Effect of Energy Substrates on the Development of Porcine Embryos

Chinese Journal of Zoology 2010 45 4 164 ~ 170 1 2 2 2 2 2 12 12* 1 150086 2 150030 Sus scrofa NCSU-23 5. 56 mmol / L 0. 2 mmol / L 5. 7 mmol / L mncsu-23 mncsu-23 NCSU-23 2 5 ~ 8 6 mncsu / NCSU 5 ~ 8 P < 0. 05 mncsu P < 0. 05 Q492 A 0250-3263 2010 04-164-07 Effect of Energy Substrates on the Development of Porcine Embryos FU Bo 1 FANG Qing-Chang 2 REN Liang 2 WANG Liang 2 SUN Hong-Tao 2 BIE Shu 2 MA Hong 1 2 LIU Di 1 2 * 1 Heilongjiang Academy of Agricultural SciencesHarbin 2 College of Life ScienceNortheast Agriculture UniversityHarbin 150086 150030 China Abstract This study aimed to investigate the effects of pyruvate and lactic acid on early development of porcine embryos. The NCSU-23 culture medium was modified by replacing 5. 56 mmol / L glucose with 0. 2 mmol / L pyruvate and 5. 7 mmol / L lactic acidand the modified medium was called mncsu-23. Parthenogenetic embryos and nuclear transfer embryos were transferred into either NCSU-23 or mncsu-23 medium. Embryo development to 5-8 cells was observed 2 days after egg activationand blastocyst rate and the number of nuclei in the blastocyst were determined on day 6. A higher proportion of the embryos reached 5-8 cells at 48 h and blastocysts on day 6 in mncsu / NCSU treatment first 48 h in mncsu-23 medium and thereafter in NCSU-23 medium group when compared with the control NCSU / NCSUfirst 48 h in NCSU-23 medium and thereafter also in NCSU-23 medium group P < 0. 05. The rate of blastocyst formation in mncsu-23 / mncsu-23 first 48 h in mncsu-23 medium and thereafter also in mncsu-23 medium group was the lowest P < 0. 05. Our results have demonstrated that replacing glucose with pyruvate and lactic acid during the first 48 h of IVC may be beneficial to the development of porcine embryos. Key words Pig Energy substrate Parthenogenetic embryos Nuclear transfer embryos No. 2008BADB2B02 No. 2008ZX08006-003 * E-mail liudi1963@ 163. com E-mail fubohao810@ 163. com 2009-12-22 2010-03-15

4 165 1. 1 18G in vitro culture IVC 20 ml 3 ~ 6 mm cumulus-oocyte complex TCM-199 Hank's 1 3 - cumulus-oocyte complexescocs TCM-199 Earle's + 10% pff + 0. 57 mmol / L L- L-cysteine + 10 ng / ml EGF + 10 IU / ml NCSU-23 PMSG + 10 IU / ml 2 hcg NCSU-23 5% CO 2 39 22 h PMSG hcg 22 h 1. 2 - COCs 3 NCSU- 23 Sigma 38. 5 4 min 4 10% FBS DMEM GIBCO 1 500 r / min 5 5 min 5 10 5 / ml 35 mm 38. 5 5% CO 2 80% 1 h 48 h NCSU-23 4 NCSU-23 1 Sigma Aldrich St. lousemo 1 1. 8 kv / cm 30 NCSU-23 μs 1 min 5. 56 mmol / L mncsu-23 NCSU-23 + 4 mg / ml BSA + 2 mmol / L 6-DMAP mncsu-23 + 4 mg / ml BSA + 2 mmol / L 6-DMAP 0. 2 mmol / L 5. 7 mmol / L 4 44 h COC 0. 1% 1. 3 44 h 39 3 1 min 39 5% CO 2 4 h

