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

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Havemeyer Foundation Monograph Series No. 3TRANSVAGINAL INTRAFOLLICULAR SPERM CELLINJECTION IN THE CYCLIC MAREM. Meinjtes*, B. Eilts † , R. Cochran ** , K. Graff ‡ , R. Denniston, D. Paccamonti*and R. GodkeDepartment <strong>of</strong> Animal Science; † Louisiana State University Agricultural Center, Department <strong>of</strong>Veterinary Clinical Sciences, School <strong>of</strong> Veterinary Medicine, Louisiana State University, Baton Rouge,Louisiana 70803, USAIntrafollicular insemination (IFI) is an assistedreproductive technique that has been successfullyused in human reproduction (Lecena et al. 1991;Zbella et al. 1992). However it has not beenreported in domestic animals. Intrafollicularinsemination has potential applications forcircumventing <strong>the</strong> uterine inflammation in mareswith persistent mating induced endometritis,breeding with reduced numbers <strong>of</strong> sperm cellsfrom oligospermic ejaculates or frozen semen, andresearch applications to fur<strong>the</strong>r understandfertilisation. Intrafollicular insemination may alsobe an attractive, less technically complex approachto achieve <strong>the</strong> same goal as with conventional IVF.The objective <strong>of</strong> this study was to performtransvaginal ultrasound-guided intrafollicularinsemination to evaluate <strong>the</strong> effectiveness <strong>of</strong> IFI toestablish a pregnancy.In this experiment 10 light horse mares withnormal length oestrous cycles were used in a 2 x 2factorial arrangement. The mares were teased,palpated per rectum and had <strong>the</strong> ovaries evaluatedby transrectal ultrasonography daily. All IFIprocedures were performed 9–13 h beforeovulation. Ovulation was verified by hourlytransrectal ultrasound examinations <strong>of</strong> <strong>the</strong> ovariesstarting at <strong>the</strong> time <strong>of</strong> IF. Mares in Treatment A(n=3) and Treatment B (n=2) received 3,300 iu <strong>of</strong>hCG iv at <strong>the</strong> time <strong>of</strong> IFI, and mares in TreatmentPresent address: Presbyterian Hospital, AssistedReproductive Technology Services; **Present address:Reproductive Medicine and Fertility Center, 615 E.Princeton Street, Suite 225, Orlando, Florida 32806USA; ‡ Present address: Arnold Palmer Hospital,Fertility Center, 23 West Copeland Drive, Orlando,Florida 32806, USAC (n=3) and Treatment D (n=2) were allowed toovulate naturally after IFI. The sperm used foreach mare was freshly collected from <strong>the</strong> samefertile stallion. The sperm cells used for IFI werewashed and resuspended in 1.5 ml <strong>of</strong> Hams F-10to obtain an insemination concentration <strong>of</strong> 120 x10 6 progressively motile sperm per ml. Themotility <strong>of</strong> different ejaculates ranged between 40and 80%. In addition, <strong>the</strong> sperm cells in TreatmentA and C were treated with a 3 µM concentration <strong>of</strong>calcium ionophore A32187 for 5 min to inducecapacitation. The sperm cells for Treatment B andD were not treated with calcium ionophoreA32187, but only were washed. All IFI wereperformed with <strong>the</strong> sperm cell concentrationadjusted to 120 x 10 6 progressively motile spermper ml. The IFI sperm injection procedure wasperformed under transvaginal ultrasound guidancewith a 22 g needle connected to a 2.5 ml syringecontaining <strong>the</strong> 1.5 ml sperm-cell suspension forIFI. The sperm cells remaining in <strong>the</strong> needle aftereach insemination were used to calculate <strong>the</strong> totalmotile sperm cells inseminated. The results aresummarised in Table 1. All mares in Treatment Aand B ovulated, however only one <strong>of</strong> 3 inTreatment C and one <strong>of</strong> 2 in Treatment D ovulated.None <strong>of</strong> <strong>the</strong> 10 mares having IFI were found to bepregnant when <strong>the</strong>y were examined for pregnancyusing transrectal ultrasonography.It is not clear from this study if <strong>the</strong> spermnumber per follicle was adequate, <strong>the</strong> sperm cellsremained unbound in <strong>the</strong> follicular fluid, or if <strong>the</strong>sperm cells could survive in <strong>the</strong> follicularenvironment long enough to achieve in viv<strong>of</strong>ertilisation in <strong>the</strong> follicle or oviduct. Humanpatients have become pregnant after IFinsemination with 200,000 cells (Lucena et al.39
