SummaryWe identified the most prolific sows in French Large White herds and 17 hyperprolific sows (HLW) were bought whose average litter size on 3 farrowings was 16.5 piglets born alive, i.e. a superiority of 5.3 piglets per litter over their contemporaries. In 1 to 3 subsequent pregnancies we compared ovulation and embryonic mortality rates of 10 HLW with those of 10 Large White (LW) and 7 Meishan (MS) sows. The ovulation rate of HLW was significantly higher than that of LW (-!-5.3) and MS ( + 5.7). The ovulation rate of HLW daughters was higher by 2.1 corpora lutea compared to that of LW gilts at the 3rd oestrus after puberty, which occurred at the same age in the 2 genotypes (228 days) ; MS gilts were pubescent significantly earlier (88 days) and had a significantly lower ovulation rate than both Large White groups. The embryonic mortality rate was high in HLW sows (41 p. 100), whereas that of MS sows was low (16 p. 100), compared to that of LW sows (26 p. 100). Regression of embryonic mortality rate on ovulation rate was significantly positive (!-2.5), and embryonic mortality rate remained significantly higher in both Large White groups than in MS sows when corrected for ovulation rate. It is concluded that the improvement of embryonic survival in Large White sows should be a high priority to improve the efficiency of the hyperprolific line and that the Meishan breed which is prolific owing to a low embryonic mortality may be an appropriate experimental model.

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Vitrification of bovine immature oocytes has been reported using an open pulled straw, but with limited success. In a previous report, we developed an alternative material (nylon mesh) for vitrification of large quantities of oocytes and embryos. This study was conducted to demonstrate effects of components of a cryoprotectant and a protocol of exposure for bovine immature oocytes on their subsequent in vitro maturation, fertilization and development after cryopreservation by vitrification using a nylon mesh. Bovine oocytes at the germinal vesicle stage were collected from 2-5 mm follicles in ovaries, and cumulus-oocytes complexes (COCs) were randomly assigned to treatment groups. Before vitrification, COCs were exposed to the cryoprotectant, which was composed of 40% ethylene glycol, 18% ficoll and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40) by single step or stepwise exposure. Forty COCs were transferred onto a nylon mesh (0.5 cm 2 ), which was then plunged directly into liquid nitrogen. After thawing in warm medium, vitrified COCs were in vitro-matured, fertilized and cultured. After culture for in vitro maturation, the rates in the oocytes reaching to metaphase II were 64.1% and 63.1% in the stepwise exposure to EFS40 or EFT40, respectively, which was significantly higher (P < 0.05) than the corresponding rates after a single step (22.6% and 10.0%, respectively). There was no significant effect of the two sugars on in vitro maturation after single or step-wise equilibration. Transmission electron microscopy revealed that the cytoplasm of oocytes equilibrated in a single step had many vacuoles and broken mitochondria, while oocytes equilibrated in a step-wise manner had significantly fewer abnormalities and were similar to untreated controls. Cleavage rate of thawed oocytes after IVMFC was significantly higher after stepwise exposure to EFS40 or EFT40 than that after single step exposure (37.7% and 22.2% v. 20.8% and 0%, respectively, P < 0.05).Step-wise equilibration of oocytes in EFT40 was dramatically detrimental: no cleaved embryos developed to blastocysts after a single step exposure to either vitrification solution, or stepwise exposure to EFT40. However, blastocysts were obtained following stepwise exposure to EFS40 (8%). These results suggest that stepwise equilibration and vitrification on a nylon mesh minimizes structural damage to the organelles of immature oocytes and facilitates successful cryopreservation.

The development of the open pulled straw vitrification has provided excellent results of in vitro porcine embryo development. Embryo quality evaluation after vitrification has been traditionally focused on morphological assessment performed by stereomicroscopy. The objective of this experiment was to evaluate the efficiency of the stereomicroscopic evaluation of vitrified-warmed (V) porcine blastocysts. Unhatched blastocysts were obtained after slaughter from Large-White gilts (n = 9). Blastocysts (n = 75) were vitrified and warmed using the protocol described by Cuello et al. (2004 Theriogenology 61, 353-361). After warming, vitrified blastocysts were cultured for 24 h. Then blastocysts were morphologically assessed for their progression and morphology by stereomicroscopy. Blastocysts that reformed their blastocoelic cavities showing an excellent appearance were considered viable. Some of the viable blastocysts kept their zonae pellucidae (V viable expanded blastocysts) and others hatched during the in vitro culture (V viable hatched blastocysts). The remaining blastocysts were classified as degenerated embryos. A group of fresh blastocysts was not vitrified and cultured in vitro for 24 h (control group). All of the control blastocysts were considered viable by stereomicroscopy. Some fresh, V viable expanded, V viable hatched, and V degenerated blastocysts (n = 13, n = 19, n = 9, and n = 9, respectively) were processed for ultrastructural study by light and transmission electron microscopy or stained with Hoechst-33342 and TUNEL for cell death evaluation (n = 16, n = 21, n = 11, and n = 6, respectively). All V hatched blastocysts showed ultrastructure similar to that of control hatched blastocysts. However, 26.3% of the V viable expanded blastocysts revealed important ultrastructural alterations in comparison with control expanded blastocysts. These observations suggest that stereomicroscopic evaluation was not efficient enough for V expanded blastocysts. As expected, degenerated blastocysts showed ultrastructural disintegration and disorganization. Hatched V blastocysts did not differ (P < 0.05) from control hatched blastocysts with regard to the total cell number and ratio of death cells (173 � 4.8 vs. 202.1 � 10.9 and 2.8 � 0.5% vs. 1.9 � 0.3%, respectively). However, V expanded blastocysts a had higher (P < 0.01) cell death level (4.3 � 3.4%) than that observed in the control expanded blastocysts (1.1 � 0.3%). Degenerated embryos showed the lowest (P < 0.01) total cell number (45.7 � 4.0). The 66.7% of the degenerated blastocysts exhibited wide TUNEL-labeled areas, and the remaining 33.3% showed TUNEL label over 19.4 � 6.3% of the cells. In conclusion, the hatching rate assessed by stereomicroscopy is a more efficient parameter than assessing the in vitro viability (ratio of blastocysts that reformed their blastocoelic cavities after warming) for estimating the quality of V blastocysts. This work was supported by CICYT (AGL2004-07546) and S�neca (01287/PD/04).