Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 3CHROMATIN CONFIGURATION, MEIOTICCOMPETENCE AND SUCCESS OF INTRA-CYTOPLASMICSPERM INJECTION IN HORSE OOCYTES COLLECTED BYFOLLICULAR ASPIRATION OR SCRAPINGM. E. Dell’Aquila, M. Masters<strong>on</strong>*, F. Maritato and K. Hinrichs †Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Animal Producti<strong>on</strong>, Secti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> Reproducti<strong>on</strong>, University <str<strong>on</strong>g>of</str<strong>on</strong>g> Bari, Valenzano, Bari, Italy;*Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Clinical Studies, Tufts University School <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine, Graft<strong>on</strong>, Massachusetts,USA; † Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Physiology and Pharmacology, College <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine, TexasA&M University, College Stati<strong>on</strong>, Texas, USAReported rates <str<strong>on</strong>g>of</str<strong>on</strong>g> oocyte maturati<strong>on</strong> andfertilisati<strong>on</strong> vary greatly am<strong>on</strong>g laboratories. Thismay be related to <str<strong>on</strong>g>the</str<strong>on</strong>g> populati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytesselected for use, as <str<strong>on</strong>g>the</str<strong>on</strong>g> maturati<strong>on</strong> rate <str<strong>on</strong>g>of</str<strong>on</strong>g> horseoocytes is dependent <strong>on</strong> diameter and <str<strong>on</strong>g>the</str<strong>on</strong>g> degree <str<strong>on</strong>g>of</str<strong>on</strong>g>viability <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> follicle from which <str<strong>on</strong>g>the</str<strong>on</strong>g> oocyteoriginates (Hinrichs and Williams 1997; Hinrichsand Schmidt 2000). Higher maturati<strong>on</strong> rates arefound in oocytes from large follicles, and inoocytes from atretic follicles. Cumulus expansi<strong>on</strong>is a marker for oocyte atresia, <str<strong>on</strong>g>the</str<strong>on</strong>g>refore assessment<str<strong>on</strong>g>of</str<strong>on</strong>g> cumulus morphology is an important parameterin selecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes for study. Germinal vesiclechromatin c<strong>on</strong>figurati<strong>on</strong> also differs with follicleatresia and follicle size, and is str<strong>on</strong>gly associatedwith <str<strong>on</strong>g>the</str<strong>on</strong>g> meiotic competence <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> oocyte(Hinrichs and Williams 1997; Hinrichs andSchmidt 2000).Methods for collecti<strong>on</strong> and selecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> horseoocytes have not been standardised am<strong>on</strong>glaboratories. The 2 most comm<strong>on</strong> methods forhorse oocyte collecti<strong>on</strong> are aspirati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> follicularfluid or opening follicles and scraping <str<strong>on</strong>g>the</str<strong>on</strong>g>granulosa layer. Oocytes in viable follicles may bemore closely attached to <str<strong>on</strong>g>the</str<strong>on</strong>g> follicle wall, whereasoocytes from atretic follicles may be loose within<str<strong>on</strong>g>the</str<strong>on</strong>g> follicular fluid (Hawley et al. 1995;Mlodawska and Okolski 1997). Thus, it is possiblethat <str<strong>on</strong>g>the</str<strong>on</strong>g>se 2 methods <str<strong>on</strong>g>of</str<strong>on</strong>g> oocyte recovery result incollecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> different populati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes, withaspirati<strong>on</strong> favouring collecti<strong>on</strong> oocytes fromatretic follicles and scraping favouring collecti<strong>on</strong><str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes from viable follicles. The type <str<strong>on</strong>g>of</str<strong>on</strong>g>oocytes obtained may affect <str<strong>on</strong>g>the</str<strong>on</strong>g> observed resultsafter maturati<strong>on</strong> or fertilisati<strong>on</strong>, and may be afactor in <str<strong>on</strong>g>the</str<strong>on</strong>g> reported differences in results using<str<strong>on</strong>g>the</str<strong>on</strong>g>se procedures am<strong>on</strong>g laboratories. This studywas c<strong>on</strong>ducted to determine if <str<strong>on</strong>g>the</str<strong>on</strong>g> meiotic anddevelopmental competence <str<strong>on</strong>g>of</str<strong>on</strong>g> horse oocytes couldbe dependent <strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g> method <str<strong>on</strong>g>of</str<strong>on</strong>g> oocyte collecti<strong>on</strong>.Horse oocytes were recovered fromslaughterhouse ovaries by aspirati<strong>on</strong> or scraping.Only follicles visible <strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g> surface <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> ovaryand >10 mm diameter were used. Aspirati<strong>on</strong> wasperformed using an 18 ga needle attached to avacuum pump. For scraping, follicles were openedwith a