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Morphological and molecular signs of injury and cell death and subsequent regeneration following vitrification of porcine blastocysts were evaluated by light (LM) and transmission electron microscopy (TEM) as well as TUNEL/propidium iodide (PI) nuclear staining followed by confocal microscopy (CSM). In vivo derived blastocysts were assigned to one of the following four groups: Controls-(1) fixed immediately after collection (C0h) and (2) after 24 hr culture in vitro (C24h) and vitrified embryos-(3) fixed immediately after vitrification and warming (V0h), and (4) after 24 hr of culture upon warming after vitrification (V24h). Observation by LM and TEM showed that the V0h embryos displayed collapse of the blastocoele cavity (BC) and cell swelling, a general distension or shrinkage of mitochondria and massive increase in the amount of vesicles, vacuoles, and secondary lysosomes (SLs). Approximately 2/3 of the V24h embryos had recovered, whereas the remaining 1/3 were degenerated. Recovered embryos displayed almost normal blastocyst morphology, except for a widening of the perivitelline space, accumulation of debris and partial distension of mitochondria, whereas degenerated embryos were disintegrated into a poorly defined mass of cells and debris including cells with abundant degeneration of mitochondria and other organelles. Both recovered and degenerated embryos displayed a persistent abundance of presence of small membrane bounded vesicles, vacuoles, and SLs. Evaluation of TUNEL/PI stained embryos showed only occasional appearance of TUNEL positive nuclei with typical apoptotic morphology in controls (C0h 0.67%, C24h 1.22%) and in the V0h embryos (0.93%). The percentage of apoptotic nuclei in embryos at V24h was significantly higher than in all other groups (2.64%). Vitrified embryos showed significantly increased appearance of DNA fragmented nuclei without typical morphological features of apoptosis (V0h 1.43%, V24h 4.30%) compared with controls (C0h 0.26%, C24h 0.45%). The observed morphological changes and increased DNA fragmentation observed immediately after vitrification and warming probably reflects a direct damaging effect of vitrification. During 24 hr of culture a portion of the embryos was able to regenerate and along with the regenerative process, apoptosis--a possible pathway for elimination of damaged cells--became evident.

-In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepesbuffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida. pig / embryo / morula / OPS vitrification / cryopreservationRésumé -Naissance de porcelets après transfert de morulae dans la zone pellucide intacte et vitrification par la méthode OPS. Chez la truie, 5 à 6 jours après l'insémination, il est récolté des embryons aux stades morula et blastocyste associés. Le but de ce travail est de transférer des morulae vitrifiées et d'obtenir des porcelets. Des morulae (200) sont vitrifiées par la méthode OPS après 2 étapes d'équilibration dans un mélange égal d'éthylène glycol, de DMSO et de sucrose dilués dans du TCM199 Hépès + 20 % de sérum de veau nouveau-né (TCM). Pour le réchauffement, les paillettes sont sorties de l'azote et plongées immédiatement dans du TCM + sucrose, l'élimination des cryoprotecteurs se faisant en 3 étapes. Pour évaluer la viabilité des morulae vitrifiées/réchauffées, 10 receveuses reçoivent chacune 20 morulae après transfert chirurgical. Le pourcentage de mises-bas Reprod. Nutr. Dev. 41 (2001) 267-272 267

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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SUMMARYIn the Ile-de-France ewe which has a marked sexual season, oestrus and ovulation can be efficiently controlled by progestagen-PMSG treatment, regardless of the month of the year. However, the fertility after artificial insemination at the induced oestrus increases with the interval from parturition to AI, in A reciprocal egg transfer experiment between nursing and dry ewes showed that the quality of the eggs otained from nursing ewes could be implicated as a cause of the decrease in fertility. But it appears also from this transfer experiment that the quality of the uterine environment is undoubtedly unfavorable in nursing ewes : a higher embryonic mortality occurs between 1 8 and 50 days when eggs from dry ewew are transferred to nursing ewes than when transferred to other dry ewes. In addition, a greater number of blastocysts show a delay in development days after ovulation, and the viability of fertilized eggs is reduced in nursing ewes.The endocrine balance which precedes and follows ovulation is different in nursing ewes compared to dry ewes : the pre-ovulatory discharge of oestrogens which reflects the follicular stimulation does not evolve in the same way, nor reach the same concentrations in nursing ewes. The level of plasma progesterone is correlated with the level of plasma oestradiol 17! during the 24 hours before the LH peak. Also, the plasma concentration of progesterone between the ioth and iqth days of pregnancy is lower in nursing ewes than in dry ewes.A decrease in the interval between lambings rapidly increases the number of young born per female per year. But, the breeding activity after parturition is only possible when ovarian activity and oestrous behaviour have been resumed. The

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