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Background <p>Gamete (sperm–oocyte) co-incubation duration during in vitro fertilization (IVF) is a key determinant of embryo developmental competence in buffalo. This study evaluated the effect of gamete co-incubation duration (2-h intervals from 2 to 24&#xa0;h) on embryo development and blastocyst competence.</p> Methods <p>A total of 3680 cumulus–oocyte complexes (COCs) were recovered from 1163 buffalo ovaries. Grade A and B COCs (<i>n</i> = 2390) were matured in vitro, and 2324 matured oocytes were subjected to IVF using different gamete co-incubation durations, followed by culture of 2012 presumptive embryos for 7 days. Cleavage, blastocyst development, and developmental competence were assessed using RT-qPCR.</p> Results <p>Cleavage rate was significantly affected (<i>P</i> &lt; 0.05), increasing from 65.2% at 2&#xa0;h to 100% at 8&#xa0;h and remaining comparable between 10 and 24&#xa0;h (81.7–97.1%). Blastocyst development increased progressively, reaching the highest rates at 16–20&#xa0;h (53.2–54.9%; <i>P</i> &gt; 0.05), before declining significantly at 22&#xa0;h (32.7%) and 24&#xa0;h (28.0%). Embryonic arrest was significantly lower in the 16–20&#xa0;h groups. Blastocysts produced after 16–20&#xa0;h exhibited higher (<i>P</i> &lt; 0.05) expression of HSPB1, SOD2, GPX3, GDF9, BMP15, STAR, and BCL2, with lower BAX and CASP3 expression, indicating enhanced developmental competence.</p> Conclusions <p>Although blastocysts developed following as little as 2&#xa0;h of gamete co-incubation, a duration of 16&#xa0;h produced outcomes comparable to the conventional 20-h protocol. Therefore, 16–20&#xa0;h represents the optimal gamete co-incubation window for maximizing blastocyst yield and developmental competence in buffalo IVF.</p>

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Gamete co-incubation duration modulates blastocyst developmental competence in buffalo through regulation of growth factors, steroidogenesis and oxidative stress pathways

  • 期刊论文

  • 发表日期：

    2026年7月23日

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  • Garbhit Sharma,
  • Anand Kumar Pandey,
  • Hitesh Jangra,
  • Sandeep Kumar,
  • Pradeep Saini,
  • Rajesh Kumar,
  • Divya Choudhary

摘要

Background

Gamete (sperm–oocyte) co-incubation duration during in vitro fertilization (IVF) is a key determinant of embryo developmental competence in buffalo. This study evaluated the effect of gamete co-incubation duration (2-h intervals from 2 to 24 h) on embryo development and blastocyst competence.

Methods

A total of 3680 cumulus–oocyte complexes (COCs) were recovered from 1163 buffalo ovaries. Grade A and B COCs (n = 2390) were matured in vitro, and 2324 matured oocytes were subjected to IVF using different gamete co-incubation durations, followed by culture of 2012 presumptive embryos for 7 days. Cleavage, blastocyst development, and developmental competence were assessed using RT-qPCR.

Results

Cleavage rate was significantly affected (P < 0.05), increasing from 65.2% at 2 h to 100% at 8 h and remaining comparable between 10 and 24 h (81.7–97.1%). Blastocyst development increased progressively, reaching the highest rates at 16–20 h (53.2–54.9%; P > 0.05), before declining significantly at 22 h (32.7%) and 24 h (28.0%). Embryonic arrest was significantly lower in the 16–20 h groups. Blastocysts produced after 16–20 h exhibited higher (P < 0.05) expression of HSPB1, SOD2, GPX3, GDF9, BMP15, STAR, and BCL2, with lower BAX and CASP3 expression, indicating enhanced developmental competence.

Conclusions

Although blastocysts developed following as little as 2 h of gamete co-incubation, a duration of 16 h produced outcomes comparable to the conventional 20-h protocol. Therefore, 16–20 h represents the optimal gamete co-incubation window for maximizing blastocyst yield and developmental competence in buffalo IVF.