Novel Synthetic oviductal fluid for Conventional Freezing 1 (SCF1) culture medium improves development and cryotolerance of in vitro produced Holstein embryos

Abstract

In vitro produced (IVP) embryos hold great promise in the cattle industry; however, suboptimal in vitro culture conditions induce metabolic dysfunction, resulting in poor development and low cryotolerance of IVP embryos. This limits the use of IVP embryos in the cattle industry for embryo transfer and commercial scale-up. Previous studies have reported the use of individual metabolic regulators in culture media to improve blastocyst development rates and cryopreservation. In this study, we hypothesized that using a combination of select regulators, chosen for their unique synergistic potential, would alleviate metabolic dysfunction and improve the development of in vitro produced embryos to make them more closely resemble in vivo derived embryos. To test this, we first compared lipid content between Holstein and Jersey embryos produced in vivo and in vitro, then systematically determined the combination of metabolic regulators that led to the greatest improvements in embryonic development, lipid content, mitochondrial polarity, and cryotolerance. We also tested different slow freezing techniques to further improve cryotolerance, and finally validated our results via a clinical trial. Overall, we found that the use of multiple metabolic regulators in one culture media, which we refer to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1), and an optimized slow freezing technique resulted in improved pregnancy rates for frozen IVP embryos compared to embryos cultured in a synthetic oviductal fluid media. Additionally, there was no difference in pregnancy rate between frozen and fresh IVP embryos cultured in SCF1. This suggests that optimizing culture conditions and slow freezing technique can produce cryotolerance IVP and should allow further dissemination of this assisted reproductive technology.

Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step

“…In addition, it did not quantify changes in embryos after treatment with sucrose medium. To overcome these limitations, further studies on conception rates in farmer’s fields by freeze-thawing bovine embryos pretreated with sucrose and direct embryo transfer are needed, and this study will help inform future frozen embryo transfer studies because embryo survival and conception rates are closely related ( 14 , 52 ).…”

“…Sucrose is a non-penetrating cryoprotectant used for removing intracellular water and protecting cells from osmotic shock in freezing media ( 7 , 13 ). Bovine embryos were reportedly pre-equilibrated with sucrose before slow freezing; however, pretreatment did not improve embryo re-expansion and apoptosis rates ( 14 ). Bui-Xuan-Nguyen ( 15 ) pretreated bovine embryos with sucrose before slow freezing in a medium containing glycerol; however, viability did not improve.…”

“…The use of slow-freezing media supplemented with sucrose to freeze embryos is a well-known technique ( 17–19 ). However, there are limited reports on sucrose pre-equilibration using bovine embryos ( 14 , 15 ). In this study, we aimed to evaluate embryo viability after slow freezing and thawing by inducing artificial shrinkage of the blastocoel cavity using different concentrations of sucrose medium before equilibration in a freezing medium containing EG.…”

Effects of β-Nicotinamide Mononucleotide, Berberine, and Cordycepin on Lipid Droplet Content and Developmental Ability of Vitrified Bovine Oocytes

“…Hara [14] reported that the developmental ability of cryopreserved porcine oocytes could be improved by removing lipids after centrifugation. Despite the potential benefits, centrifugation and micromanipulation are considered too complex and timeconsuming for commercial use [15] and can negatively impact the subsequent development of oocytes [16], limiting their widespread application. As a result, researchers are focusing on using substances to enhance lipid metabolism, decrease lipid droplet content, and improve the cryopreservation outcome.…”