F. Guignot

Le début de la cryoconservation d’embryons de mammifères remonte aux années 70. Depuis, les techniques se sont multipliées et diversifiées pour essayer de répondre aux problèmes que posaient certaines espèces, certains stades de développement embryonnaire et les embryons fragilisés comme les embryons produits in vitro, issus de clonage ou encore les embryons biopsiés. Sont principalement utilisées, la congélation lente qui est basée sur l’équilibration progressive entre les cryoprotecteurs et le compartiment aqueux de l’embryon, la vitrification qui transforme rapidement la phase liquide du cytoplasme embryonnaire en phase solide amorphe appelée «état vitreux», et l’OPS (Open Pulled Straw) qui est une vitrification très rapide. Chaque technique a ses avantages et ses inconvénients, mais la congélation lente est actuellement la technique de référence sur le terrain en ovin, caprin et bovin avec des embryons produits in vivo. Par contre, pour les porcins, seule la technique d’OPS perm...

The aim of this study was to evaluate the relationships between the rate and extent of post-mortem pH changes and the colour, the cooking loss and the eating quality of veal. The experiment used 12 calves aged 18 weeks. Variations in ultimate pH were induced by adrenalin administration (0.1-0.4 mg/kg liveweight) to six of the animals. Measurements were made on the Longissimus thoracis muscle. pH and osmotic pressure were measured at 0.5 h, 4 h and 29 h after slaughter. Pigment content, drip loss and cooking loss were measured at 29 h after slaughter, and colour was measured at 2 days and 9 days after slaughter. Cooking loss, tenderness, juiciness and flavour of roasts were assessed at 9 days after slaughter. Correlations between colour traits and pH values were higher with ultimate pH than with pH at 0.5 h or 4 h after slaughter. Lightness, redness and reflectance decreased when the ultimate pH increased. Drip loss was correlated with the rate of pH fall (r = -0.80, P < 0.01 with pH at 4 h), while cooking loss was correlated with ultimate pH (r = -0.94, P < 0.01). Ultimate pH and the sensory quality traits were linearity and positively correlated (r = 0.83) for tenderness, 0.81 for juiciness and 0.71 for flavour, respectively).

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

This study constitutes an experimental reproduction of Black Belly sheep in order to investigate the Bluetongue disease or catarrhal fever. A total of 32 multiparous Black Belly ewes were superovulated with porcine FSH during the last three days of progestagen ...

Background Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re–expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC...

La production d'embryons in vivo et le transfert de ceux-ci dans une receveuse font partie des techniques de pointe de la maitrise de la reproduction et de la diffusion du progres genetique. Trois applications pour lesquelles notre laboratoire a ete sollicite entre 2003 et 2009 chez les petits ruminants, chevre et brebis, de differentes races, sont detaillees dans cette communication. La premiere est la constitution d'une banque d'embryons pour conserver la race caprine Creole et les races ovines Merinos de Rambouillet et Booroola. Respectivement, 256, 40 et 427 embryons ont ete cryoconserves. La seconde est la creation d'un troupeau experimental ovin Black Belly de genotype rare en France a partir de quelques individus seulement: 264 embryons ont ete collectes sur 30 femelles et 145 petits sont nes de 230 embryons transferes. La troisieme est l'evaluation de l'efficacite du transfert d'embryons dans la lutte contre la transmission mere-jeune du CAEV. A p...

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 μm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.

Chez les petits ruminants, le cout de la transplantation embryonnaire est un facteur limitant essentiel de l'utilisation de cette methode. Des techniques rapides a mettre en oeuvre, telles que la vitrification des embryons et leur transfert direct sans observation apres degel, peuvent contribuer a reduire les couts de la transplantation embryonnaire et accroitre ainsi le developpement de cette methode chez la brebis et la chevre. Afin d'evaluer l'efficacite des ces techniques, 2 experiences ont ete realisees. Dans une premiere experience, la viabilite des embryons vitrifies/decongeles a ete comparee aux resultats obtenus apres transfert d'embryons frais chez la brebis ou apres transfert d'embryons congeles (par congelation lente) chez la chevre. Les taux de mise-bas et de survie embryonnaire ne different pas significativement en fonction du traitement des embryons (chez la brebis: embryons frais 72 % et 60 % vs embryons vitrifies 72 % et 50 %; chez la chevre: embryons congeles 69 % et 55 % vs embryons vitrifies 48 % et 39 %). Dans la seconde experience, la possibilite de transferer directement apres degel les embryons vitrifies ou congeles (sans retrait des cryoprotecteurs et sans evaluation morphologique de la qualite des embryons apres degel) a ete testee par comparaison avec la technique traditionnelle de transfert d'embryons vitrifies ou congeles (retrait des cryoprotecteurs et evaluation de la qualite des embryons). Il n'a pas ete observe d'effet significatif de la methode de transfert sur les taux de mise-bas et de survie embryonnaire (chez la brebislembryons vitrifies: transfert traditionnel 67 % et 49 % vs transfert direct 75 % et 53 %; chez la chevre/embryons vitrifies: transfert traditionnel 23 % et 15 % vs transfert direct 38 % et 26 %; chez la chevre/embryons congeles: transfert traditionnel 74 % et 45 % vs transfert direct 71 % et 57 %).

