IGF1、CoQ10、MT联合添加缓解热应激对牛IVF囊胚的影响

doi:10.11843/j.issn.0366-6964.2024.06.019

摘要/Abstract

摘要：

旨在探明胰岛素样生长因子1(insulin-like growth factors 1，IGF1)、辅酶Q10(coenzyme Q10，CoQ10)及褪黑素(melatonin，MT)联合添加对牛卵母细胞和胚胎发育以及热应激囊胚的影响。本研究于屠宰场采集牛离体卵巢，于实验室抽取卵丘卵母细胞复合体(cumulus-oocyte complexes，COCs)，在牛卵母细胞体外成熟(in vitro maturation，IVM)液及牛胚胎体外培养(in vitro culture，IVC)液中添加IGF1、CoQ10及MT，检测各添加方式对牛卵母细胞发育能力、牛卵母细胞活性氧(reactive oxygen species，ROS)水平及线粒体膜电位(mitochondrial membrane potential，ΔΨm)的影响；在囊胚期施加41 ℃热应激，检测热应激及联合添加对牛IVF囊胚发育能力及凋亡水平的影响，并利用qRT-PCR检测囊胚中胚胎质量相关基因mRNA表达水平。各组试验均重复3次。结果表明，与未添加组(CT-0组)相比，联合添加IGF1、CoQ10及MT组(ICM组)卵母细胞成熟率((90.40±2.06)% vs. (65.41±0.63)%)、卵裂率((93.33±1.96)% vs. (59.77±2.93)%)及囊胚率((51.43±5.34)% vs. (26.92±3.24)%)均显著提高(P＜0.05)；ICM组牛卵母细胞ROS水平显著降低；ΔΨm显著提高(P＜0.05)。与热应激组(HS组)相比，联合添加IGF1、CoQ10及MT(ICM+HS组)显著提高了囊胚扩张率((62.00±2.97)% vs. (30.77±8.66)%，P＜0.05)，抑制了热应激囊胚细胞凋亡，并提高了IGFBP3、ATP1A1、DSC2及IFNT2的mRNA表达水平(P＜0.05)。综上表明，联合添加IGF1、CoQ10及MT有效提高牛卵母细胞发育能力，降低ROS水平，提高ΔΨm。热应激降低牛囊胚扩张率，促进牛囊胚细胞凋亡，影响囊胚质量，而联合添加IGF1、CoQ10及MT缓解了热应激损伤。

关键词: IGF1, CoQ10, 褪黑素, 热应激, 奶牛, 胚胎

Abstract:

The aim of this study was to investigate the effects of combination of insulin-like growth factor 1 (IGF1), coenzyme Q10 (CoQ10), and melatonin (MT) on development of bovine oocytes, embryos and heat stressed blastocysts. In this study, bovine ovaries were collected from the abattoir and cumulus-oocyte complexes (COCs) were extracted in the laboratory. IGF1, CoQ10 and MT were added to bovine oocyte in vitro maturation (IVM) solution and bovine embryo in vitro culture (IVC) solution. The developmental ability, level of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) of bovine oocytes from each treatment were detected. By applying 41 ℃ heat stress at the blastocyst stage, the effects of heat stress and combined supplementation on the developmental ability and apoptosis level of bovine IVF blastocysts were determined, and the mRNA expression levels of embryo quality related genes were detected by qRT-PCR. Each experimental group was repeated 3 times. The results showed that the combination of IGF1, CoQ10 and MT (ICM group) significantly increased oocyte maturation rate ((90.40±2.06)% vs. (65.41±0.63)%), cleavage rate ((93.33±1.96)% vs. (59.77±2.93)%) and blastocyst rate ((51.43±5.34)% vs. (26.92±3.24)%)) (P < 0.05) compared with the non added group (CT-0 group). In the ICM group, the ROS level of bovine oocytes was significantly decreased and the ΔΨm of bovine oocytes was significantly increased (P < 0.05). Compared with the heat stress group (HS group), the combination of IGF1, CoQ10 and MT (ICM+HS group) significantly increased expanding rate ((62.00±2.97)% vs. (30.77±8.66)%, P < 0.05) of heat stressed blastocysts, inhibited apoptosis of heat stressed blastocyst cells, and increased mRNA expression levels of IGFBP3, ATP1A1, DSC2, and IFNT2 (P < 0.05) of heat stressed blastocysts. In summary, the combination of IGF1, CoQ10 and MT can effectively enhance the developmental ability, reduce the ROS level and increase the ΔΨm of bovine oocytes. Heat stress reduced the expanding rate of bovine blastocysts, promoted apoptosis of bovine blastocyst cells and damaged the quality of blastocysts, while the combination of IGF1, CoQ10 and MT alleviated the damage from heat stress.

