307 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING FLOW-CYTOMETRICALLY SORTED SPERMATOZOA

Abstract

There are two practical ways to predetermine the sex of mammalian offspring: sexing pre-implantation embryos and sexing spermatozoa. The only successful and non-invasive method of sexing spermatozoa is quantifying sperm DNA with fluorescing DNA-binding dye, followed by flow cytometry and cell sorting. Our investigations aimed to develop a technology for in vitro embryo production in cattle using fresh and/or frozen-thawed spermatozoa sexed by flow cytometry. Sperm was sorted in a MoFloSX^® cytometer using the method of XY, Inc. (Fort Collins, CO, USA; Research Collaboration Agreement). After sorting, the sperm was either used for IVF or frozen and stored in liquid nitrogen. Immature oocytes, recovered from slaughterhouse ovaries, after 22 to 23 h of IVM in TCM-199 containing 20% estrous cow serum and additional granulosa cells (Katska et al. 1998, J. Anim. Feed Sci. 7, 353–362), were fertilized in vitro with fresh or frozen-thawed X and Y fractions of spermatozoa. Simultaneously control, unsorted, fresh and frozen-thawed sperm was used for IVF. The standard protocol of sperm capacitation (Katska and Rynska 1998 Theriogenology 50, 213–222) was applied for both control sperm and fresh fractions of sexed sperm. Briefly, sperm was separated by Percoll gradient centrifugation, washed, and introduced into drops of Tyrode's albumin-lactate-pyruvate (TALP)-IVF (containing 10 μg heparin mL⁻¹ and mixture of penicillamine, hypotaurine, and epinephrine) at a concentration 1 to 2 × 10⁶ spermatozoa mL⁻¹ of medium. Frozen fractions of sorted spermatozoa were centrifuged after thawing (500 g for 10 min) and immediately introduced into the IVF drops at 2 to 3 × 10⁶ spermatozoa mL⁻¹ of medium. Embryos resulting after IVF were co-cultured with Vero cells in B2 medium supplemented with 2.5% fetal calf serum for 8 to 10 days, (i.e., to the hatched blastocyst stage). A total of 2074 IVM oocytes were fertilized with both fresh and frozen-thawed sexed and control sperm of 5 bulls. There were significant differences (P < 0.01) in cleavage rates among fresh control sperm (120/256; 46.9%), the X fraction (66/254; 26.0%), and the Y fraction (58/230; 25.2%). Similar differences in cleavage rates (P < 0.01) were shown for frozen-thawed control sperm (156/335; 46.6%), the X fraction (137/498; 27.5%), and the Y fraction (118/501; 23.6%). No differences were observed in efficiency of embryo development to the blastocyst stage between the fresh control (25.8%) and the Y fraction (25.9%), or among the frozen control (16.7%) and the X fraction (13.1%) or the Y fraction (16.9%). However, significant differences (P < 0.05) were shown between blastocyst rates with the fresh X fraction (10.6%) and the control. Our results suggest that there were differences due to sperm sorting but no differences in efficiency of both fresh and frozen-thawed X and Y fractions of spermatozoa.

Research was supported by the State Committee for Scientific Research as a project 3PO6D 044 23.