# A Follicle Size Window of Competence for In Vitro Embryo Production in High-Producing Dairy Cows: Evidence from OPU-IVP Performance and Follicular Fluid Profiling

## TL;DR

This study finds that medium-sized follicles (6.0–9.9 mm) in high-producing dairy cows yield the best embryos in vitro, offering a new strategy to improve breeding efficiency.

## Contribution

The study identifies a specific follicle size window (6.0–9.9 mm) that optimizes in vitro embryo production in high-yielding dairy cows.

## Key findings

- Oocytes from medium-sized follicles (6.0–9.9 mm) showed the highest maturation, fertilization, and blastocyst rates.
- Large follicles (10.0–20.0 mm) had increased oxidative stress and lower embryo production efficiency.
- Metabolomic profiling revealed that medium follicles are enriched in pathways like pyruvate and arachidonic acid metabolism.

## Abstract

Ovum pick-up followed by in vitro embryo production (OPU-IVP) is an important technology for accelerating genetic gain in dairy cattle. In high-producing cows subjected to hormonal stimulation, however, the association between follicle size and oocyte developmental competence remains equivocal. Here, 109 high-yielding Holstein cows were used to determine how follicular diameter—small (2.0–5.9 mm), medium (6.0–9.9 mm), and large (10.0–20.0 mm)—affects OPU-IVP performance and to investigate potential underlying mechanisms by integrating follicular fluid (FF) hormone profiles, oxidative stress indices, and untargeted metabolomics. Small follicles yielded the greatest number of oocytes and the highest proportion of morphologically normal oocytes. Nevertheless, oocytes recovered from medium-sized follicles exhibited superior developmental competence, with the highest rates of maturation, fertilization, and progression to transferable embryos, achieving an overall blastocyst rate of approximately 41.88% compared with 29.11% for oocytes from small follicles. In contrast, large follicles produced fewer oocytes, showed a higher proportion of degenerated oocytes, and were associated with increased lipid peroxidation. Collectively, these results identify medium-sized follicles as the optimal developmental window for oocyte retrieval in high-producing dairy cows. Emphasizing collection within this follicle size range may enhance embryo yield, minimize procedural losses, and improve the efficiency and sustainability of breeding programs.

A key objective of the dairy industry is to balance genetic progress with reproductive efficiency. Ovum pick-up followed by in vitro embryo production (OPU-IVP) is a pivotal technology for accelerating genetic gain. However, the relationship between follicle size and oocyte developmental competence in high-producing dairy cows under hormonal stimulation remains to be fully elucidated. This study systematically evaluated the effects of follicle diameter ovum pick-up on OPU-IVP outcomes and the underlying follicular fluid (FF) microenvironment. A total of 109 high-yielding Holstein cows were subjected to ovarian stimulation and OPU. Follicles were categorized as small (2.0–5.9 mm), medium (6.0–9.9 mm), or large (10.0–20.0 mm). Oocyte recovery, quality, and developmental competence were assessed. FF was analyzed for hormonal profiles, including anti-Müllerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and progesterone (PROG); oxidative stress markers, including malondialdehyde (MDA), glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC); and untargeted metabolomics (n = 10 per group). Consistently, oocytes from medium follicles exhibited superior developmental competence, achieving the highest maturation (89.93%), cleavage (72.19%), and blastocyst rates (41.88%). In contrast, large follicles had a low recovery rate (32.64%), a high proportion of degenerated oocytes (32.00%), and reduced embryonic efficiency. Metabolomic profiling revealed distinct microenvironmental differences, with medium follicles enriched in pathways like pyruvate metabolism and arachidonic acid metabolism indicating an optimal metabolic state. Hormonally, AMH decreased while E2 and PROG increased with follicle size. Large follicles exhibited significantly elevated MDA levels, indicating oxidative stress, without a concurrent rise in antioxidant capacity. In conclusion, while small follicles provide an abundant source of morphologically good oocytes, medium follicles (6.0–9.9 mm) represent a distinct “window of competence” for OPU-IVP, characterized by a follicular microenvironment most conducive to embryo production. Excessive reliance on large follicle aspiration should be avoided due to signs of over-maturity and oxidative damage. These findings provide a physiological basis for optimizing OPU strategies to enhance IVP efficiency in high-producing dairy cows.