237 SHORT-TERM TREATMENT OF 6-DMAP AND DEMECOLCINE IMPROVE DEVELOPMENTAL COMPETENCE OF PORCINE PARTHENOGENETIC EMBRYOS

Abstract

Artificial activation is one of the essential factors for successful development of embryos derived from parthenogenetic activation or somatic cell nuclear transfer (SCNT). This study was carried out to confirm whether the short-term treatment of 6-dimethylaminopurine (6-DMAP) or demecolcine would improve developmental competence of porcine parthenogenetic activated embryos and find out the optimal activation protocol. Ovaries were collected from gilts at a local slaughterhouse and cumulus–oocyte complexes were cultured for 44 h in the tissue culture medium-199 with 10% of porcine follicular fluid. Matured oocytes were chemically activated with 10 min treatment of 0.2 mm thimerosal (Thi) followed by 30 min treatment of 8 mm dithiothrethiol (DTT). Additional treatments of 6-DMAP (2 mm) and/or demecolcine (0.4 μg mL^–1) for a short term (40 min) or a long term (3 h) were conducted. Activated embryos were cultured in PZM-3. Cleavage rate, blastocyst (BL) formation rate and total cell number of BL were assessed at Day 2, 7 of culture, respectively. All data were subjected to one-way ANOVA followed by Tukey’s test using Prism version 4.0 (Graphpad Software, San Diego, CA, USA) to determine differences among experimental groups. To begin with, we evaluated that effect of short-term treatment of 6-DMAP or demecolcine. Short-term treatment of 6-DMAP improved developmental competence of activated embryos compared to long-term treatment (47.0 ± 1.0% v. 50.5 ± 1.5% for cleavage rate, 5 ± 3% v. 3 ± 1% for BL formation rate, 70 ± 13 v. 37 ± 6 for total cell numbers). Moreover, short-tem treatment of demecolcine also inhibited second polar body extrusion effectively compared with nontreated control (14 ± 2.1 v. 8.3 ± 4.1%). We confirmed that short-term treatment of 6-DMAP or demecolcine is valid. To optimize activation procedure with short-term treatment, development of embryos derived from each treatment were compared among nontreated control (C), 6-DMAP only treatment (6O), demecolcine only (DO) treatment, and 6-DMAP + demecolcine treatment (6 + D) groups. Cleavage rates of 4 groups were similar (53.5 ± 3.0, 62.1 ± 2.9, 53.6 ± 3.0 and 57.0 ± 3.0%, respectively). However, 6 + D group shows significantly greater BL formation rate (18.0 ± 2.5%) compared with C, 6O, or DO groups (9.4 ± 1.9, 14.0 ± 2.3 and 4.3 ± 1.3%, respectively). Total cell number of BL also increased in 6 + D group (52.8 ± 13.0) compared with C, 6O, or DO groups (47 ± 21.5, 31.5 ± 4.22 and 45.2 ± 13.7, respectively). In conclusion, we elucidated that short-term treatment of 6-DMAP or demecolcine improves developmental competence of embryos compared with long-term treatment. We recommend a combination of Thi/DTT with short-term treatment of both 6-DMAP and demecolcine for optimal activation procedure of porcine embryos.

This study was supported by BK21, KOSEF (#M10625030005-07N250300510), HANWHA L&C Corp, and Gyeonggido Veterinary Service.