Protein in culture and endogenous lipid interact with embryonic stages in vitro to alter calf birthweight after embryo vitrification and warming

Abstract

Short-term protein removal in vitro improves long-term blastocyst competence to survive vitrification. We investigated the mechanisms and effects underlying protein removal. Day-6 morulae and early blastocysts were cultured individually with and without protein for 24h. Development and lipid content were analysed in expanded blastocysts derived from morulae (M-XB) and from early blastocysts (EB-XB). Expression of genes involved in lipid metabolism, stress responses and apoptosis was analysed in fresh and vitrified-warmed M-XB produced with and without protein. Pregnancy rates, birth rates and birthweight (BW) were recorded after transfer of embryos. Day-7 EB-XB production rates (with, 66.9±6.2 and without, 68.8±6.0 protein) were higher than M-XB rates (with, 21.4±4.6 and without, 9.4±4.6 protein; P<0.005). EB-XB showed fewer lipids than M-XB (P=0.03). In fresh M-XB, expression of sterol regulatory element binding protein (SREBP1) was lower with (4.1±2.2) than without (13.6±2.2) protein, contrary to results obtained for Patatin-like phospholipase domain containing 2, Hormone-sensitive lipase and Bcl-2-associated X protein (P<0.05). Protein did not affect pregnancy rates and birth phenotypes (P>0.05). However, BW was higher (P<0.01) in calves born from vitrified M-XB (48.6±3.4kg) than from EB-XB (39.8±2.9kg). Such effects were more pronounced in females (P<0.001). Calves from fresh embryos did not show BW differences. These results indicate that embryonic kinetics and vitrification impact birth phenotypes, at least in females. Alterations might involve exogenous protein and mobilisation of lipid stocks.

The Metabolic Signature of In Vitro Produced Bovine Embryos Helps Predict Pregnancy and Birth after Embryo Transfer

“…In domestic species, lipids are stored in blastomeres as trygliceride [ 48 , 49 ] with higher stocks in IVP embryos than in vivo collected embryos [ 43 ]. Lipid granules decrease through blastulation concurrent with an increase in lipolytic gene expression [ 40 , 50 , 51 ]. The lipid breakdown in embryos is consistent with the embryonic ability to develop in a medium deprived of exogenous substrate [ 52 ], and lipid granules decrease in our protein-free SCS [ 40 ].…”

Single-Cell Transcriptome Profiling Revealed That Vitrification of Somatic Cloned Porcine Blastocysts Causes Substantial Perturbations in Gene Expression

“…To date, the transcription level of genes in porcine oocytes and early embryos by vitrification is mainly examined by real-time quantitative PCR (qRT-PCR). As revealed by using qRT-PCR, vitrification will affect the apoptosis-related genes TNF-α, caspase-8, caspase-9, and Bcl-2 (Chen Y. N. et al, 2018); oxidative stress-related genes SOD1 and SOD2 (Castillo-Martin et al, 2015); lipolysis-related genes LIPE, PLPLA (Gomez et al, 2017) and IGF2, IGF2R (Bartolac et al, 2018); and other genes in porcine embryos. However, qRT-PCR can only detect a small number of genes, while traditional high-throughput sequencing requires a large number of cells.…”

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

“…An alternative explored by several authors is the deprivation of serum over the last 24 h of embryo culture 12 . It is known that serum deprivation represents a stimulus for lipolysis leading to an increase in free fatty acids, and their consumption through β-oxidation for energy production 35,36 . The addition of lipolytic agents is another non-invasive technique allowing the reduction of the level of intracellular lipid content.…”