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

Havemeyer Foundation Monograph Series No. 3CHROMATIN CONFIGURATION, MEIOTICCOMPETENCE AND SUCCESS OF INTRA-CYTOPLASMICSPERM INJECTION IN HORSE OOCYTES COLLECTED BYFOLLICULAR ASPIRATION OR SCRAPINGM. E. Dell’Aquila, M. Masterson*, F. Maritato and K. Hinrichs †Department <strong>of</strong> Animal Production, Section <strong>of</strong> Reproduction, University <strong>of</strong> Bari, Valenzano, Bari, Italy;*Department <strong>of</strong> Clinical Studies, Tufts University School <strong>of</strong> Veterinary Medicine, Grafton, Massachusetts,USA; † Department <strong>of</strong> Veterinary Physiology and Pharmacology, College <strong>of</strong> Veterinary Medicine, TexasA&M University, College Station, Texas, USAReported rates <strong>of</strong> oocyte maturation andfertilisation vary greatly among laboratories. Thismay be related to <strong>the</strong> populations <strong>of</strong> oocytesselected for use, as <strong>the</strong> maturation rate <strong>of</strong> horseoocytes is dependent on diameter and <strong>the</strong> degree <strong>of</strong>viability <strong>of</strong> <strong>the</strong> follicle from which <strong>the</strong> oocyteoriginates (Hinrichs and Williams 1997; Hinrichsand Schmidt 2000). Higher maturation rates arefound in oocytes from large follicles, and inoocytes from atretic follicles. Cumulus expansionis a marker for oocyte atresia, <strong>the</strong>refore assessment<strong>of</strong> cumulus morphology is an important parameterin selection <strong>of</strong> oocytes for study. Germinal vesiclechromatin configuration also differs with follicleatresia and follicle size, and is strongly associatedwith <strong>the</strong> meiotic competence <strong>of</strong> <strong>the</strong> oocyte(Hinrichs and Williams 1997; Hinrichs andSchmidt 2000).Methods for collection and selection <strong>of</strong> horseoocytes have not been standardised amonglaboratories. The 2 most common methods forhorse oocyte collection are aspiration <strong>of</strong> follicularfluid or opening follicles and scraping <strong>the</strong>granulosa layer. Oocytes in viable follicles may bemore closely attached to <strong>the</strong> follicle wall, whereasoocytes from atretic follicles may be loose within<strong>the</strong> follicular fluid (Hawley et al. 1995;Mlodawska and Okolski 1997). Thus, it is possiblethat <strong>the</strong>se 2 methods <strong>of</strong> oocyte recovery result incollection <strong>of</strong> different populations <strong>of</strong> oocytes, withaspiration favouring collection oocytes fromatretic follicles and scraping favouring collection<strong>of</strong> oocytes from viable follicles. The type <strong>of</strong>oocytes obtained may affect <strong>the</strong> observed resultsafter maturation or fertilisation, and may be afactor in <strong>the</strong> reported differences in results using<strong>the</strong>se procedures among laboratories. This studywas conducted to determine if <strong>the</strong> meiotic anddevelopmental competence <strong>of</strong> horse oocytes couldbe dependent on <strong>the</strong> method <strong>of</strong> oocyte collection.Horse oocytes were recovered fromslaughterhouse ovaries by aspiration or scraping.Only follicles visible on <strong>the</strong> surface <strong>of</strong> <strong>the</strong> ovaryand >10 mm diameter were used. Aspiration wasperformed using an 18 ga needle attached to avacuum pump. For scraping, follicles were openedwith a scalpel blade and <strong>the</strong> granulosa cell layerwas removed using a bone curette. Recoveredoocytes were classified as having compact,expanded or partial cumuli; obviouslydegenerating oocytes were discarded. InExperiment 1, oocytes were denuded and fixedimmediately after collection, and were stainedwith Hoechst 33258 to assess whe<strong>the</strong>r <strong>the</strong>collection method influenced <strong>the</strong> initial chromatinconfiguration <strong>of</strong> oocytes. This was done both inMay and in October. In Experiment 2, in vitromaturation rates <strong>of</strong> oocytes recovered byaspiration or scraping were compared. IVM wasperformed as previously described (Dell’Aquila etal. 1996), with <strong>the</strong> medium supplemented with20% (v/v) oestrous mare serum. Oocytes werecultured for 28–30 h at 38.5°C under 5%CO 2 inair. In Experiment 3, oocytes were matured in vitroas for Experiment 2, but with 20% follicular fluidin place <strong>of</strong> <strong>the</strong> mare serum, which increases <strong>the</strong>rate <strong>of</strong> male pronucleus formation in oocyteshaving compact cumuli (Dell’Aquila et al. 1997).37