Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Veto cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 + 45.6 nuclei compared to 137.5 + 32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freczing/thawing, delipidated (n=73), control (n=67) and shmn (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing

La production in vitro et la cryoconservation de l'embryon chez les bovins

no. spécial "Reproduction des ruminants"National audienc

La production in vitro et la cryoconservation de l'embryon chez les bovins

no. spécial "Reproduction des ruminants"National audienc

Freezing bovine IVP embryos: Presence of serum and two step dilution improves in vitro survival

National audienc

Oocyte and embryo production and quality after OPU-IVF in dairy heifers given diets varying in their n-6/n-3 fatty acid ratio

Dietary fat supplementation can improve oocyte quality in ruminants. The influence of the type of dietary fat on the number and quality of oocytes collected by ovum pick-up and on the production of embryos in vitro was investigated in Holstein heifers. Heifers were given hay plus one of two dietary supplements for 42 days. The supplements were linseed (L, rich in linolenic acid, C18:3n-3, n = 9) or soya bean (S, rich in linoleic acid, C18:2n-6, n = 9). Oocytes were collected by ovum pick-up (OPU) for 6 wks (2 sessions/wk) and morphologic quality assessed. Half the oocytes were frozen and the other half was used to produce embryos. Blood samples were analyzed for: insulin, insulin-like growth factor-1, glucose, non-esterified fatty acids, β-hydroxy butyrate and urea and the proportions of fatty acids. Neither growth rate nor plasma hormone and metabolite concentrations were affected by dietary supplement. However, L significantly increased the proportion of plasma C18:3n-3 while S significantly increased the proportion of C18:2n-6(P < 0.001). Neither oocyte characteristics (number, their quality and number fertilized and cleaved per heifer per session) nor embryo characteristics (number and quality per heifer per session) and embryo development stages were affected by dietary treatment. Real-time RT-PCR was performed on immature and mature cumulus-oocyte complexes (COC). Prostaglandin E synthase-1 expression increased in L compared to S heifers. In conclusion, the type of fatty acid did not modify the numbers of oocytes and embryos produced by OPU-IVF and their quality in dairy heifers. Upregulation of prostaglandin E synthase-1 may ensure sufficient PGE(2) production for oocyte maturation even when its precursor is low

Improved cryopreservation of in vitro-produced bovine embryos using a chemically defined freezing medium

International audienceThis study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in bovine embryo cryopreservation solutions. During the experiment, fetal calf serum (FCS) and bovine serum albumin (BSA) were used as references. A combination of a thermodynamic approach using differential scanning calorimetry and a biological approach using in vitro-produced bovine embryo slow-freezing was used to characterize cryopreservation solutions containing CRYO3, FCS and BSA. The CRYO3 and fetal calf serum (FCS) slow-freezing solutions were made from Dulbecco's phosphate-buffered saline containing 1.5 m ethylene glycol, 0.1 m sucrose and 20% (v.v(-1)) of CRYO3 or FCS. The bovine serum albumin (BSA) solution was made by adding 0.1 m sucrose to a commercial solution containing 1.5 m ethylene glycol and 4 g L(-1) BSA. These solutions were evaluated using three characteristics: the end of melting temperature, the enthalpy of crystallization (thermodynamic approach) and the embryo survival and hatching rates after in vitro culture (biological approach). The CRYO3 and FCS solutions had similar thermodynamic properties. In contrast, the thermodynamic characteristics of the BSA solution were different from those of the FCS and CRYO3 solutions. Nevertheless, the embryo survival and hatching rates obtained with the BSA and FCS solutions were not different. Similar biological properties can thus be obtained with slow freezing solutions that have different physical properties within a defined range. The embryo survival rate after 48 h of in vitro culture obtained with the CRYO3 solution (81.5%) was higher than that obtained with the BSA (42.2%, P = 0.000 12) and FCS solutions (58%, P = 0.016). Similarly, the embryo hatching rate after 72 h of in vitro culture was higher with the CRYO3 solution (61.1%) than with the BSA (31.1%, P = 0.0055) and FCS solutions (36%, P = 0.018). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in in vitro-produced bovine embryo cryopreservation solutions

Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

International audienc