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The proper culturing of Zona-free embryos increases their developmental viability
Niu Yingjie 1 #,Wang Chengfei 1,Chi Daming 1,Zeng Yaqiong 1,Li Jing 2,Li Song 2,Liu Honglin 1,Dai Yunping 2,Li Juan 1 *
1.College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095
2.State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094
*Correspondence author
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Funding: This research was supported by the grants from the Ph.D. Programs Foundation of Ministry of Education of China （No.B0201300687), Natural Science Foundation of Jiangsu Province）
Opened online:20 November 2014
Accepted by: none
Citation: Niu Yingjie,Wang Chengfei,Chi Daming.The proper culturing of Zona-free embryos increases their developmental viability[OL]. [20 November 2014] http://en.paper.edu.cn/en_releasepaper/content/4618411
 
 
As the important structure of oocyte and early embryo, zona pellucid causes adverse effect to the development of the embryo because of thickness and hardening during development. The present study examined the effect of zona pellucid for porcine embryo development and the influence of electric stimulation and culture systems (WOWs type) for ZP-free embryo development. ZP-free oocytes were parthenogenetically activated (PA) with a single direct current (DC) pulse of 0.86 kV/cm for 80 μs, while ZP-intact oocytes activated with a single DC of 1.26 kV/cm or 0.86 kV/cm for 80 μs, followed by 4 h treatment with porcine zygote medium 3 (PZM-3) added 5 μg/ml cytochalasin B and 10 μg/ml cycloheximide. The results showed that the developmental competence of ZP-free and ZP-intact PA embryos was corresponding when proper electrical strengths with a DC for 80 μs of 0.86 kV/cm and 1.26 kV/cm were executed respectively. To investigate the effect of WOWs' shape and size on the developmental viability of ZP-free PA embryos, the matured ZP-free PA oocytes were randomly grouped and cultured into three type WOWs: S-W: the small bowl-shaped WOW; L-W: the large bowl-shaped WOW; VF-W: the V-shaped but flat bottom WOW. The rate of 2-cell was no different in the three types WOWs (p > 0.05), but more embryos cultured in S-W (76.4±4.4%) will developed into 4-cell stage compared to embryos cultured either in L-W or in VF-W (61.6±2.1% or 63.0±4.7%). The blastocyst rate showed a significantly decreasing tendency from 50.2±6.4% (S-W) to 31.2±3.4 %(L-W) and 13.0±5.1 %(VF-W). To assess the effect of ZP on the hatching ability and the survival viability, blastocysts from ZP-free and ZP-intact pig PA embryos were collected on Day 7 and were transferred into a new Nunc 4-well dish in PZM-3. The survival viability of ZP-free blastocyst cultured to day 8, 9 and 10 was comparable to ZP-intact blastocyst. But the ZP-intact blastocyst hatched rate was very low with only 17.9±5.7%. Accordingly, the presence of zona pellucida is not essential under certain in-vitro culture situations using the WOW culture system and may harmer the hatching viability of ZP-intact porcine blastocysts cultured in vitro.
Keywords:ZP-free; WOWs; developmental competence; hatching viability
 
 
 

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