Key words: IGF1, CoQ10, melatonin, heat stress, dairy cow, embryo

中图分类号:

S823.3

张航, 张培培, 杨柏高, 冯肖艺, 牛一凡, 余洲, 曹建华, 万鹏程, 赵学明. IGF1、CoQ10、MT联合添加缓解热应激对牛IVF囊胚的影响[J]. 畜牧兽医学报, 2024, 55(6): 2474-2485.

Hang ZHANG, Peipei ZHANG, Baigao YANG, Xiaoyi FENG, Yifan NIU, Zhou YU, Jianhua CAO, Pengcheng WAN, Xueming ZHAO. Combination of IGF1, CoQ10 and MT Alleviated the Effects of Heat Stress on Bovine IVF Blastocysts[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2474-2485.

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组别 Group	体外成熟处理 In vitro maturation treatment	体外培养处理 In vitro culture treatment	热应激处理 Heat stress treatment
CT-0	—	—	—
CT-I	IGF1	—	—
IC	IGF1+CoQ10	IGF1+CoQ10	—
IM	IGF1+MT	IGF1+MT	—
ICM	IGF1+CoQ10+MT	IGF1+CoQ10+MT	—
HS	IGF1	—	41 ℃ 12 h
ICM+HS	IGF1+CoQ10+MT	IGF1+CoQ10+MT	41 ℃ 12 h

基因 Gene	引物(5′→3′) Primer	长度/bp Size	序列号 GenBank Accession No.
IGFBP3	F: AAAATCGCCCTTGCTTGGTG	188	NM_174556.1
	R: CTCGGTCGTGGCTGTGTTT
ATP1A1	F: CCTAGCTCCTTCCTCTTCGCTC	181	NM_001076798.1
	R: CTTGTCCCCATGCTCTGAAACA
DSC2	F: AAGCATACGACCCGGAAACA	86	NM_001166526.1
	R: TTCATCCACATCGACCCACC
IFNT2	F: CTCTCCTCATCCCTGTCTGC	83	NM_001015511.4
	R: GATCCTTCTGGAGCTGGTTG
β-actin	F: ACTTGCGCAGAAAACGAGAT	121	NM_173979.3
	R: CACCTTCACCGTTCCAGTTT

组别 Group	成熟率/% Maturation rate	卵裂率/% Cleavage rate	囊胚率/% Blastocyst rate
CT-0	65.41±0.63(121/185)^d	59.77±2.93(52/87)^d	26.92±3.24(14/52)^c
CT-I	71.01±0.72(120/169)^c	70.33±3.19(64/91)^c	37.50±2.50(24/64)^b
IC	85.60±2.12(107/125)^b	87.50±5.39(63/72)^ab	47.62±2.81(30/63)^a
IM	84.38±2.65(108/128)^b	86.49±3.25(64/74)^b	45.31±4.22(29/64)^a
ICM	90.40±2.06(113/125)^a	93.33±1.96(70/75)^a	51.43±5.34(36/70)^a

组别 Group	卵裂率/% Cleavage rate	囊胚率/% Blastocyst rate	囊胚扩张率/% Expanding rate
CT-I	75.37±6.29(101/134)^b	31.69±0.48(32/101)^b	82.76±3.91(24/29)^a
HS	75.63±2.95(90/119)^b	32.22±1.92(29/90)^b	30.77±8.66(4/13)^c
ICM+HS	93.45±3.50(157/168)^a	49.68±2.04(78/157)^a	62.00±2.97(31/50)^b

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