scalpel blade and <str<strong>on</strong>g>the</str<strong>on</strong>g> granulosa cell layerwas removed using a b<strong>on</strong>e curette. Recoveredoocytes were classified as having compact,expanded or partial cumuli; obviouslydegenerating oocytes were discarded. InExperiment 1, oocytes were denuded and fixedimmediately after collecti<strong>on</strong>, and were stainedwith Hoechst 33258 to assess whe<str<strong>on</strong>g>the</str<strong>on</strong>g>r <str<strong>on</strong>g>the</str<strong>on</strong>g>collecti<strong>on</strong> method influenced <str<strong>on</strong>g>the</str<strong>on</strong>g> initial chromatinc<strong>on</strong>figurati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes. This was d<strong>on</strong>e both inMay and in October. In Experiment 2, in vitromaturati<strong>on</strong> rates <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes recovered byaspirati<strong>on</strong> or scraping were compared. IVM wasperformed as previously described (Dell’Aquila etal. 1996), with <str<strong>on</strong>g>the</str<strong>on</strong>g> 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 <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> mare serum, which increases <str<strong>on</strong>g>the</str<strong>on</strong>g>rate <str<strong>on</strong>g>of</str<strong>on</strong>g> male pr<strong>on</strong>ucleus formati<strong>on</strong> in oocyteshaving compact cumuli (Dell’Aquila et al. 1997).37
Equine Embryo TransferOocytes in metaphase II were submitted tointracytoplasmic sperm injecti<strong>on</strong> (ICSI), aspreviously described (Dell’Aquila et al. 1997).Frozen-thawed sperm, prepared by swim-up, wereused for injecti<strong>on</strong>. Oocytes were not chemicallyactivated after ICSI.The oocyte recovery rate was significantlyhigher for scraping than for aspirati<strong>on</strong> (83% vs48%). Oocytes collected by scraping had asignificantly higher proporti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> intact compactcumuli (72/107, 67% vs 69/209, 33%,respectively), and a significantly lower proporti<strong>on</strong><str<strong>on</strong>g>of</str<strong>on</strong>g> partial cumuli (16/107, 15% vs 111/209, 53%for scraping and aspirati<strong>on</strong>, respectively). Oocytescollected by scraping during <str<strong>on</strong>g>the</str<strong>on</strong>g> breeding seas<strong>on</strong>(May) had a higher proporti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> diffusechromatin within <str<strong>on</strong>g>the</str<strong>on</strong>g> germinal vesicle than didoocytes collected by aspirati<strong>on</strong> (10/43, 23% vs8/113, 7%, respectively), but this difference wasnot seen in oocytes collected in October.Experiment 2 (maturati<strong>on</strong>) was c<strong>on</strong>ducted in <str<strong>on</strong>g>the</str<strong>on</strong>g>fall and winter. The rates <str<strong>on</strong>g>of</str<strong>on</strong>g> maturati<strong>on</strong> tometaphase II were 56/101, 55.4 % and 65/106,61.4% for oocytes collected by scraping and thosecollected by aspirati<strong>on</strong> respectively. These rateswere not significantly different. The rates <str<strong>on</strong>g>of</str<strong>on</strong>g>pr<strong>on</strong>ucleus formati<strong>on</strong> after ICSI for oocytesrecovered by scraping or by aspirati<strong>on</strong> were 50/99,52.6% vs 50/85, 68.5% respectively; <str<strong>on</strong>g>the</str<strong>on</strong>g>se rateswere not significantly different.These findings dem<strong>on</strong>strate that follicleaspirati<strong>on</strong> results in loss <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> cumulus in <str<strong>on</strong>g>the</str<strong>on</strong>g>majority <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes, which agrees with previousreports (Hinrichs 1991; Alm et al. 1997). During<str<strong>on</strong>g>the</str<strong>on</strong>g> breeding seas<strong>on</strong>, aspirati<strong>on</strong> and scraping resultin collecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> populati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes havingdiffering germinal vesicle chromatinc<strong>on</strong>figurati<strong>on</strong>s, with scraping resulting in morediffuse chromatin, a c<strong>on</strong>figurati<strong>on</strong> which isassociated with follicle viability (Hinrichs andWilliams 1997). The observed difference inchromatin c<strong>on</strong>figurati<strong>on</strong> in scraped oocytesbetween seas<strong>on</strong>s is likely due to <str<strong>on</strong>g>the</str<strong>on</strong>g> increase inprevalence <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> diffuse chromatin c<strong>on</strong>figurati<strong>on</strong>during <str<strong>on</strong>g>the</str<strong>on</strong>g> breeding seas<strong>on</strong> (Hinrichs and Schmidt2000). These findings also indicate that whenfollicles >10 mm diameter are used, similarmaturati<strong>on</strong> and fertilisati<strong>on</strong> results may beobtained when oocytes are collected by aspirati<strong>on</strong>or by scraping. Selecti<strong>on</strong> against small follicles(
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