Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process

Abstract

Currently, in vitro embryo production (IVP) is successfully commercially applied in cattle. However, the high sensitivity of embryos to cryopreservation in comparison to in vivo (IVD) embryos slows the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to in vitro culture conditions. The objective of this study was to evaluate the lipid composition of biopsied and sexed embryos, produced either in vivo or in vitro from the same Holstein heifers before and after a slow freezing protocol. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry, which enabled the detection of 496 features. Our results highlighted a lipid enrichment of IVP embryos in triglycerides and oxidised glycerophospholipids and a reduced abundance in glycerophospholipids. The slow freezing process affected the lipid profiles of IVP and IVD embryos similarly. Lysophosphatidylcholine content was reduced when embryos were frozen/thawed. In conclusion, the embryonic lipid profile is impacted by IVP and slow freezing protocols but not by sex. Lysophosphatidylcholine seemed highly sensitive to cryopreservation and might contribute to explain the lower quality of frozen embryos. Further studies are required to improve embryo freezability by modulating the lipidome.

Effects of the donor factors and freezing protocols on the bovine embryonic lipid profile

“…Experimental heifers were subjected to estrus synchronization, as described by Janati Idrissi et al. [ 3 ]. Briefly, synchronization treatments were performed by insertion of an intravaginal progesterone releasing device (Prid ® Delta, 1.55 g, Ceva, Libourne, France) and this was followed, 6 days later, by 2 mL intramuscular injection of a prostaglandin F2 alpha analog (Estrumate ® , MSD Santé Animale — Intervet, Beaucouzé, France; equivalent to 0.5 mg cloprostenol).…”

“…For Holstein heifers, ovarian stimulation was performed as previously described [ 3 ] 36 h after the removal of the dominant follicles. Briefly, a new intravaginal progesterone device (Prid ® Delta, 1.55 g, Ceva, Libourne, France) was inserted, and the ovarian stimulation was performed by five intramuscular injections of decreasing porcine follicle stimulating hormone/porcine luteinizing hormone (pFSH/pLH) doses (Stimulfol ® , Reprobiol, Ouffet, Belgium), every 12 h, over 2.5 days.…”

“…In vitro development was performed as previously described [ 3 ]. Whatever the groups, 82–90% of the recovered COCs were selected.…”

“…Lipid composition is an important factor in ensuring proper embryonic development and thus achieving high pregnancy rates [ 1 , 2 ]. Moreover, it has been shown that the embryonic origin (vivo or vitro) as well as embryo freezing had a significant impact on the lipid profile of bovine embryos [ 3 ]. In fact, in vitro-produced embryos seemed to present an overabundance of oxidized glycerophospholipids and triglycerides, while the in vivo ones had an overabundance of phospholipids.…”

“…In fact, the lipid phase transition, followed by separation, is one of the major causes of cryo-damages in lipid-rich oocytes and embryos [ 21 , 23 ]. It has also been shown that embryo and oocyte cryopreservation protocols (slow freezing and vitrification) lead to a decrease in the phospholipid and LPC content [ 3 , 24 ]. However, LPCs are precursors of lysophosphatidic acid, which is a modulator of embryos quality markers, such as insulin-like growth factor 2 receptor and placental associated 8 as well as pluripotency factors like sex-determining region Y box 2 and octamer-binding transcription factor 4, indicating that a decrease in LPC content can affect bovine embryo quality and can lead to a dysregulation of pluripotency pathway [ 25 , 26 ].…”