Effect of Energy Substrates on the Development of Porcine Embryos

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1 Chinese Journal of Zoology ~ * Sus scrofa NCSU mmol / L 0. 2 mmol / L 5. 7 mmol / L mncsu-23 mncsu-23 NCSU ~ 8 6 mncsu / NCSU 5 ~ 8 P < mncsu P < Q492 A 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 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 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 < 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 < 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. 2008ZX * liudi1963@ 163. com fubohao810@ 163. com

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

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

4 mncsu-23 / NCSU-23 mncsu-23 / mncsu h 5 ~ h 11 mncsu mncsu 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 ± 2. 3 b ± 3. 4 b ± 1. 5 a mncsu-23 / mncsu ± 1. 1 a ± 2. 6 c ± 3. 8 a mncsu-23 / NCSU ± 2. 5 a ± 3. 7 a ± 2. 6 a P < P > Different letters in the same row mean significant difference between the treatments P < and the same letter in the same row means the absence of significant difference between treatments P > 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 ± 3. 6 b ± 1. 4 b ± 1. 5 a mncsu-23 / NCSU ± 2. 5 a ± 1. 1 a ± 2. 4 a mncsu- 23 / NCSU-23 5 ~ 8 7 NCSU-23 / NCSU-23 mncsu-23 / NCSU- 23 NCSU-23 / NCSU Rieger 2 mncsu 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 2 Hoechst Enucleated oocytes stained with Hoechst A B C + = 100 μm A Phase contrast

5 168 Chinese Journal of Zoology mmol / L 5. 7 mmol / L 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

6 4 169 mncsu-23 5 ~ 8 NCSU- 23 mncsu-23 NCSU h Rieger D Park YHong JYong Het al. 20 embryos. Zygote Reprod Bureau MBailey J LSirard M A. Influence of oviductal cells and conditioned medium on porcine gametes. Zygote Macháty ZDay B NPrather R S. Development of early porcine embryos in vitro and in vivo. Biol Reprod Sturmey R GLeese H J. Energy metabolism in pig oocytes and early embryos. Reproduction Nichol RHunter R HGardner D Ket al. Concentrations of energy substrates in oviductal fluid and blood plasma of pigs during the peri-ovulatory period. J Reprod Fertil Kim H SLee G SHyun S Het al. Improved in vitro development of porcine embryos with different energy substrates and serum. Theriogenology Thompson J GSimpson A CPugh P Aet al. Requirement for glucose during in vitro culture of sheep preimplantation embryos. Mol Reprod Dev Houghton F DThompson J GKennedy C Jet al. Oxygen consumption and energy metabolism of the early mouse embryo. Mol Reprod Dev Relationship between energy metabolism and development of early mammalian embryos. Theriogenology Effect of exogenous carbohydrates in a serum-free culture medium on the development of in vitro matured and fertilized porcine 11Wales R GWhittingham D G. Metabolism of specifically labelled pyruvate by mouse embryos during culture from the two-cell stage to the blastocyst. Aust J Biol Sci Lane MGardner D K. Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo. Biol 13Kikuchi KOnishi AKashiwazaki Net al. Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol Reprod Yoshioka KSuzuki CTanaka Aet al. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod Scott LWhittingham D G. Influence of genetic background and media components on the development of mouse embryos in vitro. Mol Reprod Dev Kishi JNoda YNarimoto Ket al. Block to development in cultured rat 1cell embryos is overcome using medium HECM-1. Hum Reprod Miyoshi KFunahashi HOkuda Ket al. Development

7 170 Chinese Journal of Zoology 45 of rat one-cell embryos in a chemically defined medium effects of glucosephosphate and osmolarity. J Reprod Fertil Kim J HNiwa KLim J Met al. Effects of phosphate energy substratesand amino acids on development of in vitro-maturedin vitro-fertilized bovine oocytes in a chemically definedprotein-free culture medium. Biol Reprod Thompson J GSimpson A CPugh P Aet al. Requirement for glucose during in vitro culture of sheep preimplantation embryos. Mol Reprod Dev Wilding MFiorentino ADe Simone M Let al. Energy substratesmitochondrial membrane potential and human preimplantation embryo division. Reprod Biomed Online journal@ ioz. ac. cn Word ~ Wu P Zhou K Y. General condition of systemetics study on Tesudines. Chinese Journal of Zoology Jiang Z G. Conservation Biology. Hangzhou Zhejiang Science and Technology Press Yang T. On the leeches from Wuling Mountains area in south China Song D X. Invertebrates of Wuling Mountains Area Southwestern China. Beijing Science Press

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