First and second cycle nuclear transfer in cattle: Comparison of efficiency in embryo development and ongoing pregnancies

204

Theriogenology

FIRST AND SECOND CYCLE NUCLEAR TRANSFER IN CATI’LEz COMPARISON OF EFFICIENCY IN EMBRYO DEVELOPMENT AND ONGOING PREGNANCIES F.J. ~ctorsl, A. Delvall, L.C. Smith2, K. Touatil, J-F. Beckersl and F. Ectorsl 1IRSIA Research Unit, Sart-Tilman, Belgium 2CRRA Saint-Hyacinthe, Qu&ec Embryo reconstruction by transplantation of 1 blastomem from an undifferentiated embryo into a maturated and enucleated oocyte has been proposed as a means to obtain genetically identical animals. Due to various methodological (enucleation, electrofusion) or regulatory parameters (oocyte maturation, embryo development), the overall efficiency of the method is low. As mcloning constitutes a way to increase the number of embryos available for transfer, the aim of this work was to compare the rates of in vitro development and ongoing pregnancies after cloning and recloning. The enucleation was performed after 24-26h IVM by aspirating a small amount of cytoplasm adjacent to the first polar body. To confirm enucleation, the aspirated cytoplasm was checked by epifluomscence for the presence of the meiotic spindle. Enucleated oocytes were then returned to the maturation medium until 40h post-onset of IVM. Parent donor embryos (32cell) were used on D5 after IVF (DO = IV-F) while reconstructed embryos for recloning were used on D6 (DO = day of enucleation). Fusion was induced at 44 to 46h post-onset of IVM by a single electrical pulse of 2.7kWcm for 50 flsec. Couplets were then cocultured with bovine epithelial oviductal cells in Menezo B2 medium. Two reconstructed embryos at the blastocyst stage (D7) were transferred non-surgically to recipient heifers 7d a&r estrus. Pregnancies were diagnosed on D35 by pregnancy associated glycoprotein (PAG) determinations and were confirmed after 90d of gestation by rectal palpation. Table 1.

Percentages of embryos developing after first or second cycle nuclear transfers:

lst cycle cleaved (D3) 8-tell (D3) morula (D6) 2ti cycle

cleaved (D3) 8-&l (D3) blastocysts (D7)

Mean fSEM 76f 17 22f 10 15 f 12

Range 41 to 92 8to36 Oto36

Number ((l;$;;)

Replicates 11

(33/228)

ff

79f7 30f9 15 f 12

66to89 18to43 Oto41

(237/303) t 8

I:;;::{

Table 2.

Percentages of pregnancies following transfer of 2 blastocysts from tirst and second cycle nuclear transfers: 1st cycle D35 D90 Term Newbornb 50.0 50.0 35.7 21.4 % 7114 No 7114 5/14 6r2.8

62.5 40.0 % 62.5 No 5/8 215a 518 a: 3 recipients remain pregnant b: No calves I No of transferred embryos. Zrd cycle

20.0 2lloa

In contrast to the findings of Stice and Keefer (Biol. Reprod., 48, 715-719, 1993), similar percentages of development in vitro and of pregnancy were obtained after cloning and recloning. While the number of newborn obtained remains low in both groups, these results indicate. that the development potential of blastomem nuclei from first cycle nuclear transfer embryos is not significantly affected by a recloning protocol.