胎牛血清在猪孤雌囊胚玻璃化冷冻后恢复培养中的作用研究

doi:10.16431/j.cnki.1671-7236.2023.02.018

摘要/Abstract

摘要： 【目的】研究胎牛血清(fetal bovine serum,FBS)在猪孤雌囊胚玻璃化冷冻后恢复培养中的作用。【方法】本试验以体外培养第5天的猪孤雌激活囊胚为材料,将新鲜和冷冻囊胚分别在含10% FBS(V/V)的胚胎培养液中继续培养48 h,即分为新鲜组(Fresh)、新鲜+FBS组(Fresh+FBS)、冷冻组(Vitrified)、冷冻+FBS组(Vitrified+FBS)。观察各组囊胚的扩张和孵化能力,检测胚胎的细胞膜损伤、凋亡细胞数目、总细胞数目、胞内活性氧(ROS)水平、线粒体活性以及发育相关基因的表达水平。【结果】与Fresh和Vitrified组相比,Fresh+FBS和Vitrified+FBS组的完全扩张率、孵化率和囊胚细胞总数均显著提高(P＜0.05),细胞膜损伤率和细胞凋亡率均显著降低(P＜0.05)。与Fresh组相比,Vitrified组ROS水平显著升高(P<0.05),Fresh+FBS和Vitrified+FBS组ROS水平均显著降低(P<0.05)。Vitrified+FBS组的线粒体活性显著高于Vitrified组(P<0.05)、显著低于Fresh+FBS组(P<0.05),与Fresh组无显著差异(P＞0.05)。相比于Fresh组,Vitrified组POU结构域与类转录因子1(POU5F1)表达水平显著上升(P＜0.05)、过氧化氢酶(CAT)表达水平显著下降(P＜0.05)。增殖细胞核抗原(PCNA)、DNA甲基转移酶3A(DNMT3A)、超氧化物歧化酶1(SOD1)、BCL2相关X蛋白(BAX)/BCL2L1的表达水平在Fresh和Vitrified组之间均无显著性差异(P＞0.05)。Fresh+FBS和Vitrified+FBS组中PCNA、SOD1和CAT的表达水平显著高于Fresh和Vitrified组(P<0.05)。【结论】体外培养第5天的新鲜和冷冻猪孤雌囊胚在10% FBS中继续培养,其发育能力及胚胎质量均得到明显改善。

关键词: 猪; 囊胚; 玻璃化冷冻; 胎牛血清; 胚胎质量

Abstract: 【Objective】 The aim of this study was to evaluate the effects of fetal bovine serum (FBS) supplementation during postwarming culture on vitrified porcine parthenogenetic blastocysts.【Method】 The porcine parthenogenetic blastocysts on the 5th day of in vitro culture were used in this experiment,and the fresh and vitrified blastocysts were cultured in the embryo culture medium containing 10% FBS (V/V) for 48 h.This experiment was divided into four groups:Fresh,Fresh+FBS,Vitrified and Vitrified+FBS groups.The blastocysts expansion and hatching rates,the percentages of membrane damage and apoptosis and total cell number were determined.Moreover,the intracellular reactive oxygen species (ROS) levels,mitochondrial activity and expression levels of genes related embryo development in the blastocysts were detected.【Result】 Compared with the Fresh and Vitrified groups, the complete expansion rate, hatching rate and total number of blastocyst cells in the Fresh+FBS and Vitrified+FBS groups were significantly increased (P<0.05), while the cell membrane damage rate and apoptotic cell rate were significantly decreased (P<0.05). Compared with the Fresh group, ROS levels were significantly increased in the Vitrified group, while ROS levels were significantly decreased in both the Fresh+FBS and Vitrified+FBS groups (P<0.05). The mitochondrial activity of Vitrified+FBS group was significantly higher than that of Vitrified+FBS group and significantly lower than that of Fresh+FBS group (P<0.05), but there was no significant difference between Vitrified+FBS group and Fresh group (P>0.05). Compared with the Fresh group, POU class 5 homeobox 1(POU5F1) gene expression level in Vitrified group was significantly increased (P<0.05) and catalase (CAT)gene expression level was significantly decreased (P<0.05). There were no significant differences in the expression levels of proliferating cell nuclear antigen (PCNA), DNA methyltransferase 3A (DNMT3A), superoxide dismutase 1 (SOD1) and BCL2-associated X protein (BAX):BCL2L1 between the Fresh and Vitrified groups (P>0.05). The expression levels of PCNA, SOD1 and CAT genes in Fresh+FBS and Vitrified+FBS groups were significantly higher than those in Fresh and Vitrified+FBS groups (P<0.05).【Conclusion】 This study found that fresh and vitrified parthenogenes blastocysts were cultured in 10% FBS on the 5th day of in vitro culture, and their development ability and quality of embryo were significant improved.

