Acta Veterinaria et Zootechnica Sinica ›› 2025, Vol. 56 ›› Issue (9): 4432-4451.doi: 10.11843/j.issn.0366-6964.2025.09.025
• Animal Biotechnology and Reproduction • Previous Articles Next Articles
WANG Rui(
), HENG Nuo, HU Yingfan, WANG Huan, ZHU Ni, HE Wei, XUAN Xiuli, HU Zhihui, XIONG Keng, GONG Jianfei, HAO Haisheng, ZHU Huabin, ZHAO Shanjiang*()
Received:2025-03-11
Online:2025-09-23
Published:2025-09-30
Contact:
ZHAO Shanjiang
E-mail:ruiwang202010@163.com;zhaoshanjiang@caas.cn
CLC Number:
WANG Rui, HENG Nuo, HU Yingfan, WANG Huan, ZHU Ni, HE Wei, XUAN Xiuli, HU Zhihui, XIONG Keng, GONG Jianfei, HAO Haisheng, ZHU Huabin, ZHAO Shanjiang. Mechanisms of Developmental Competence Differences of Oocytes from Different Grades of Bovine Cumulus-Oocyte Complexes after in Vitro Maturation[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4432-4451.
Table 1
Primers for the ddPCR and qRT-PCR"
| 引物名称 Primer name | 引物序列(5′→3′) Sequence | 产物长度/bp PCR size | 退火温度/℃ Annealing temperature |
| GAPDH-Forward | CACCCTCAAGATTGTCAGCA | 103 | 58 |
| GAPDH-Reverse | GGTCATAAGTCCCTCCACGA | ||
| PKM-Forward | CCCACAGTCGGGATAACCTTG | 127 | 58 |
| PKM-Reverse | TCCAGAGGAACTCCGACG | ||
| IDH3α-Forward | TGCAATCTTCGAGTCGGTTCA | 130 | 60 |
| IDH3α-Reverse | CTTCGCAGCGTGGTCAAAAA | ||
| β-actin-Forward | GCCCTGAGGCTCTCTTCCA | 101 | 60 |
| β-actin-Reverse | GCGGATGTCGACGTCACA | ||
| NDUFA4L2-Forward | CGATGATCGGCTTCATTGGC | 103 | 59 |
| NDUFA4L2-Reverse | GGGTTGTTCTTTCGGTCCCA |
Table 2
Comparison of in vitro developmental ability of oocytes from first-, second- and third-, fourth-grade COCs"
| 组别 Group | 体外培养总数/枚 Total number of embryos in vitro | 卵裂数/枚(%±SEM) Cleavage number | 囊胚数/枚(%±SEM) Number of blastocysts |
| 一二级COCs First- and second-grade COCs | 365 | 285(78.09±3.01)a | 93(32.56±4.33)a |
| 三四级COCs Third- and fourth-grade COCs | 317 | 192(60.59±6.54)b | 43(22.11±2.79)b |
Table 3
Smart-seq data quality analysis"
| 样本 Sample | Raw reads数量 Raw reads number | Clean reads数量 Clean reads number | Effective reads比率/% Effective reads rate | Mapped reads比率/% Mapped reads rate | Q30值/% Q30 value |
| GV1-1 | 45 674 378 | 43 982 102 | 96.29 | 94.42 | 95.75 |
| GV1-2 | 51 129 184 | 49 398 606 | 96.62 | 94.28 | 95.90 |
| GV1-3 | 54 554 672 | 52 536 610 | 96.30 | 93.81 | 95.99 |
| GV3-1 | 67 726 306 | 65 549 142 | 96.79 | 94.24 | 95.85 |
| GV3-2 | 48 507 034 | 46 525 878 | 95.92 | 93.56 | 95.79 |
| GV3-3 | 46 170 534 | 44 167 230 | 95.66 | 93.49 | 95.50 |
| MII1-1 | 54 309 472 | 53 026 116 | 97.64 | 95.64 | 96.20 |
| MII1-2 | 52 572 712 | 50 965 326 | 96.94 | 94.99 | 95.96 |
| MII1-3 | 58 974 948 | 57 600 648 | 97.67 | 95.27 | 96.35 |
| MII3-1 | 61 658 056 | 59 849 842 | 97.07 | 95.19 | 96.09 |
| MII3-2 | 65 222 820 | 62 237 638 | 95.42 | 93.64 | 95.80 |
| MII3-3 | 69 933 254 | 67 284 420 | 96.21 | 93.76 | 95.96 |
Fig. 1
PCA analysis of bovine oocyte transcripts A.PCA analysis of transcripts of first-, second-grade oocytes and third-, fourth-grade oocytes before maturation; B. PCA analysis of transcripts of first-, second-grade oocytes and third-, fourth-grade oocytes after maturation(22-24 h in vitro)"
Fig. 2
Comparison of significant DEGs between groups before and after maturation(22-24 h in vitro) A.Volcano plot comparing significant DEGs between groups before maturation; B. Volcano plot comparing significant DEGs between groups after maturation(22-24 h in vitro); C. Veen plot comparing significant DEGs between groups before maturation (left image) and after maturation (right image)"
Fig. 3
Significant DEGs GO enrichment plot A. GO enrichment plot of significant DEGs in first-, second-grade oocytes and third-, fourth-grade oocytes before maturation; B. GO enrichment plot of significant DEGs in first-, second-grade oocytes and third-, fourth-grade oocytes after maturation (22-24 h in vitro)"
Fig. 4
Significant DEGs KEGG enrichment plot A.KEGG enrichment plot of significant DEGs in first-, second-grade oocytes and third-, fourth-grade oocytes before maturation; B. KEGG enrichment plot of significant DEGs in first-, second-grade oocytes and third-, fourth-grade oocytes after maturation(22-24 h in vitro)"
Fig. 5
PKM gene digital PCR verification results A. Copy number scatter plots of PKM gene in DNA solutions of first-, sceond- and third-, fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h; B. Copy number bar chart of PKM gene in DNA solutions of first-, sceond- and third-, fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h; C. Copy number bar chart of PKM gene in DNA solution of first-sceond-grade oocytes matured in vitro for 0, 12, and 22-24 h; D. Copy number bar chart of PKM gene in DNA solution of third- and fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h"
Fig. 6
