N OCIETY' - the Society for Reproductive Biology

Effect of calcium on the in vitro fertilisation of in vitro maturedporcine oocytes by frozen epididymal spermatozoa.A. Preshaw, W.M.C. Maxwell and G. EvansDepartment of Animal Science, University of Sydney, NSW 2006, AustraliaCONFOCAL ANALYSIS OF DECONDENSATION AND PRONUCLEAR FORMATION IN HUMANSPERM INJECTED INTO MURINE OOCYTESSandra Galea, Orly Lacham Kaplan and Alan Trounson.Institute of Reproduction and Development, Monash University, 27-32 Wright street, Clayton Victoria Australia.IntroductionThe use of frozen epididymal sperm for in vitro fertilisation can result in low fertilisation rates despite highmotility of sperm (1). Intracellular calcium increases during the acrosome reaction of sperm but its role incapacitation is not fully understood (2). This study was undertaken to determine if doubling calciumconcentration and increasing sperm concentration could increase fertilisation rates of frozen-thawedepididymal sperm.Methods and MaterialsSperm were aspirated from slaughterhouse epididymides, by retrograde injection of air through the vasdeferens, and then frozen in a TFE diluent (3) at a concentration of 500 x 10 6 sperm/ml. Oocytes wereaspirated from 2-4 mm follicles on pre-pubertal slaughterhouse ovaries and matured for 44 hr. Oocytes werethen fertilised in 100 III droplets of TALP-PVA (4) + 4 roM caffeine containing 4.66 or 9.32 roM CaCh and62.5 x 10 3 , 125 X 10 3 , 250 X 10 3 or 500 x 10 3 sperm/ml. Oocyte maturation and fertilisation were carried outat 39°C in 5% CO 2 in air. Oocytes were removed 17 h after insemination, stained with Hoechst 33342 andassessed for maturation and fertilisation under UV light. Six replicates were run and the data were analysedby ANOVA and are presented in Tables 1 and 2.Table 1. Mean monospermic, polyspermic and total fertilisation rates of oocytes in media with differingCaCI 2 content ( a11 f our sperm concentratIOns . )Calcium No of Mean monospermic Mean polyspermic Mean totalconcentration (roM) oocytes fertilisation fertilisation fertilisation(% ±SE) (% + SE) (% + SE)4.66 650 20.8 + 2.2 a 6.4 + 2.0 a 27.3 + 2.8 a9.32 717 35.0 ± 2.2 b 15.2 + 2.0 b 50.2 + 2.8 bMeans WIth dIfferent superscnpts in the same columns are significantly different (P