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

Equine Embryo TransferEMBRYONIC DEVELOPMENT OF EQUINE OOCYTESFERTILISED BY ICSIC. Galli, G. Crotti, G. Mari* and G. LazzariLaborotorio di Tecnologie della Riproduzione, C.I.Z.- A.I.A. Via Porcellasco 7/f, 26100 Cremona, Italy;*Dipartmento Clinico Veterinario, Universita degli Studi di Bologna, Via Tolara di Sopra 50, OzzanoEmilia, 40064 Bologna, ItalyINTRODUCTIONThe application <strong>of</strong> in vitro embryo productiontechnology in <strong>the</strong> horse is very limited because <strong>the</strong>overall procedure is much less efficient as comparedfor example to <strong>the</strong> bovine where at present manythousands <strong>of</strong> calves are born worldwide following<strong>the</strong> transfer <strong>of</strong> in vitro produced embryos. In <strong>the</strong>horse successful birth <strong>of</strong> foals following assistedreproductive techniques are still occasional (Palmeret al. 1991; Squires et al. 1996; Cochran et al. 1998;McKinnon et al. 1998), anatomical andphysiological peculiarities are <strong>the</strong> main constraint to<strong>the</strong> development <strong>of</strong> assisted reproductive techniquesin this species. The potential application in <strong>the</strong> horsewill be for assisted reproduction <strong>of</strong> infertile orsubfertile mare and stallions ra<strong>the</strong>r than for <strong>the</strong>increase <strong>of</strong> <strong>the</strong> number <strong>of</strong> <strong>of</strong>fspring obtained fromany given donor.The 3 steps involved in <strong>the</strong> production <strong>of</strong>embryos in vitro (maturation, fertilisation andculture) have been studied by several laboratories.In vitro maturation is <strong>the</strong> more efficient step asassessed by <strong>the</strong> transfer <strong>of</strong> in vitro matured oocytesto <strong>the</strong> oviduct for fertilisation and development(Zhang et al. 1989). In vitro fertilisation is <strong>the</strong> lessreproducible step and it is now overcome by <strong>the</strong>use <strong>of</strong> intracytoplasmic sperm injection (ICSI).Embryo culture is <strong>the</strong> less studied aspect because<strong>the</strong> embryos are usually transferred to <strong>the</strong> oviductas soon as possible after fertilisation. An efficientsystem that would allow <strong>the</strong> development <strong>of</strong> invitro produced embryo to blastocyst it would behighly desirable both because it would allow <strong>the</strong>non surgical transfer and possibly <strong>the</strong> freezing <strong>of</strong>such embryos.The aim <strong>of</strong> this study was to improve <strong>the</strong> basicknowledge on <strong>the</strong> influence <strong>of</strong> follicle size andcumulus morphology on <strong>the</strong> embryo development<strong>of</strong> horse oocytes fertilised by ICSI and cultured in<strong>the</strong> surrogate oviduct <strong>of</strong> <strong>the</strong> sheep.MATERIALS AND METHODSOocytes were collected from ovaries <strong>of</strong>slaughtered mares during <strong>the</strong> period December1999–February 2000. The ovaries were collectedin plastic bags with saline solution and transportedto <strong>the</strong> laboratory in <strong>the</strong>rmostatic boxes kept at24–26°C within 4 h from slaughter. Theconnective tissue capsule was removed from <strong>the</strong>ovaries to help locate <strong>the</strong> follicles. A slit was madein each follicle with a scalpel blade and a Volkmanspoon was used to scrape <strong>the</strong> inside <strong>of</strong> each folliclelarge than an estimated 0.5 cm. The oocytesrecovered were divided in two groups according to<strong>the</strong> size <strong>of</strong> <strong>the</strong> follicle above (large follicles) orbelow (small follicles) 2 cm <strong>of</strong> diameter. Withineach group <strong>the</strong> oocytes were divided according to<strong>the</strong> cumulus morphology, compacted andexpanded. The oocytes belonging to <strong>the</strong> 2 classes<strong>of</strong> follicle size were matured separately in vitro for26 h (oocytes with expanded cumulus) or 30 h(oocytes with compacted cumulus). Maturationmedium consisted <strong>of</strong> TCM 199 with 10% FCS,ITS (insulin transferring and selenium, Sigma),epidermal growth factor analogue (50 ng/ml,Sigma), 0.5 IU <strong>of</strong> FSH LH (Pergovet, Serono), 17ßoestradiol (2 µg/ml). Incubation was performed at38°C in 5% CO 2. At <strong>the</strong> end <strong>of</strong> maturation oocyteswere decumulated mechanically beforemicroinjection.One straw <strong>of</strong> frozen semen <strong>of</strong> proven fertilityfrom <strong>the</strong> same batch was used for ICSi andprepared immediately before use. After thawingmotile spermatozoa were separated on 45–90%32