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

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Havemeyer Foundation Monograph Series No. 310080n=12% conceived6040n=14n=34n=820n=25025 million 25 million 5 million 5 million 5 million 300 million 5 million0.5 ml 0.5 ml 0.1 ml 0.1 ml 0.1 ml 0.5 ml 0.1 mlConventional AI Ipsilateral UTJ Ipsilateral UTJ Cranial to cervix Contralateral UTJ Epididymal UTJ Epididymal UTJn=12n=19Fig 1: Conception rates achieved in mares inseminated hysteroscopically and conventionally with low numbers <strong>of</strong>frozen-thawed ejaculated and epididymal spermatozoa.inseminated once with frozen-thawed ejaculatedspermatozoa from fertile stallions at a fixed timeafter an ovulation-inducing dose <strong>of</strong> gonadotropin.Not only were high conception rates obtained usingboth conventional uterine body and hysteroscopicuterotubal insemination techniques, <strong>the</strong> number <strong>of</strong>frozen-thawed spermatozoa inseminated was only5–10% <strong>of</strong> <strong>the</strong> minimum number <strong>of</strong> frozen thawedspermatozoa used conventionally in commercialinsemination programmes.When <strong>the</strong> insemination dose was 25 millionmotile spermatozoa, <strong>the</strong> hysteroscopic uterotubalmethod <strong>of</strong> insemination held no advantage over<strong>the</strong> conventional uterine body inseminationtechnique. When <strong>the</strong> insemination dose wasreduced to only 5 million spermatozoa in 0.1 mlextender, however, <strong>the</strong> hysteroscopic uterotubalmethod showed a definite advantage overconventional deposition <strong>of</strong> <strong>the</strong> inseminate justcranial to <strong>the</strong> cervix.Not surprisingly, only one mare conceivedafter depositing <strong>the</strong> low dose <strong>of</strong> frozen-thawedspermatozoa onto <strong>the</strong> uterotubal papillacontralateral to <strong>the</strong> side <strong>of</strong> ovulation. This reflects<strong>the</strong> fragility <strong>of</strong> <strong>the</strong> frozen-thawed cells and <strong>the</strong>irpoor ability to migrate around <strong>the</strong> uterus to achievea satisfactory sperm reservoir at <strong>the</strong> site <strong>of</strong>fertilisation.The low number <strong>of</strong> pregnancies obtained inmares inseminated with frozen-thawed epididymalspermatozoa was disappointing. The epididymalspermatozoa showed an appreciably lower motilitythan <strong>the</strong> ejaculated spermatozoa, both before andafter freezing, and <strong>the</strong>y exhibited a higherproportion <strong>of</strong> acrosome-reacted spermatozoa afterfreezing. Thus, it appears that <strong>the</strong> longevity,viability and fertilising potential <strong>of</strong> epididymalspermatozoa is much less than ejaculatedspermatozoa after freezing and thawing, and thisdeficiency cannot be overcome by hysteroscopicdeposition <strong>of</strong> <strong>the</strong> sample close to <strong>the</strong> site <strong>of</strong>fertilisation.The most important outcome <strong>of</strong> <strong>the</strong> study wasthat high conception rates can be achieved inmares inseminated with low numbers <strong>of</strong> frozenthawedejaculated spermatozoa at a fixed timeafter <strong>the</strong> administration <strong>of</strong> an ovulation inducingagent.REFERENCESKloppe, L.H., Varner. D.D., Elmore. R.G., Bretzlaff,K.N. and Shull. J.W. (l988) Effect <strong>of</strong> inseminationtiming on <strong>the</strong> 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 <strong>of</strong> unfrozen and frozen stallionspermatand pacMorris, L.HHysterospermapreovulPace, M.M.insemincompon5
Equine Embryo TransferHYSTEROSCOPIC INSEMINATION OF NON FROZENAND FROZEN UNSEXED AND SEXED EQUINESPERMATOZOAA. C. Lindsey, L. H. A. Morris*, W. R. Allen*, J. L. Schenk † , J. K. Graham,J. E. Bruemmer and E. L. SquiresAnimal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado80523, USA; *Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB89BH, UK; † XY, Inc., ARBL Building, Foothills Research Campus, Fort Collins, Colorado 80523, USAA safe and reliable method for preconceptual sexselection <strong>of</strong> <strong>of</strong>fspring has been sought for decadesin humans, livestock, and even companionanimals. With a method developed some 10 yearsago (Johnson et al. 1989), effective preselection <strong>of</strong>sex has been accomplished in many species <strong>of</strong>livestock, as well as in humans (Johnson 1991;Cran et al. 1997; Seidel et al. 1997; Fugger 1999).Sex preselection has also been successful in horses(Buchanan et al. 2000), but due to <strong>the</strong> limitednumber <strong>of</strong> spermatozoa available after <strong>the</strong> sortingprocess, traditional breeding doses are notavailable. Therefore, low-dose inseminationtechniques must be improved in order to maximise<strong>the</strong> efficiency <strong>of</strong> sex-sorted spermatozoa.Hysteroscopic insemination has recently beenshown to produce acceptable pregnancy rateswhen using only one million freshly collectedmotile spermatozoa (Morris et al. 2000). With<strong>the</strong>se encouraging results, it has been hypo<strong>the</strong>sisedthat hysteroscopic insemination could be aneffective and practical method to achievepregnancies using low numbers <strong>of</strong> sex-sortedspermatozoa. The objectives <strong>of</strong> Experiment 1were: 1) to compare pregnancy rate with 5 x 10 6spermatozoa inseminated deep in <strong>the</strong> uterine hornaided by ultrasonography, or deposited onto <strong>the</strong>uterotubal papilla with <strong>the</strong> use <strong>of</strong> a flexible videoendoscope;and 2) to determine if hysteroscopicinsemination <strong>of</strong> sexed spermatozoa can result insatisfactory pregnancy rate.Semen was collected from 2 stallions <strong>of</strong>acceptable fertility. Oestrus was synchronised(June–July) in 40 mares, ages 3–10, byadministering 10 ml <strong>of</strong> altrenogest orally for 10days, followed by 250 µg cloprostenol im on Day11. Mares were given 3,000 iu hCG iv at <strong>the</strong> time<strong>of</strong> insemination and assigned to one <strong>of</strong> 3 groups:Group 1 mares (n=10) were inseminated with 5 x10 6 Percoll-washed spermatozoa deposited deepinto <strong>the</strong> uterine horn with <strong>the</strong> aid <strong>of</strong>ultrasonography. Group 2 mares (n=10) wereinseminated with 5 x 10 6 Percoll-washedspermatozoa deposited onto <strong>the</strong> uterotubal papillaevia hysteroscopic insemination. Group 3 mares(n=20) were inseminated using <strong>the</strong> hysteroscopictechnique with 5 x 10 6 sex-sorted spermatozoa.Spermatozoa were stained with Hoechst 33342and sorted into X and Y chromosome-bearingpopulations based on DNA content using an SXMoFlo sperm sorter. Pregnancy was determinedultrasonographically at 16 days post ovulation.Hysteroscopic insemination resulted in morepregnancies than did <strong>the</strong> ultrasound-guidedTABLE 1: Pregnancy rate resulting from hysteroscopic or deep intrauterine inseminationTreatment Mares inseminated Pregnant (16 d) Pregnancy rate (%)Non-sorted sperm, ultrasound guided 10 0 0 aNon-sorted sperm, hysteroscopic 10 5 50 bSex-sorted sperm, hysteroscopic 20 5 25 aba,b Values without common superscripts differ (P
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