Key words: pig; blastocyst; vitrification; fetal bovine serum; embryo quality

中图分类号:

S828

相德才, 李水英, 张斌, 张彦, 梁家充, 吕春荣, 洪琼花, 权国波, 吴国权. 胎牛血清在猪孤雌囊胚玻璃化冷冻后恢复培养中的作用研究[J]. 中国畜牧兽医, 2023, 50(2): 606-615.

XIANG Decai, LI Shuiying, ZHANG Bin, ZHANG Yan, LIANG Jiachong, LYU Chunrong, HONG Qionghua, QUAN Guobo, WU Guoquan. The Study on the Role of Fetal Bovine Serum Supplementation During Postwarming Culture on Vitrified Porcine Parthenogenetic Blastocysts[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 606-615.

参考文献

[1] DU X,ZHUAN Q,CHENG K,et al.Cryopreservation of porcine embryos:Recent updates and progress[J]. Biopreservation and Biobanking,2021,19(3):210-218.
[2] BEEBE L F S,BOUWMAN E G,MCILFATRICK S M,et al.Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container[J]. Theriogenology,2011,75(8):1453-1458.
[3] MEN H,ZHAO C,SI W,et al.Birth of piglets from in vitro-produced,zona-intact porcine embryos vitrified in a closed system[J]. Theriogenology,2011,76(2):280-289.
[4] CUELLO C,MARTINEZ C A,NOHALEZ A,et al.Effective vitrification and warming of porcine embryos using a pH-stable,chemically defined medium[J]. Scientific Reports,2016,6(1):33915.
[5] JIN B,HIGASHIYAMA R,NAKATA Y,et al.Rapid movement of water and cryoprotectants in pig expanded blastocysts via channel processes:Its relevance to their higher tolerance to cryopreservation[J]. Biology of Reproduction,2013,89(4):87.
[6] 曹俊国,魏海军,许保增.哺乳动物生殖细胞冷冻保存对其DNA甲基化影响的研究进展[J].中国畜牧兽医,2017,44(3):819-824. CAO J G,WEI H J,XU B Z.Research progress on the relationship between cryopreservation of mammalian germ cells and their DNA methylation[J]. China Animal Husbandry ＆ Veterinary Medicine,2017,44(3):819-824.(in Chinese)
[7] ESTUDILLO E,JIMÉNEZ A,BUSTAMANTE-NIEVES P E,et al.Cryopreservation of gametes and embryos and their molecular changes[J]. International Journal of Molecular Sciences,2021,22(19):10864.
[8] HOCHI S,ABDALLA H,HARA H,et al.Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification[J]. Theriogenology,2010,73(8):1139-1145.
[9] SUCCU S,PASCIU V,MANCA M E,et al.Dose-dependent effect of melatonin on postwarming development of vitrified ovine embryos[J]. Theriogenology,2014,81(8):1058-1066.
[10] MUN S,SIM B,YOON S,et al.Dual effect of fetal bovine serum on early development depends on stage-specific reactive oxygen species demands in pigs[J]. PLoS One,2017,12(4):e175427.
[11] ASADA M,ISHIBASHI S,IKUMI S,et al.Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes[J]. Theriogenology,2002,58(6):1199-1208.
[12] CUELLO C,SANCHEZ-OSORIO J,ALMIÑANA C,et al.Superfine open pulled straws vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation[J]. Reproduction,Fertility and Development,2010,22(5):808.
[13] MISUMI K,HIRAYAMA Y,EGAWA S,et al.Successful production of piglets derived from expanded blastocysts vitrified using a micro volume air cooling method without direct exposure to liquid nitrogen[J]. Journal of Reproduction and Development,2013,59(6):520-524.
