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

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.