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

Havemeyer Foundation Monograph Series No. 3EFFECTS OF OOCYTE MATURITY AND METHODSOF COLLECTION, CULTURE AND INSEMINATIONON EQUINE OOCYTE TRANSFERE. M. Carnevale, L. J. Maclellan, T. J. Scott, M. A. Coutinho da Silva,C. F. Scoggin and E. L. SquiresAnimal Reproduction 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 maturation is completed invitro prior to transfer. Development <strong>of</strong> techniquesthat would eliminate in vitro culture and allow <strong>the</strong>use <strong>of</strong> immature oocytes and low numbers <strong>of</strong>sperm would permit assisted reproductivetechniques to be used when an incubator and/orculture expertise are not available, whenpreovulatory oocytes are not available (egeuthanasia <strong>of</strong> mare), or when sperm numbers arelow (eg subfertile stallions, sexed sperm, frozensperm). Objectives <strong>of</strong> 2 experiments were tocompare embryo development rates for: 1) oocytescompleting maturation in vitro or within <strong>the</strong>oviduct; 2) intraoviductal versus intrauterineinsemination <strong>of</strong> recipients; 3) oocytes maturedwithin <strong>the</strong> follicle versus in vitro; and 4) oocytescollected from live mares versus fromslaughterhouse ovaries.In Experiment 1, oocytes were recovered usingtransvaginal, ultrasound-guided follicularaspirations (TVA) from naturally cycling donormares 24 to 26 h after hCG when follicles reached35 mm. Multiple oocytes (1–4) were transferredsurgically into oviducts <strong>of</strong> 4 or 5 recipients pergroup. Recipient’s oocytes were collected prior totransfers. Three groups <strong>of</strong> transfers werecompared: 1) transfers <strong>of</strong> oocytes cultured in vitr<strong>of</strong>or 12 to 14 h post collection with uterineinsemination <strong>of</strong> recipients 2 h post surgery; 2)transfers <strong>of</strong> oocytes into oviducts within 1 h <strong>of</strong>collection, with completion <strong>of</strong> oocyte maturationoccurring within oviducts, and uterineinsemination <strong>of</strong> recipients 14–16 h post surgery;and 3) transfers <strong>of</strong> 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 <strong>of</strong> 5% CO 2and air at 38.5°C. Recipients were inseminatedinto <strong>the</strong> uterine body with 2 x 10 9 progressivelymotile sperm from one <strong>of</strong> 2 stallions <strong>of</strong> knownfertility. Approximately 1 h prior to intraoviductalinsemination (Group 3), semen was collected froma stallion 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<strong>the</strong>tic oviduct fluid (H-SOFwith 0.6% BSA); 500,000 progressively motilesperm were transferred with oocytes into eachrecipient’s oviduct. In Experiment 1, numbers <strong>of</strong>embryos detected on Day 16 <strong>of</strong> gestation 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 <strong>of</strong>maturation within <strong>the</strong> oviduct, and spermdeposited within <strong>the</strong> oviduct were capable <strong>of</strong>fertilising oocytes.In Experiment 2, oocytes were collected byTVA from live mares or from slicingslaughterhouse ovaries. Four groups <strong>of</strong> oocyteswere transferred into oviducts <strong>of</strong> recipients:1) oocytes matured in vivo and collected by TVAfrom preovulatory follicles <strong>of</strong> oestrous mares32–36 h after administration <strong>of</strong> hCG; 2) immatureoocytes collected from dioestrous mares between 5and 10 days after aspiration or ovulation by TVAand matured in vitro for 36–38 h; 3) immatureoocytes collected from dioestrous mares between5–10 days after aspiration or ovulation by TVAand transferred into a recipient’s oviduct