Forskolin improves the cryosurvival of in vivo-derived porcine embryos at very early stages using two vitrification methods.

This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10 μM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96 h, 72 h and 24 h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos.

Gomis J ，Cuello C ，Sanchez-Osorio J ，Gil MA ，Parrilla I ，Angel MA ，Vazquez JM ，Roca J ，Martinez EA ... -  《-》

Factors affecting the success rate of porcine embryo vitrification by the Open Pulled Straw method.

The objectives of this study were: (1) to evaluate the influence of porcine embryo developmental stage on in vitro embryo development after vitrification, (2) to study the efficiency of the one-step dilution procedure, compared with conventional warming, for vitrified embryos at different stages of development, and (3) to determine the influence of the embryo donor on the in vitro survival of vitrified embryos at morulae and blastocyst stages. Two to four cell embryos, morulae and blastocysts were collected by laparotomy from weaned crossbred sows (n=55). Vitrification and conventional warming were performed using the OPS procedure with Superfine Open Pulled Straws (SOPS). For one-step dilution, embryos were placed in 800 microl TCM199-HEPES containing 20% of new born calf serum and 0.13 M sucrose for 5 min. To evaluate development, two to four cell embryos, morulae and blastocysts were cultured in vitro for 120, 48 and 24h, respectively. Some fresh embryos from each developmental stage were not vitrified and cultured as controls. Embryos were morphologically evaluated for their developmental capacity during the in vitro culture by stereomicroscopy. The total cell number of embryos was assessed by Hoechst-33342 staining and fluorescence microscope observation. There was a significant effect of the stage of development on the in vitro survival, perihatching rate and the number of cells of embryos after vitrification and warming (Experiment 1; p<0.001). The survival and perihatching rates of two to four cell embryos were lower than those obtained for morulae and blastocysts (p<0.001). No differences (p>0.05) in survival rates were found between vitrified and fresh blastocysts. The warming procedure did not affect the development and total cell number of vitrified two to four cell embryos, morulae or blastocysts (Experiment 2). However, donor had a significant effect (p<0.001) on the in vitro development and the number of cells of morulae and blastocysts after vitrification and warming (Experiment 3). In conclusion, the embryo developmental stage and the embryo donor were important factors that affected the development of porcine embryos after OPS-vitrification and warming. OPS-vitrification and the one-step dilution are efficient procedures to be used with intact porcine morulae and blastocysts.

Sanchez-Osorio J ，Cuello C ，Gil MA ，Almiñana C ，Parrilla I ，Caballero I ，Garcia EM ，Vazquez JM ，Roca J ，Martinez EA ... -  《-》

Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures.

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.

Cuello C ，Gil MA ，Parrilla I ，Tornel J ，Vázquez JM ，Roca J ，Berthelot F ，Martinat-Botté F ，Martínez EA ... -  《THERIOGENOLOGY》

Vitrification and warming of in vivo-derived porcine embryos in a chemically defined medium.

The aim of this study was to design a protocol for vitrification and warming of porcine embryos in a chemically defined medium. A total of 663 morulae and blastocysts were collected from weaned crossbred sows (Large White-Landrace) 5 to 6 d after estrus and vitrified with the Superfine Open Pulled Straw method. In Experiment 1, embryos were vitrified using as a basic medium TCM-199-HEPES supplemented with 20% newborn calf serum (NBCS) or with 0, 0.1%, 0.5%, or 1% polyvinyl alcohol (PVA). Nonvitrified embryos were used as a fresh control group. Survival and hatching rates were evaluated after 72 h of in vitro culture to assess embryo viability. In addition, some hatched blastocysts derived from morulae and blastocysts were processed to determine the total cell number and the cell proliferating index as measures of their quality. Within each stage of embryo development, the different vitrification groups and the fresh control group showed similar high embryo survival (range, 70.5+/-7.1% to 84.9+/-8.1% and 85.3+/-8.1% to 98.4+/-8.2% for morulae and blastocysts, respectively) and hatching rate (range, 46.3+/-10.1% to 66.7+/-11.2% and 73.7+/-11.3% to 89.4+/-11.2% for morulae and blastocysts, respectively) and quality after in vitro culture. In Experiment 2, embryos were vitrified using 0.1% PVA and warmed with TCM-199-HEPES-0.13 M sucrose supplemented with 20% NBCS or either 0 or 0.1% PVA. Nonvitrified embryos were used as a fresh control group. As in Experiment 1, no significant differences were detected in embryo survival (range, 67.9+/-6.6% to 74.5+/-6.6% and 91.9+/-7.0% to 99.5+/-6.3% for morulae and blastocysts, respectively) and hatching rate (range, 47.0+/-7.2% to 64.8+/-9.9% and 89.4+/-7.4% to 98.2+/-6.9% for morulae and blastocysts, respectively) and quality among the warming groups or among vitrified and fresh control embryos. In both experiments, the developmental embryo stage influenced the survival and hatching rates, as well as the number of cells (P<0.01), with the blastocyst stage yielding the best results. In conclusion, PVA can be used as a substitute for serum in vitrification and warming solutions without detrimental effects on the in vitro development of in vivo-derived porcine morulae and blastocysts.

Sanchez-Osorio J ，Cuello C ，Gil MA ，Parrilla I ，Maside C ，Almiñana C ，Lucas X ，Roca J ，Vazquez JM ，Martinez EA ... -  《-》

Effects of lipid polarisation on survival of in vivo-derived porcine zygotes vitrified by the superfine open pulled-straw method.

Gomis J ，Cuello C ，Sanchez-Osorio J ，Gil MA ，Parrilla I ，Angel MA ，Vazquez JM ，Roca J ，Martinez EA ... -  《-》