D. Le Bourhis

Reproductive technologies and genomic selection in dairy cattle.

Genomic tools are now available for most livestock species and are used routinely for genomic selection (GS) in cattle. One of the most important developments resulting from the introduction of genomic testing for dairy cattle is the application of reasonably priced low-density single nucleotide polymorphism technology in the selection of females. In this context, combining genome testing and reproductive biotechnologies in young heifers enables new strategies to generate replacement and elite females in a given period of time. Moreover, multiple markers have been detected in biopsies of preimplantation stage embryos, thus paving the way to develop new strategies based on preimplantation diagnosis and the genetic screening of embryos. Based on recent advances in GS, the present review focuses on new possibilities inherent in reproductive technologies used for commercial purposes and in genetic schemes, possible side effects and beneficial impacts on reproductive efficiency. A particular focus is on the different steps allowing embryo genotyping, including embryo micromanipulation, DNA production and quality assessment.

Spatial and temporal changes of Decorin, Type I collagen and Fibronectin expression in normal and clone bovine placenta

INTRODUCTION Alteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after somatic cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies. METHODS Genotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin, Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and somatic cloning pregnancies. RESULTS The expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta. DISCUSSION In bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes. CONCLUSIONS These observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy.

78 SUPPLEMENTATION WITH CARNOSINE DURING IN VITRO CULTURE IMPROVES THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

The objective of the present study was to determine the effect of supplementation of culture medium with carnosine (β-alanyl-l-histidine; Sigma, St-Quentin Fallavier, France), a reactive oxygen species scavenger, on in vitro bovine embryo development and survival following cryopreservation. Abattoir-derived bovine oocytes (4 replicates) were in vitro matured and fertilized with frozen-thawed semen of one bull, according to our standard procedures. In Experiment 1, 20h after IVF, groups of presumptive zygotes were cultured in 30μL of SOF BSAaa+1% oestrus cow serum with 0 (control; n=205) or 5μgmL-1 of carnosine (n=209) under humidified air with 5% CO2, 5% O2, and 88% N2. Cleavage rates were determined on Day 2, and the blastocyst rates and grade were assessed on Day 7 according to IETS classification. Day 7 grade 1 expanded blastocysts (n=25 control and n=27 carnosine) were frozen in 1.5M ethylene glycol+0.1M sucrose. Embryos were thawed and then cultured for 72h in SOF-BSAaa+1% oestrus cow serum for re-expansion and hatching rate assessments at +24h, +48h, and +72h post-thawing. In Experiment 2, presumed zygotes were cultured in SOF BSAaa+1% oestrus cow serum with 0 (control; n=48) or 5μgmL-1 of carnosine (n=48) in a WOW dish and observed with Time Laps Cinematography (Primo Vision®, VitroLife, Göteborg, Sweden). Images were recorded every 15min for up to 168h post-insemination. For embryos that reached the blastocyst stage, mean timing of the first cleavage (C1; 2-cell stage), second cleavage (C2; 4-cell stage), second cleavage to compaction (C3), and blastocoel cavity appearance (B4) were recorded. Chi-square test for Experiment 1 and Students t-test for Experiment 2 were used, and differences were considered significant at P<0.05. In Experiment 1, no differences were observed in cleavage rate, blastocyst rate on Day 7, and grade 1 blastocyst rate between both control and carnosine groups (84.0±4.2v.85.2±3.8, P=0.7; 46.9±7.1v. 45.0±7.5, P=0.7; 24.1±2.0v. 24.0±6.5, P=0.6; respectively). After thawing, the re-expansion at +24h was not different between groups (74.1v. 48.0% for carnosine and control groups, respectively; P=0.06). However, at +48h and +72h, the survival rate of carnosine treated blastocysts was significantly higher than that of blastocysts in the control group: 70.4±4.5% v. 40.0±3.8% and 59.3±3.8% v. 24.0±3.6%, respectively. Results from Experiment 2 indicated no difference between control and carnosine groups for C1 (32.1±3.9 v. 33.8±6.1; P=0.3), C2 (8.2±8.9 v. 8.9±0.9; P=0.07), and B4 (147.0±9.5 v. 145.4±11.6; P=0.6), whereas C3 was significantly different within groups: 59.9±9.6v. 51.8±6.7 (P=0.008). In conclusion, bovine blastocysts derived from zygotes cultured in the presence of 5μgmL-1 carnosine possess a significantly faster kinetic from 4-cell stage to compaction and show a higher post-thawing viability. However, further analyses are still needed to clarify the relationship between the reactive oxygen species intracellular levels after carnosine treatment and in vitro bovine embryo quality.

180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liege, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P   0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

41 NUCLEAR REMODELING IS AFFECTED BY TREATMENT OF BOVINE OOCYTES WITH A PROTEASOME INHIBITOR BEFORE NUCLEAR TRANSFER

Complete reprogramming of somatic cell nuclei after nuclear transfer (NT) depends on extensive remodeling of chromatin by factors present in the recipient cytoplast. M-Phase Promoting Factor (MPF) activity, responsible for nuclear remodeling in metaphase II recipients, may be lowered by oocyte enucleation and handling prior to NT. Then, a partial nuclear envelope breakdown or incomplete premature chromosome condensation (PCC) may be, in turn, associated with an inefficient reprogramming. The aim of the present study was to maintain the bovine recipient cytoplast at a high level of MPF activity during the fusion procedure by using a proteasome inhibitor, MG132, and to assess the consequences on nuclear remodeling and developmental potential. Bovine COCs were in vitro-matured for 23 h. Matured oocytes were denuded, and then incubated in TCM-199 for 45 min and enucleated in the presence (treated group) or absence (control group) of 5 µm MG132. Embryos were reconstructed by fusion with adult fibroblasts and activated in 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. In Experiment 1, MPF activity was analyzed immediately after fusion/activation by measuring the phosphorylation of exogenous histone H1, and Cyclin B expression was assessed by Western blotting. In Experiment 2, microtubules revealed by immunofluorescense with anti-tubulin antibody and chromatin stained with 10 µg mL–1 propidium iodide were analyzed by confocal microscopy 1 h after fusion/activation. In Experiment 3, NT embryos activated for 5 h were cultured in vitro for 7 days. Rate of development and cell counts in both groups were then recorded at the blastocyst stage. Remarkably, in Experiment 1, a high MPF activity was found in only 50% of the control oocytes, but MG132 treatment did not enhance this rate. On the other hand, cyclin B persisted for 2 h after activation in treated oocytes whereas it had dropped in controls. Experiment 2 revealed a higher rate of PCC in the treated embryos (n = 51) than in control embryos (n = 54): 96.0% v. 24.0% (chi-square, P < 0.001). Moreover, microtubules reorganized in a metaphasic spindle in embryos undergoing PCC, whereas cytoplasmic microtubules were observed in the others. In Experiment 3, cleavage and blastocyst rates were not significantly different between the treated (n = 92) and the control groups (n = 105): 83.7% and 53.3% v. 78.1% and 50.5%, respectively. However, the mean cell number in treated embryos (n = 27) was significantly higher than in controls (n = 20): 134 ± 25 v. 109 ± 43 (P < 0.05). This study suggests that MG132 treatment improved the maintenance of oocyte factors responsible for PCC in bovine NT embryos, although it did not modify MPF activity, thus questioning the role of MPF in the induction of PCC. Accordingly, PCC may be important for blastocyst quality and nuclear reprogramming in NT embryos. Full-term development of MG132-derived embryos is under investigation.