260 ULTRARAPID NON-ELECTOPHORETIC PCR-SEXING OF BOVINE EMBRYOS

Abstract

In recent years, many improvements have been made in bovine embryo sexing. The challenge has been to create a fast, yet simple and user-friendly assay that can be performed with inexpensive equipment. The latter characteristics are featured in the widely used nonelectrophoretic Ampli-Y^™ PCR assay (Finnzymes Oy) in which tubes with male embryo biopsies result in pink fluorescence under UV light (Bredbacka et al. 1995 Theriogenology 44, 167–176). However, until now the post-biopsy assay time has remained at about 1.5 h, which is impractical under field conditions. The recent developments of polymerases, thermal cyclers, and reaction tubes have presented an opportunity to improve the speed of sex analysis. This study aimed at optimizing a faster protocol for nonelectrophoretic sexing by taking advantage of the new developments. The Ampli-Y kit was the starting point for the new assay. The polymerase was replaced by the extremely fast Phusion^™ DNA Polymerase (Finnzymes), in which a unique dsDNA-binding domain is fused to a Pyrococcus-like proofreading polymerase. The oligonucleotide primers were redesigned to allow higher annealing temperature and, hence, faster thermal cycling. The reaction tubes were replaced with ultra-thin walled PCR tubes (UTW^™, Finnzymes) allowing quicker sample thermal response. For thermal cycling, the MJ Research Minicycler was replaced with the 24-well Piko^™ Thermal Cycler (Finnzymes) enabling faster ramp rates. Different PCR protocols were tested by adding 100 pg of male or female DNA into the reaction vessels. The running time of the resulting optimized PCR protocol (initial denaturation 30 s at 98°C, followed by 38 cycles of 0 s at 95°C and 30 s at 72°C) was 35 min. The optimized PCR protocol was tested with Day 7 bovine morulae and blastocysts produced by standard IVP technology. Each of the 24 embryos was split into four biopsies. Of each embryo, one biopsy was subjected to electrophoretic based sexing (Bredbacka and Peippo 1992 Agric. Sci. Finl. 2, 233–238), one was subjected to sexing by the standard Ampli-Y^™ assay, and the remaining two biopsies were each subjected to the optimized protocol. Some single blastomeres that detached from the embryo during biopsying were also assayed using the new protocol. Biopsies and blastomeres were lysed in PCR buffer containing polymerase K for 4 min at 55°C followed by 2 min at 98°C. The embryos were diagnosed as 15 males and 9 females with all assays giving the same result for each embryo. Male samples tested with the optimized protocol had all clear signals easily distinguishable from the female samples. The male single blastomeres showed fluorescence levels equal to that of the male biopsies indicating high sensitivity of the new assay. In conclusion, the optimized sexing protocol offers a significant time saving. Following biopsy and a 6 to 7 min lysis step, the results are obtained in 35 (±1) min, about 1 h shorter than with the original Ampli-Y^™ protocol.