NDUFA4L2 and IDH3α genes digital PCR verification results A. Copy number scatter plots of NDUFA4L2 gene in DNA solutions of first-, second- and third-, fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h; B. Copy number scatter plots of IDH3α gene in DNA solutions of first-, second- and third-, fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h; C. Copy number bar chart of NDUFA4L2 gene in DNA solutions of first-, second- and third-, fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h; D. Copy number bar chart of NDUFA4L2 gene in DNA solutions of first-, second-grade oocytes matured in vitro for 0, 12, and 22-24 h; E. Copy number bar chart of NDUFA4L2 gene in DNA solutions of third- and fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h; F. Copy number bar chart of IDH3α gene in DNA solutions of first-, second- and third-, fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h; G. Copy number bar chart of IDH3α gene in DNA solutions of first-, second-grade oocytes matured in vitro for 0, 12, and 22-24 h; H. Copy number bar chart of IDH3α gene in DNA solutions of third- and fourth-grade oocytes matured in vitro for 0, 12, and 22-24 h"
Fig. 7
PKM gene qRT-PCR verification results A. qRT-PCR detection results of PKM in first-, sceond- and third-, fourth-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3);B. qRT-PCR detection results of PKM in first-, second-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3);C. qRT-PCR detection results of PKM in third- and fourth-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3)"
Fig. 8
IDH3α and NDUFA4L2 genes qRT-PCR verification results A. qRT-PCR detection results of IDH3α in first-, second- and third-, fourth-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3);B. qRT-PCR detection results of IDH3α in first-, second-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3);C. qRT-PCR detection results of IDH3α in third- and fourth-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3);D. qRT-PCR detection results of NDUFA4L2 in first-, second- and third-, fourth-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3);E. qRT-PCR detection results of NDUFA4L2 in first-, second-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3);F. qRT-PCR detection results of NDUFA4L2 in third- and fourth-grade cumulus cells matured in vitro at 0, 12, and 22-24 h(n=3)"
Fig. 9
OPLS-DA and displacement assay analysis of metabolites in mature culture medium A. OPLS-DA analysis of mature culture medium for first- and second-stage (TR1) and third- and fourth-stage (TR3) cumulus oocyte complexes in positive ion mode; B. Displacement assay analysis of mature culture medium for first- and second-stage (TR1) and third- and fourth-stage (TR3) cumulus oocyte complexes in positive ion mode; C. OPLS-DA analysis of mature culture medium for first- and second-stage (TR1) and third- and fourth-stage (TR3) cumulus oocyte complexes in negative ion mode; D. Displacement assay analysis of mature culture medium for first- and second-stage (TR1) and third- and fourth-stage (TR3) cumulus oocyte complexes in negative ion mode"
Fig. 10
The significant differential metabolites between groups A. The volcano plot of significant differential metabolites in in vitro maturation culture medium of first- and second-grade (TR1) and third- and fourth-grade (TR3) cumulus oocyte complexes under positive and negative ion modes; B. The column chart of significant differential metabolites in in vitro maturation culture medium of first- and second-grade (TR1) and third- and fourth-grade (TR3) cumulus oocyte complexes under positive and negative ion modes"
Fig. 11
Enrichment analysis of significant differential metabolites A. Pathway analysis of significantly upregulated differential metabolites in the maturation buffer of first-, second- and third-, fourth-grade cumulus oocyte complexes; B. Pathway analysis of significantly downregulated differential metabolites in the maturation buffer of first-, second- and third-, fourth-grade cumulus oocyte complexes"
Fig. 12
Significant differential metabolites related pathways diagram (https://www.kegg.jp/kegg/compound/) A. Metabolic pathway diagram of tricarboxylic acid cycle; B. Metabolic pathway diagram of glycolysis/gluconeogenesis"
Fig. 13
Analysis of relative abundance of differential metabolites in oocyte maturation fluid A. Relative abundance map of α-D-glucose in oocyte maturation fluid; B. Relative abundance map of pyruvate in oocyte maturation fluid; C. Relative abundance map of lactic acid in oocyte maturation fluid"
Table 4
The effect of adding sodium pyruvate on the developmental ability of third- and fourth-grade oocytes"
| 组别 Group | 培养COCs总数/枚 Total number of COCs cultivated | 卵裂数/枚 ((mean±SEM)%) Cleavage number | 囊胚数/枚 ((mean±SEM)%) Number of blastocysts |
| 一二级COCs First- and second-grade COCs | 412 | 313 (75.15±4.46) | 105 (33.02±2.76) |
| 三四级COCs+丙酮酸钠 Third- and fourth-grade COCs +sodium pyruvate | 322 | 234 (72.54±4.79)a | 71 (30.63±4.15)a |
| 三四级COCs Third- and fourth-grade COCs | 311 | 192 (61.71±4.75)b | 44 (22.72±2.39)b |
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