</strong> and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado80523, USA; *Equine Fertility Unit, Mertoun Paddocks, Woodditt<strong>on</strong> Road, Newmarket, Suffolk CB89BH, UK; † XY, Inc., ARBL Building, Foothills Research Campus, Fort Collins, Colorado 80523, USAA safe and reliable method for prec<strong>on</strong>ceptual sexselecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g>fspring has been sought for decadesin humans, livestock, and even compani<strong>on</strong>animals. With a method developed some 10 yearsago (Johns<strong>on</strong> et al. 1989), effective preselecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>sex has been accomplished in many species <str<strong>on</strong>g>of</str<strong>on</strong>g>livestock, as well as in humans (Johns<strong>on</strong> 1991;Cran et al. 1997; Seidel et al. 1997; Fugger 1999).Sex preselecti<strong>on</strong> has also been successful in horses(Buchanan et al. 2000), but due to <str<strong>on</strong>g>the</str<strong>on</strong>g> limitednumber <str<strong>on</strong>g>of</str<strong>on</strong>g> spermatozoa available after <str<strong>on</strong>g>the</str<strong>on</strong>g> sortingprocess, traditi<strong>on</strong>al breeding doses are notavailable. Therefore, low-dose inseminati<strong>on</strong>techniques must be improved in order to maximise<str<strong>on</strong>g>the</str<strong>on</strong>g> efficiency <str<strong>on</strong>g>of</str<strong>on</strong>g> sex-sorted spermatozoa.Hysteroscopic inseminati<strong>on</strong> has recently beenshown to produce acceptable pregnancy rateswhen using <strong>on</strong>ly <strong>on</strong>e milli<strong>on</strong> freshly collectedmotile spermatozoa (Morris et al. 2000). With<str<strong>on</strong>g>the</str<strong>on</strong>g>se encouraging results, it has been hypo<str<strong>on</strong>g>the</str<strong>on</strong>g>sisedthat hysteroscopic inseminati<strong>on</strong> could be aneffective and practical method to achievepregnancies using low numbers <str<strong>on</strong>g>of</str<strong>on</strong>g> sex-sortedspermatozoa. The objectives <str<strong>on</strong>g>of</str<strong>on</strong>g> Experiment 1were: 1) to compare pregnancy rate with 5 x 10 6spermatozoa inseminated deep in <str<strong>on</strong>g>the</str<strong>on</strong>g> uterine hornaided by ultras<strong>on</strong>ography, or deposited <strong>on</strong>to <str<strong>on</strong>g>the</str<strong>on</strong>g>uterotubal papilla with <str<strong>on</strong>g>the</str<strong>on</strong>g> use <str<strong>on</strong>g>of</str<strong>on</strong>g> a flexible videoendoscope;and 2) to determine if hysteroscopicinseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> sexed spermatozoa can result insatisfactory pregnancy rate.Semen was collected from 2 stalli<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g>acceptable fertility. Oestrus was synchr<strong>on</strong>ised(June–July) in 40 mares, ages 3–10, byadministering 10 ml <str<strong>on</strong>g>of</str<strong>on</strong>g> altrenogest orally for 10days, followed by 250 µg cloprostenol im <strong>on</strong> Day11. Mares were given 3,000 iu hCG iv at <str<strong>on</strong>g>the</str<strong>on</strong>g> time<str<strong>on</strong>g>of</str<strong>on</strong>g> inseminati<strong>on</strong> and assigned to <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> 3 groups:Group 1 mares (n=10) were inseminated with 5 x10 6 Percoll-washed spermatozoa deposited deepinto <str<strong>on</strong>g>the</str<strong>on</strong>g> uterine horn with <str<strong>on</strong>g>the</str<strong>on</strong>g> aid <str<strong>on</strong>g>of</str<strong>on</strong>g>ultras<strong>on</strong>ography. Group 2 mares (n=10) wereinseminated with 5 x 10 6 Percoll-washedspermatozoa deposited <strong>on</strong>to <str<strong>on</strong>g>the</str<strong>on</strong>g> uterotubal papillaevia hysteroscopic inseminati<strong>on</strong>. Group 3 mares(n=20) were inseminated using <str<strong>on</strong>g>the</str<strong>on</strong>g> hysteroscopictechnique with 5 x 10 6 sex-sorted spermatozoa.Spermatozoa were stained with Hoechst 33342and sorted into X and Y chromosome-bearingpopulati<strong>on</strong>s based <strong>on</strong> DNA c<strong>on</strong>tent using an SXMoFlo sperm sorter. Pregnancy was determinedultras<strong>on</strong>ographically at 16 days post ovulati<strong>on</strong>.Hysteroscopic inseminati<strong>on</strong> resulted in morepregnancies than did <str<strong>on</strong>g>the</str<strong>on</strong>g> ultrasound-guidedTABLE 1: Pregnancy rate resulting from hysteroscopic or deep intrauterine inseminati<strong>on</strong>Treatment Mares inseminated Pregnant (16 d) Pregnancy rate (%)N<strong>on</strong>-sorted sperm, ultrasound guided 10 0 0 aN<strong>on</strong>-sorted sperm, hysteroscopic 10 5 50 bSex-sorted sperm, hysteroscopic 20 5 25 aba,b Values without comm<strong>on</strong> superscripts differ (P