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. 3TRANSVAGINAL INTRAFOLLICULAR SPERM CELLINJECTION IN THE CYCLIC MAREM. Meinjtes*, B. Eilts † , R. Cochran ** , K. Graff ‡ , R. Dennist<strong>on</strong>, D. Paccam<strong>on</strong>ti*and R. GodkeDepartment <str<strong>on</strong>g>of</str<strong>on</strong>g> Animal Science; † Louisiana State University Agricultural Center, Department <str<strong>on</strong>g>of</str<strong>on</strong>g>Veterinary Clinical Sciences, School <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine, Louisiana State University, Bat<strong>on</strong> Rouge,Louisiana 70803, USAIntrafollicular inseminati<strong>on</strong> (IFI) is an assistedreproductive technique that has been successfullyused in human reproducti<strong>on</strong> (Lecena et al. 1991;Zbella et al. 1992). However it has not beenreported in domestic animals. Intrafollicularinseminati<strong>on</strong> has potential applicati<strong>on</strong>s forcircumventing <str<strong>on</strong>g>the</str<strong>on</strong>g> uterine inflammati<strong>on</strong> in mareswith persistent mating induced endometritis,breeding with reduced numbers <str<strong>on</strong>g>of</str<strong>on</strong>g> sperm cellsfrom oligospermic ejaculates or frozen semen, andresearch applicati<strong>on</strong>s to fur<str<strong>on</strong>g>the</str<strong>on</strong>g>r understandfertilisati<strong>on</strong>. Intrafollicular inseminati<strong>on</strong> may alsobe an attractive, less technically complex approachto achieve <str<strong>on</strong>g>the</str<strong>on</strong>g> same goal as with c<strong>on</strong>venti<strong>on</strong>al IVF.The objective <str<strong>on</strong>g>of</str<strong>on</strong>g> this study was to performtransvaginal ultrasound-guided intrafollicularinseminati<strong>on</strong> to evaluate <str<strong>on</strong>g>the</str<strong>on</strong>g> effectiveness <str<strong>on</strong>g>of</str<strong>on</strong>g> 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 <str<strong>on</strong>g>the</str<strong>on</strong>g> ovaries evaluatedby transrectal ultras<strong>on</strong>ography daily. All IFIprocedures were performed 9–13 h beforeovulati<strong>on</strong>. Ovulati<strong>on</strong> was verified by hourlytransrectal ultrasound examinati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> ovariesstarting at <str<strong>on</strong>g>the</str<strong>on</strong>g> time <str<strong>on</strong>g>of</str<strong>on</strong>g> IF. Mares in Treatment A(n=3) and Treatment B (n=2) received 3,300 iu <str<strong>on</strong>g>of</str<strong>on</strong>g>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> IFI, and mares in TreatmentPresent address: Presbyterian Hospital, AssistedReproductive Technology Services; **Present address:Reproductive Medicine and Fertility Center, 615 E.Princet<strong>on</strong> 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 <str<strong>on</strong>g>the</str<strong>on</strong>g> samefertile stalli<strong>on</strong>. The sperm cells used for IFI werewashed and resuspended in 1.5 ml <str<strong>on</strong>g>of</str<strong>on</strong>g> Hams F-10to obtain an inseminati<strong>on</strong> c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 120 x10 6 progressively motile sperm per ml. Themotility <str<strong>on</strong>g>of</str<strong>on</strong>g> different ejaculates ranged between 40and 80%. In additi<strong>on</strong>, <str<strong>on</strong>g>the</str<strong>on</strong>g> sperm cells in TreatmentA and C were treated with a 3 µM c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>calcium i<strong>on</strong>ophore A32187 for 5 min to inducecapacitati<strong>on</strong>. The sperm cells for Treatment B andD were not treated with calcium i<strong>on</strong>ophoreA32187, but <strong>on</strong>ly were washed. All IFI wereperformed with <str<strong>on</strong>g>the</str<strong>on</strong>g> sperm cell c<strong>on</strong>centrati<strong>on</strong>adjusted to 120 x 10 6 progressively motile spermper ml. The IFI sperm injecti<strong>on</strong> procedure wasperformed under transvaginal ultrasound guidancewith a 22 g needle c<strong>on</strong>nected to a 2.5 ml syringec<strong>on</strong>taining <str<strong>on</strong>g>the</str<strong>on</strong>g> 1.5 ml sperm-cell suspensi<strong>on</strong> forIFI. The sperm cells remaining in <str<strong>on</strong>g>the</str<strong>on</strong>g> needle aftereach inseminati<strong>on</strong> were used to calculate <str<strong>on</strong>g>the</str<strong>on</strong>g> totalmotile sperm cells inseminated. The results aresummarised in Table 1. All mares in Treatment Aand B ovulated, however <strong>on</strong>ly <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> 3 inTreatment C and <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> 2 in Treatment D ovulated.N<strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> 10 mares having IFI were found to bepregnant when <str<strong>on</strong>g>the</str<strong>on</strong>g>y were examined for pregnancyusing transrectal ultras<strong>on</strong>ography.It is not clear from this study if <str<strong>on</strong>g>the</str<strong>on</strong>g> spermnumber per follicle was adequate, <str<strong>on</strong>g>the</str<strong>on</strong>g> sperm cellsremained unbound in <str<strong>on</strong>g>the</str<strong>on</strong>g> follicular fluid, or if <str<strong>on</strong>g>the</str<strong>on</strong>g>sperm cells could survive in <str<strong>on</strong>g>the</str<strong>on</strong>g> follicularenvir<strong>on</strong>ment l<strong>on</strong>g enough to achieve in viv<str<strong>on</strong>g>of</str<strong>on</strong>g>ertilisati<strong>on</strong> in <str<strong>on</strong>g>the</str<strong>on</strong>g> follicle or oviduct. Humanpatients have become pregnant after IFinseminati<strong>on</strong> with 200,000 cells (Lucena et al.39