Holding immature equine oocytes for up to 3 nights prior to ICSI under practical shipping conditions (10-22 °C) is compatible with blastocyst development.

Research Overview

• This study investigates the effects of holding immature horse eggs (oocytes) at different temperatures for up to three nights before fertilization through intracytoplasmic sperm injection (ICSI), a common equine reproductive technique.
• Results show that storing oocytes between about 10 °C and 22 °C for up to 3 nights still allows successful embryo development, which supports practical oocyte shipping for equine breeding.

Background and Purpose

• ICSI is widely used in equine breeding to produce embryos in vitro.
• Oocytes are often recovered by veterinarians and shipped to specialized labs; however, shipment delays can occur.
• Previous research suggested that holding oocytes at 15 °C helps maintain their ability to develop, but common shipping is done around 22 °C.
• The study tested if lower temperatures would better preserve oocyte viability during potential shipment delays.

Methods

• Oocytes (total n=664) were collected from slaughterhouse ovaries on 19 collection days.
• On each day, oocytes were randomly split into two temperature conditions:
  • T15 (target ~15 °C) held in passive cooling devices with actual temperatures ranging 10-20 °C.
  • T22 (target ~22 °C) held in devices with actual temperatures ranging 15-22 °C.
• Each collection day was randomly assigned to one of three holding durations:
  • 20 hours (1 night)
  • 44 hours (2 nights)
  • 68 hours (3 nights)
• This yielded six total treatment groups (2 temperatures x 3 durations).
• Oocytes were exposed to ambient temperatures during holding to mimic shipping conditions.
• After holding, all oocytes underwent in vitro maturation and then ICSI using sperm from one stallion.
• No oocytes were discarded at any stage to ensure consistent comparison across treatments.
• Key outcomes measured included:
  • Maturation rate: percentage of oocytes that matured after holding.
  • Cleavage rate: percentage of injected oocytes that started dividing.
  • Blastocyst formation rate: percentage of injected oocytes reaching blastocyst stage (early embryo).
  • Overall efficiency calculated as maturation rate × blastocyst rate.

Results

• Maturation rates ranged from 27% to 43% across groups.
• Cleavage rates varied from 66% to 83%.
• Blastocyst rates per injected oocyte were between 18% and 34%.
• Overall efficiency ranged from 7% to 12% (maturation × blastocyst rate).
• Statistical analysis showed no significant differences in maturation, cleavage, or blastocyst rates related to temperature (15 °C vs 22 °C) or duration (1 to 3 nights).
• When combining temperatures:
  • 1 night holding: 40% maturation rate, 24% blastocyst rate
  • 2 nights holding: 34% maturation, 26% blastocyst
  • 3 nights holding: 30% maturation, 27% blastocyst
• This suggests little to no decline in developmental competency of oocytes held up to 3 nights.

Conclusions and Implications

• Equine oocytes can be stored for up to three nights at temperatures varying between about 10 °C and 22 °C without impairing their ability to mature and develop into blastocysts after ICSI.
• This finding supports the practicality of shipping immature oocytes in passive cooling containers, even when there are delays or variable temperatures during transport.
• Such flexibility offers greater convenience for veterinarians and breeding programs by enabling oocyte recovery at remote locations and delayed processing without reducing embryo production success.
• The study provides important data for optimizing equine assisted reproductive techniques and improving outcomes in commercial and clinical settings.