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.

The objectives of the present study were to evaluate the effect of conjugated linoleic acid (CLA t10, c12, C18:2), linolenic acid (C18:3) and docosahexaenoic acid (DHA, C22:6) supplementation on in vitro bovine embryo development, embryo survival after cryopreservation, gene expression and AMPKalpha phosphorylation. Control groups with modified synthetic oviduct fluid (mSOF)+/-100microM beta-mercaptoethanol (beta-ME) were performed. The effects of co-culture with bovine oviduct epithelial cell (Boec) monolayers, serum supplementation and embryo development in the ewe oviduct, on gene expression were also examined. Experiments 1 and 2: a lower d 7 embryo survival was found with 100microM C22:6 and 100microM C18:2 supplementation compared to 1microM C22:6 and 100microM beta-ME supplementation (P<0.05). C18:3 supplementation had no effect on d 7 embryo survival, but 100microM C18:3 increased d 8 embryo survival compared to 100microM beta-ME supplementation (P<0.05). Experiments 3 and 4: stearoyl-CoA desaturase 1 (SCD1) and sterol regulatory element-binding transcription factor 1 (SREBP1) mRNA decreased after 10microM C22:6 supplementation compared to all other supplementations (P<0.05). A lower fatty acid desaturase 2 (FADS2) transcript level was found with 100microM C18:2, 10microM C22:6 and 10microM C18:3 supplementations compared to groups without fatty acid supplementation (P<0.05). Acetyl-CoA-carboxylase (ACC), fatty acid synthase (FAS), adipose differentiation-related protein (ADRP), acyl-CoA synthetase long-chain family member 1 (ACSL1), diacylglycerol O-acyltransferase 1 (DGAT1), carnitin palmitoyltransferase-II (CPT-II) mRNAs expression and AMPKalpha phosphorylation were not modified with PUFA supplementation. Experiment 5: SCD1 and FAS mRNA decrease in Boec group compared to serum supplementation, as SCD1 mRNA in ewe oviduct group (P<0.05). In conclusion, this study showed that a PUFA supplementation with C18:2, C18:3 or C22:6 in bovine culture development for 6 days and co-culture with Boec down-regulate mRNA expression of proteins involved in lipid metabolism in d 7-8 embryo (SCD1 and FADS2 desaturases), probably through SREBP1 mRNA regulation after 10microM C22:6 supplementation, indicating a modification of saturated/unsaturated fatty acid balance in bovine blastocyst.

The post mortem evolution of water distribution was studied in muscle tissue from veal calves in two experiments. Myofilament spacing, extracellular space and rate and extent of pH fall were determined in Psoas major muscle in Experiment 1. Extracellular space and rate and extent of pH fall were determined in Longissimus dorsi, Psoas major and Trapezius muscles in Experiment 2. In Experiment 2, the variability of ultimate pH was increased by using adrenalin injections. The myofilament spacing decreased after slaughter when pH reached values around 5·9. In both experiments, the extracellular space began to increase soon after slaughter in close relation with the pH changes. The size of the ultimate extracellular space was significantly correlated with the rate of pH fall, but not with the ultimate pH.

In equine species, embryo cryopreservation is not as widely developed as in some other species. Slow freezing has been applied to equine embryos but with relatively low success rates. This higher sensitivity to conventional freezing procedures may be explained by the presence of a capsule surrounding the equine embryo that may impair cryoprotectant penetration. Recently, good in vitro embryo survival rate was obtained after open pulled straw (OPS) vitrification (Moussa et al. 2005 Theriogenology 64, 1619–1632). The aim of the present study was to evaluate in vivo survival of vitrified embryos five days after surgical transfer into Welsh pony mares. Morulae (M), early blastocysts (EB), and blastocysts (B) ranging from 140 to 320 μm in diameter were collected (n = 20) in a Ringer lactate solution on Day 6.75 after ovulation. Before vitrification, embryos were assessed morphologically and their size was measured (McKinnon and Squires 1988 J. Am. Vet. Med. Assoc. 192, 401–406). Then, em...