166 Chinese Journal of Zoology 45 NCSU-23 + 4 mg / ml BSA 48 h 5 ~ 8 144 h mncsu-23 + 4 mg / ml BSA 6 3. 7% 39 5% CO 2 DPBS 3 5 min 1. 4 4 10 μg / ml Hoechst33342 1 ~ 2 d DPBS 5 min 1 ml / 10 μg / ml CB / 4 5 μg / ml Hoechest33342 NCSU- 23 Hepes 10% ~ 6 20% 1. 7 1. 7. 1 NCSU- 23 mncsu-23 5% CO 2 - NCSU-23 / NCSU-23 NCSU-23 + 4 mg / ml BSA NCSU-23 48 h NCSU- mncsu-23 + 4 mg / ml BSA 39 5% CO 2 1. 5 h 1. 5 39 mncsu-23 144 h 3 1 min mncsu-23 / NCSU-23 mncsu-23 48 h NCSU-23-144 h 1. 7. 2 1. 8 kv / cm 30 μs 1 min NCSU-23 + 4 mg / ml BSA mncsu-23 + 4 mg / ml BSA 23 144 h mncsu-23 / mncsu-23 mncsu-23 48 h NCSU-23 mncsu-23 5% CO 2 5 4 h NCSU-23 NCSU-23 / NCSU-23 + 4 mg / ml BSA + 2 mmol / L 6-DMAP NCSU-23 48 h NCSU-23 mncsu-23 + 4 mg / ml BSA + 2 mmol / L 6-DMAP 144 h mncsu-23 / NCSU-23 39 5% CO 2 mncsu-23 48 h 4 h 6-DMAP 1 h NCSU-23 144 h 1. 8 Mean ± SE 1. 6 SPSS 17. 0 ANONA NCSU-23 + 4 mg / ml BSA mncsu-23 + 4 mg / ml BSA 3 SPSS 17. 0 Independent-Samples t-test NCSU-23 3 P < 0. 05 NCSU-23 mncsu-23

4 167 2 mncsu-23 / NCSU-23 mncsu-23 / mncsu-23 48 h 5 ~ 8 144 h 11 mncsu-23 2. 1 mncsu-23 1 2 mncsu-23 / NCSU-23 mncsu-23 / mncsu-23 5 ~ 8 NCSU-23 / NCSU-23 3 Table 1 1 Effect of energy substrate in culture system on the development of parthenogenetic embryos in vitro Type of culture No. of treated oocytes 5 ~ 8 No. of 5-8 cells % No. of blastocysts % No. of blastocyst cells NCSU-23 / NCSU-23 150 27 18. 0 ± 2. 3 b 60 40 ± 3. 4 b 36. 5 ± 1. 5 a mncsu-23 / mncsu-23 160 45 28. 1 ± 1. 1 a 33 20. 7 ± 2. 6 c 37. 5 ± 3. 8 a mncsu-23 / NCSU-23 155 45 29. 0 ± 2. 5 a 103 66. 6 ± 3. 7 a 38. 0 ± 2. 6 a P < 0. 05 P > 0. 05 Different letters in the same row mean significant difference between the treatments P < 0. 05 and the same letter in the same row means the absence of significant difference between treatments P > 0. 05. The same is applied below. Table 2 2 Effect of energy substrate in culture system on the development of somatic nuclear transferred embryos in vitro Type of culture 5 ~ 8 % No. of treated No. of 5-8 cell oocytes % No. of blastocysts No. of blastocyst cells NCSU-23 / NCSU-23 120 14 18. 3 ± 3. 6 b 7 10. 0 ± 1. 4 b 36. 0 ± 1. 5 a mncsu-23 / NCSU-23 122 24 35. 1 ± 2. 5 a 14 19. 6 ± 1. 1 a 39. 6 ± 2. 4 a 2. 2 2 2 2 mncsu- 23 / NCSU-23 5 ~ 8 7 NCSU-23 / NCSU-23 mncsu-23 / NCSU- 23 NCSU-23 / NCSU-23 3 8 Rieger 2 mncsu-23 48 h 9 3 mncsu-23 / NCSU-23

168 Chinese Journal of Zoology 45 10 4 2 0. 2mmol / L 5. 7 mmol / L 11-12 0. 2 mmol / L 1 Fig. 1 Porcine parthenogenetic embryos at blastocysts stage A NCSU-23 / NCSU-23 B mncsu-23 / mncsu-23 C mncsu-23 / NCSU-23. Bar = 100 μm Fig. 2 2 Hoechst Enucleated oocytes stained with Hoechst A B C + = 100 μm A Phase contrast images B Epifluorescent images C Phase contrast images + epifluorescent images. Bar = 100 μm 3 Fig. 3 Porcine nuclear transfer embryos at blastocysts stage A mncsu-23 / NCSU-23 B NCSU-23 / NCSU-23. Bar = 100 μm

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