Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos

Abstract

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non‐surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm<B < 700 µm and C ≥ 700 µm). Six embryos 233–1360 µm in diameter were punctured via piezo manipulator and/or laser pulse before cryopreservation. All embryos were cryopreserved on a Cryotop®. Frozen‐thawed embryos were cultured for 3h and transferred to the recipient mares. After one week, pregnancy was diagnosed by ultrasonography. Two of six embryos resulted in a positive pregnancy, the result of pregnancy in group A and B was positive, but in group C was negative, and further investigation is necessary for ≥700 µm embryos. The results showed laser‐assisted puncture could be helpful to extract blastocoel fluid and replace it with cryoprotectant. This is the first positive pregnancy report in laser puncture‐assisted frozen‐thawed equine embryo (>300 µm). However, more research is required to find the best method for embryos ≥700 µm.

First Kiso pony foal produced via transfer of long-distance shipped fresh embryo to Hokkaido native pony

“…The mares were then examined daily using ultrasonography to confirm ovulation. Once ovulation was confirmed in donor mares, embryo collection was performed 7 or 8 days later using the non-surgical transcervical embryo recovery method, as reported previously [ 16 , 18 ]. The recovered embryo was washed in commercial embryo holding media (VIGRO®, Vetoquinol N. - A. Inc., Princeville, QC, Canada) before being put into 0.5 ml straw (01123300, Fujihira Industry Co., LTD, Tokyo, Japan) and sealed with polyvinylpyrrolidone (PVP) powder (FHK straw powder, 01128770, Fujihira Industry Co., LTD, Tokyo, Japan).…”