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

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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
Equine Embryo TransferUTERINE BLOOD FLOW DURING OESTROUS CYCLEAND EARLY PREGNANCY IN MARESH. Bollwein, R. Mayer and R. StollaGynaecological Animal Clinic, University <strong>of</strong> Munich, GermanyINTRODUCTIONWhile <strong>the</strong>re are several studies on uterine bloodflow during early pregnancy in o<strong>the</strong>r species(Greiss and Anderson 1970; Ford and Christenson1979; Ford et al. 1989), until now <strong>the</strong>re are noinformation about uterine perfusion duringoestrous cycle and early pregnancy in mares. In apreliminary study we could demonstrate thattransrectal Colour Doppler sonography is a usefulmethod to measure impedance to uterine bloodflow in mares (Bollwein 1998). In this study weaimed to use this technique for <strong>the</strong> examination <strong>of</strong>uterine blood flow during oestrous cycle and earlypregnancy in mares.MATERIALAND METHODSFive Trotter mares with a mean age <strong>of</strong> 9.2 years(range 6–13 years) were examined as describedbelow. Two <strong>of</strong> <strong>the</strong>m were multiparae and 3 werenullipareae. Each mare underwent transrectalDoppler investigations by <strong>the</strong> same operator over 2°oestrous cycles and 2 early pregnancies. The time <strong>of</strong>ovulation was determined from daily real-timeultrasound examinations, <strong>the</strong> last day on which <strong>the</strong>dominant follicle was visible being defined as Day1, and <strong>the</strong> first day on which <strong>the</strong> ovulatory folliclewas gone being Day 0. Examinations were carriedout daily between Day °0 (=° ovulation) and Day°3, <strong>the</strong>n every second day until Day 29 <strong>of</strong>pregnancy. During oestrous cycle investigationswere performed daily again between Day °3 andDay 0 <strong>of</strong> <strong>the</strong> next oestrous cycle.Both <strong>the</strong> left and right Aa. uterinae wereinvestigated transrectally as published earlier(Bollwein 1998). The transrectal pulsed Dopplerultrasound examinations were always carried out at<strong>the</strong> same time (ie from 16.00–22.00 h) and lastedabout 20 min for each mare. All obtained bloodflow velocity waveforms were were displayedonline and recorded on videotapes. Following <strong>the</strong>collection <strong>of</strong> all data, <strong>the</strong> Doppler calculations wereperformed <strong>of</strong>fline by using 2 similar consecutiveflow velocity waveforms with a maximumenddiastolic frequency shift. The analysis wasbased on <strong>the</strong> resistance index (RI), which iscalculated as <strong>the</strong> ratio <strong>of</strong> <strong>the</strong> difference betweenpeak systolic frequency shift (PSF) and enddiastolicfrequency shift (EDF) to peak systolicfrequency shift: RI = (PSF-EDF)/PSF). The RIincreases if <strong>the</strong> proximal conditions remain constantand <strong>the</strong> distal vascular bed constricts. Conversely, alow value for <strong>the</strong> RI indicates decreased impedanceto blood flow in <strong>the</strong> distal vasculature. The RIvalues <strong>of</strong> <strong>the</strong> two uniform consecutive pulse waveswere averaged. To study <strong>the</strong> intraobserverreproducibility <strong>the</strong> A. uterina was examined 2times, <strong>the</strong> interval between each measurement in <strong>the</strong>same vessel being approximately 5–15 min.Statistical analyses were carried out using <strong>the</strong>Statview II+Graphics statistical s<strong>of</strong>tware package(Abacus Concepts, Inc, California, USA, 1992)and <strong>the</strong> Statistical Analysis System (SAS Institute,North Carolina, USA, 1996).The resistance index<strong>of</strong> <strong>the</strong> left and right A.°uterina and <strong>of</strong> <strong>the</strong> pregnantand non pregnant A. uterina were compared usingcorrelation coefficient and paired Student’s t-test.Measurements were subjected to analysis <strong>of</strong>variance <strong>of</strong> replicate measurements, taking intoaccount <strong>the</strong> between animals variance componentand <strong>the</strong> between cycles within animal component.Intraobserver reproducibility <strong>of</strong> Dopplermeasurement results were expressed as intraclasscorrelation coefficient (Intra-CC).8
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