Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 3EFFECTS OF OOCYTE MATURITY AND METHODSOF COLLECTION, CULTURE AND INSEMINATIONON <strong>EQUINE</strong> OOCYTE TRANSFERE. M. Carnevale, L. J. Maclellan, T. J. Scott, M. A. Coutinho da Silva,C. F. Scoggin and E. L. SquiresAnimal Reproducti<strong>on</strong> and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado80523, USAAssisted reproductive techniques, such as oocytetransfer, are currently being used to obtainpregnancies from subfertile mares. For oocytetransfer, preovulatory oocytes are usually collected24 h after hCG, and maturati<strong>on</strong> is completed invitro prior to transfer. Development <str<strong>on</strong>g>of</str<strong>on</strong>g> techniquesthat would eliminate in vitro culture and allow <str<strong>on</strong>g>the</str<strong>on</strong>g>use <str<strong>on</strong>g>of</str<strong>on</strong>g> immature oocytes and low numbers <str<strong>on</strong>g>of</str<strong>on</strong>g>sperm would permit assisted reproductivetechniques to be used when an incubator and/orculture expertise are not available, whenpreovulatory oocytes are not available (egeuthanasia <str<strong>on</strong>g>of</str<strong>on</strong>g> mare), or when sperm numbers arelow (eg subfertile stalli<strong>on</strong>s, sexed sperm, frozensperm). Objectives <str<strong>on</strong>g>of</str<strong>on</strong>g> 2 experiments were tocompare embryo development rates for: 1) oocytescompleting maturati<strong>on</strong> in vitro or within <str<strong>on</strong>g>the</str<strong>on</strong>g>oviduct; 2) intraoviductal versus intrauterineinseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> recipients; 3) oocytes maturedwithin <str<strong>on</strong>g>the</str<strong>on</strong>g> follicle versus in vitro; and 4) oocytescollected from live mares versus fromslaughterhouse ovaries.In Experiment 1, oocytes were recovered usingtransvaginal, ultrasound-guided follicularaspirati<strong>on</strong>s (TVA) from naturally cycling d<strong>on</strong>ormares 24 to 26 h after hCG when follicles reached35 mm. Multiple oocytes (1–4) were transferredsurgically into oviducts <str<strong>on</strong>g>of</str<strong>on</strong>g> 4 or 5 recipients pergroup. Recipient’s oocytes were collected prior totransfers. Three groups <str<strong>on</strong>g>of</str<strong>on</strong>g> transfers werecompared: 1) transfers <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes cultured in vitr<str<strong>on</strong>g>of</str<strong>on</strong>g>or 12 to 14 h post collecti<strong>on</strong> with uterineinseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> recipients 2 h post surgery; 2)transfers <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes into oviducts within 1 h <str<strong>on</strong>g>of</str<strong>on</strong>g>collecti<strong>on</strong>, with completi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> oocyte maturati<strong>on</strong>occurring within oviducts, and uterineinseminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> recipients 14–16 h post surgery;and 3) transfers <str<strong>on</strong>g>of</str<strong>on</strong>g> sperm and oocytes (cultured 12to 14 h in vitro) into oviducts. Oocytes, cultured invitro, were placed in culture medium (TCM 199with 10% FCS, 0.2 mM pyruvate and 50 µg/mlgentamicin sulphate) in an atmosphere <str<strong>on</strong>g>of</str<strong>on</strong>g> 5% CO 2and air at 38.5°C. Recipients were inseminatedinto <str<strong>on</strong>g>the</str<strong>on</strong>g> uterine body with 2 x 10 9 progressivelymotile sperm from <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> 2 stalli<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> knownfertility. Approximately 1 h prior to intraoviductalinseminati<strong>on</strong> (Group 3), semen was collected froma stalli<strong>on</strong> and separated through a density gradient(90/45% Percoll centrifuged for 10 min at 300G).The sperm pellet was washed and resuspended inHepes-buffered syn<str<strong>on</strong>g>the</str<strong>on</strong>g>tic oviduct fluid (H-SOFwith 0.6% BSA); 500,000 progressively motilesperm were transferred with oocytes into eachrecipient’s oviduct. In Experiment 1, numbers <str<strong>on</strong>g>of</str<strong>on</strong>g>embryos detected <strong>on</strong> Day 16 <str<strong>on</strong>g>of</str<strong>on</strong>g> gestati<strong>on</strong> were notdifferent (P>0.1) for Groups 1, 2 and 3 (8/14, 57%;6/14, 43% and 3/11, 27%). Therefore, maturingoocytes successfully completed final stages <str<strong>on</strong>g>of</str<strong>on</strong>g>maturati<strong>on</strong> within <str<strong>on</strong>g>the</str<strong>on</strong>g> oviduct, and spermdeposited within <str<strong>on</strong>g>the</str<strong>on</strong>g> oviduct were capable <str<strong>on</strong>g>of</str<strong>on</strong>g>fertilising oocytes.In Experiment 2, oocytes were collected byTVA from live mares or from slicingslaughterhouse ovaries. Four groups <str<strong>on</strong>g>of</str<strong>on</strong>g> oocyteswere transferred into oviducts <str<strong>on</strong>g>of</str<strong>on</strong>g> recipients:1) oocytes matured in vivo and collected by TVAfrom preovulatory follicles <str<strong>on</strong>g>of</str<strong>on</strong>g> oestrous mares32–36 h after administrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> hCG; 2) immatureoocytes collected from dioestrous mares between 5and 10 days after aspirati<strong>on</strong> or ovulati<strong>on</strong> by TVAand matured in vitro for 36–38 h; 3) immatureoocytes collected from dioestrous mares between5–10 days after aspirati<strong>on</strong> or ovulati<strong>on</strong> by TVAand transferred into a recipient’s oviduct
Equine Embryo Transfercollecti<strong>on</strong>; and 4) immature oocytes collected fromslaughterhouse ovaries c<strong>on</strong>taining a corpus luteumand matured in vitro for 36–38 h. Recipients inGroup 1, 2 and 4 were inseminated 12–14 h priorto oocyte transfer; recipients in Group 3 wereinseminated 34–36 h after transfer. Recipientswere inseminated with 2 x 10 9 progressivelymotile sperm from <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> two stalli<strong>on</strong>s. Oocytes,matured in vitro, were placed in maturati<strong>on</strong>medium (TCM 199 with 0.2 mM pyruvate, 1 mMglutamine, 25 mM bicarb<strong>on</strong>ate, 10% FCS, 15ng/ml FSH, 1 µg/ml LH, 1 µg/ml E 2 , 50 µg/mlgentamicin) and incubated for 36–38 h at 38.5°Cin 5% CO 2 and air. Oocyte recipients were allowedto ovulate <str<strong>on</strong>g>the</str<strong>on</strong>g> preovulatory follicle; subsequently,<str<strong>on</strong>g>the</str<strong>on</strong>g> number <str<strong>on</strong>g>of</str<strong>on</strong>g> recipient ovulati<strong>on</strong>s were subtractedfrom <str<strong>on</strong>g>the</str<strong>on</strong>g> number <str<strong>on</strong>g>of</str<strong>on</strong>g> embry<strong>on</strong>ic vesicles imagedwith ultrasound.In Experiment 2, oocytes matured in vivo(9/11, 82%) resulted in higher (P0.1) embryo development rates after transfer <str<strong>on</strong>g>of</str<strong>on</strong>g>immature oocytes.In c<strong>on</strong>clusi<strong>on</strong>, oocyte transfer was a repeatablemethod for testing oocyte competence after IVM.Embryo development rates were high after transfer<str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes matured in vivo. The final stage <str<strong>on</strong>g>of</str<strong>on</strong>g>oocyte maturati<strong>on</strong> successfully occurred within<str<strong>on</strong>g>the</str<strong>on</strong>g> oviduct; however, immature oocytes did notcomplete maturati<strong>on</strong> within <str<strong>on</strong>g>the</str<strong>on</strong>g> oviduct.Pregnancies were obtained from oocytes collectedby TVA or from slaughterhouse ovaries, maturedin vitro, and transferred into recipients; however,embryo development rates were low. Transfer <str<strong>on</strong>g>of</str<strong>on</strong>g>sperm and oocytes into <str<strong>on</strong>g>the</str<strong>on</strong>g> oviduct resulted inembryo development, suggesting this technique(GIFT) has a potential use in equine assistedreproducti<strong>on</strong>.ACKNOWLEDGEMENTSThis project was funded in part through <str<strong>on</strong>g>the</str<strong>on</strong>g>Research Council <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> College <str<strong>on</strong>g>of</str<strong>on</strong>g> VeterinaryMedicine and Biomedical Sciences at ColoradoState University, Colorado Equine RacingCommissi<strong>on</strong> and by benefactors <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> programmefor Preservati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> Equine Genetics and <str<strong>on</strong>g>the</str<strong>on</strong>g> LucyWhittier Foundati<strong>on</strong>. The authors would like tothank Mary O’D<strong>on</strong>ovan, Camille Torres andJessica Valle for technical assistance and Drs.George Seidel, Jr., Marco Alvarenga, FernandaAlvarenga, and Young Ho Choi for scientificinput.48
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