[14] BARTOLAC L K,LOWE J L,KOUSTAS G,et al.A comparison of different vitrification devices and the effect of blastocoele collapse on the cryosurvival of in vitro produced porcine embryos[J]. Journal of Reproduction and Development,2015,61(6):525-531.
[15] NATURIL-ALFONSO C,SAENZ-DE-JUANO M D,PEÑARANDA D S,et al.Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos[J]. Animal Reproduction Science,2011,127(3-4):222-228.
[16] LI R,LI J,LIU Y,et al.Optimal developmental stage for vitrification of parthenogenetically activated porcine embryos[J]. Cryobiology,2012,64(1):60-64.
[17] GUO S,LIU S,BOU G,et al.Fetal bovine serum promotes the development of in vitro porcine blastocysts by activating the Rho-associated kinase signalling pathway[J]. Reproduction,Fertility and Development,2019,31(2):366.
[18] 付博,任亮,赵金凤,等.表皮生长因子和胎牛血清对猪体细胞克隆胚胎发育的影响[J].黑龙江畜牧兽医,2010,5:7-9. FU B,REN L,ZHAO J F,et al.Effect of EGF and FBS on the development of porcine cloned embryos[J]. Heilongjiang Animal Science and Veterinary Medicine,2010,5:7-9.(in Chinese)
[19] INABA Y,MIYASHITA S,SOMFAI T,et al. Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts[J]. Cryobiology,2016,72(2):86-92.
[20] CASTILLO-MARTÍN M,YESTE M,PERICUESTA E,et al.Effects of vitrification on the expression of pluripotency,apoptotic and stress genes in in vitro-produced porcine blastocysts[J]. Reproduction,Fertility and Development,2015,27(7):1072.
[21] 庄利利,胡德宝,蔡丽娟,等.哺乳动物胚胎的活性氧来源及其抗氧化机制[J].中国畜牧兽医,2013,40(3):173-176. ZHUANG L L,HU D B,CAI L J,et al.ROS sources and anti-oxidative mechanisms in mammalian embryos[J]. China Animal Husbandry＆Veterinary Medicine,2013,40(3):173-176.(in Chinese)
[22] 贾振伟.线粒体代谢功能对早期胚胎表观遗传组和发育的影响[J].中国细胞生物学学报,2020,42(4):733-740. JIA Z W.The effect of mitochondrial function on epigenome and development in preimplantation embryos[J]. Chinese Journal of Cell Biology,2020,42(4):733-740.(in Chinese)
[23] MARTINO N A,DELL’AQUILA M E,CARDONE R A,et al.Vitrification preserves chromatin integrity,bioenergy potential and oxidative parameters in mouse embryos[J]. Reproductive Biology and Endocrinology,2013,11:27.
[24] CUELLO C,SANCHEZ-OSORIO J,ALMIÑANA C,et al.Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts[J]. Cryobiology,2008,56(3):189-194.
[25] KAZEMI P,DASHTIZAD M,SHAMSARA M,et al.Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts[J]. Molecular Reproduction and Development,2016,83(8):735-742.
[26] STINSHOFF H,WILKENING S,HANSTEDT A,et al.Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level[J]. Theriogenology,2011,76(8):1433-1441.
[27] 陈亚宁,吴彩凤,戴建军,等.玻璃化冷冻对猪孤雌激活囊胚中细胞凋亡的影响[J].畜牧兽医学报,2017,48(2):243-251. CHEN Y N,WU C F,DAI J J,et al.Effect of vitrification on cell apoptotic levels of porcine parthenogenetic blastocysts[J]. Acta Veterinaria et Zootechnica Sinica,2017,48(2):243-251.(in Chinese)
[28] MEHAISEN G M K,SAEED A M,GAD A,et al.Antioxidant capacity of melatonin on preimplantation development of fresh and vitrified rabbit embryos:Morphological and molecular aspects[J]. PLoS One,2015,10(10):e139814.
[29] VINING L M,ZAK L J,HARVEY S C,et al.The role of apoptosis in cryopreserved animal oocytes and embryos[J]. Theriogenology,2021,173:93-101.