146 BOVINE EMBRYO AGGREGATION TO MULTIPLY ELITE GENOTYPES

Abstract

Each in vitro produced bovine embryo has only a limited chance of developing into a live calf. As genetically superior embryos can now be identified (Humblot et al. 2010 Vet. Med. Int.), maximizing these odds is desirable. We aim to multiply the elite genetics of selected male embryos by aggregating them with female embryos and generating germ line chimaeras. We postulate that this approach will result in functionally male cattle that produce sperm derived from the embryo of high genetic value. In this study, we evaluated the number and developmental stage of male cleavage-stage blastomeres required for efficient sex conversion and chimaerism. On Day 4, at the 12- to 16-cell stage, each presumptive male embryo donor (generated from Y-chromosome-sorted sperm) was dissociated into 1/4 (3–4 cells), 1/2 (6–8 cells) or 3/4 (9–12 cells) portions. One-quarter of each donor embryo was kept for future genotyping. Each donor portion was aggregated with a whole Day 4 presumptive female embryo generated from X-chromosome-sorted sperm from a different sire. On Day 7, blastocysts were assessed for blastomere incorporation and suitable quality embryos transferred individually to recipients. Development of individual aggregates to suitable blastocysts was not significantly different between 1/4-, 1/2-, and 3/4-aggregates or control whole male embryos (11/49 = 22%, 20/49 = 41% 15/50 = 30%, and 11/37 = 30%, respectively). However, aggregating three 1/4 embryos resulted in a 2.3-fold increase in the number of blastocysts produced per donor embryo compared to controls (11/16 = 69% v. 11/37 = 30%). Following embryo transfer of different aggregate groups (1/4, 1/2, and 3/4) and controls, there was neither a significant difference for pregnancy establishment on Day 35 (5/11 = 45%, 9/15 = 60%, 8/13 = 62%, and 5/10 = 50%, respectively), nor on Day 150 of gestation (2/11 = 18%, 3/15 = 20%, 4/13 = 31%, and 4/10 = 40%, respectively). Most of the aggregate fetuses recovered around Day 150 were phenotypically male (1/4 = 2 of 2, 1/2 = 1 of 3, and 3/4 = 4 of 4 fetuses; 7/9 = 78% overall). Expression of the female-specific mRNA for XIST (X-inactive specific transcript) was detected in liver and/or heart from aggregate male fetuses (3 from 3/4 aggregates and 1 from a 1/4 aggregate; 4/7 = 57% overall), indicating somatic cell sex chimaerism. Using sex-specific sequences of the amelogenin gene (AMELX and AMELY), no Y-chromosomal contribution was detected in liver or heart of the two phenotypically female aggregate fetuses. A recent asynchronous aggregation experiment, whereby three 1/4 male Day 5 embryos were each aggregated with a single Day 4 female embryo, resulted in a 4.6-fold increase in the number of blastocysts produced per donor embryo compared to controls (39/17 = 229% v. 22/44 = 50%). In summary, embryo aggregation more than doubled the number of blastocysts that potentially contained the desired genetics of a donor embryo. The majority of the resulting fetuses were male and developed to Day 150 of gestation at rates comparable to controls. Future studies will determine the minimum number of male blastomeres required to ensure sex conversion and colonisation of the germ line to reliably generate bull calves.

Supported by MSI C10X1002.