Equine Embryo TransferTABLE 1: Ovulation and pregnancy results in mares administered or not administered 3,300 iu hCG ivbefore intrafollicular insemination with 180 x 10 6 motile sperm cells treated with calcium ionophore A32187 (capacitated) or not treated with calcium ionophore A 32187 (not capacitated)Treatment A Treatment B Treatment C Treatment D(hCG and (hCG and not (No hCG and (No hCG and notcapacitated) capacitated) capacitated) capacitated)N 3 2 3 2Ovulated 3 2 1 1Pregnant 0 0 0 01991) so it seems that enough cells wereinseminated. However, <strong>the</strong> disparity <strong>of</strong> follicularfluid volume between <strong>the</strong> human and mare follicleresults in a greater dilution <strong>of</strong> cells in <strong>the</strong> marefollicle. Therefore, <strong>the</strong> dose <strong>of</strong> sperm cells is stillquestionable. Binding <strong>of</strong> <strong>the</strong> sperm cells togranulosa cells may have prevented <strong>the</strong> sperm cellsfrom being carried into <strong>the</strong> oviducts at ovulation.Sperm cells are known to bind to mare oviductepi<strong>the</strong>lial cells (Thomas et al. 1994) so <strong>the</strong> spermcells also may have <strong>the</strong> ability to bind to granulosacells in <strong>the</strong> follicle. It was reported that follicularfluid recovered shortly after IFI contained nosperm cells, perhaps indicating that binding <strong>of</strong> <strong>the</strong>sperm cells took place. (Alvarenga, EETVDiscussion) If too many sperm cells are bound togranulosa cells, <strong>the</strong>n insufficient sperm cells mayenter <strong>the</strong> oviduct at ovulation to achievefertilisation. Perhaps cryopreserved stallion spermcells, which have a decreased adherence to mareoviduct epi<strong>the</strong>lial cells (Ellington et al. 1999) mayactually result in higher pregnancy rates because<strong>of</strong> decreased adhesion to granulosa cells. Theaspiration <strong>of</strong> <strong>the</strong> small volume <strong>of</strong> follicular fluiddoes not inhibit ovulation or fertilisation in maresbred by normal artificial insemination procedures(Duchamp et al. 1996) and only a small amount <strong>of</strong>follicular fluid is actually needed to enter <strong>the</strong>oviduct at ovulation (Bezard et al. 1996) Sinceonly a small volume <strong>of</strong> follicular fluid actuallyenters <strong>the</strong> oviduct at ovulation, an insufficientnumber <strong>of</strong> sperm cells may have been carried into<strong>the</strong> oviduct with a low volume <strong>of</strong> follicular fluid.Sperm cells may not retain sufficient motility infollicular fluid to allow normal fertilisation ratesafter IF insemination. Unpublished work in thislaboratory has shown sperm cells have 63, 49, 36,16, 7, 2 and 1% motility after incubation in vitr<strong>of</strong>or 0, 1, 2, 3, 4, 5 and 6 h, respectively in follicularfluid at 37°C (Eilts et al. unpublished data). This isin contrast to motility <strong>of</strong> semen extended in skimmilk extender which had 80, 72, 48, 32, 25, 16 and12% after 0, 1, 2, 3, 4, 5 and 6 h, respectively.Therefore, if ovulation did not occur until 9–13 hafter IF insemination in <strong>the</strong> present study, spermmotility after IFI may not have been adequate toattain optimal fertilisation rates. It appears that <strong>the</strong>use <strong>of</strong> hCG will be desirable in future studies <strong>of</strong>IFI to ensure ovulation.Although IFI has been reported to besuccessful in human (Lecena et al. 1991; Zbella etal. 1992), a more comprehensive study attainedonly one pregnancy in 50 attempts (Nuojua-Huttunen et al. 1995). In <strong>the</strong> mare, o<strong>the</strong>rresearchers also reported that no pregnancies wereachieved after intrafollicular insemination(Alvarenga and McCue, EETV discussion).Perhaps <strong>the</strong> follicular fluid could be removed andreplaced entirely with extended sperm cells.Preliminary work to determine if IFI may bedetrimental in establishing pregnancy whennormal artificial insemination is performedconcomitant with IFI has resulted in nopregnancies (Eilts et al. unpublished data). Inconclusion, more research is needed to determineif in vivo-matured oocytes can be fertilised usingIFI.REFERENCESBezard, J., Duchamp, G., Couty, I. et al. (1996) Thefollicular-fluid is not a compulsory carrier <strong>of</strong> <strong>the</strong>mares oocyte at ovulation. Arch. Tierzucht. 39, 27.Duchamp, G., Gerard, N., Bezard, J., et al. (1996)Sampling <strong>of</strong> preovulatory follicular-fluid in mares.Arch. Tierzucht. 39, 79.Ellington, J.E., Samper, J.C., Jones, A.E. et al. (1999) Invitro interactions <strong>of</strong> cryopreserved stallionspermatozoa and oviduct (uterine tube) epi<strong>the</strong>lialcells or56, 51-6Lucena, E., RintrafollJ. reprodNuojua-Huttu(1995) In<strong>of</strong> inferti40
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