Oral propylene glycol modifies follicular fluid and gene expression profiles in cumulus-oocyte complexes and embryos in feed-restricted heifers.

Dietary supplementation with propylene glycol (PG) increases in vitro production of high-quality embryos in feed-restricted heifers. The aim of the present study was to evaluate the effects of PG in feed-restricted heifers on follicular fluid insulin and insulin-like growth factor (IGF) 1 concentrations, expression of IGF system genes in oocytes and cumulus cells and the expression of selected genes in blastocysts. Feed-restricted (R) heifers were drenched with water or PG during induced oestrous cycles (400mL of PG or water/drench, daily drenching at 1600 hours for the first 9 days of the oestrous cycle). Ovum pick-up (OPU) was performed after superovulation to produce in vitro embryos and without superovulation to recover oocytes, cumulus cells and follicular fluid. OPU was also performed in a control group (not feed restricted and no drenching). Follicular fluid IGF1 concentrations were reduced by R, and PG restored IGF1 concentrations to those seen in the control group. In cumulus cells, expression of IGF1, IGF1 receptor (IGF1R) and IGF binding protein 4 (IGFBP4) was decreased in the R group, and fully (IGF1 and IGF1R) or partially (IGFBP4) restored to control levels by PG. Blastocyst perilipin 2 (PLIN2; also known as adipophilin), Bcl-2-associated X protein (BAX), SCL2A1 (facilitated glucose/fructose transporter GLUT1), aquaporin 3 (AQP3), DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and heat shock 70-kDa protein 9 (HSPA9B) expression were decreased in R heifers; PG restored the expression of the last four genes to control levels. In conclusion, these results suggest that, during follicular growth, PG exerts epigenetic regulatory effects on gene expression in blastocyst stage embryos.

Review of bovine brucellosis surveillance in Europe in 2015.

Bovine brucellosis is a major zoonosis and is responsible for significant reproductive disorders and production losses in cattle. Surveillance and control are regulated at the European level with specific conditions to obtain and maintain the officially free status, which facilitates access to export markets. These European standards allow for harmonisation in brucellosis surveillance and diagnosis while leaving some flexibility to countries in the choice of measures to meet the desired objectives. This study reviews the bovine brucellosis surveillancesystems currently in place across the European continent, according to countries' brucellosis status, based on a survey addressed to brucellosis diagnosis expertsin the National Reference Laboratory of each country. Experts were asked toprovide synthesised surveillance data and to describe technical conditions andscreening tests carried out for the surveillance of abortions, serological testing inherds, movement controls and any other surveillance components in 2015. Results were obtained for 34 out of 37 countries (92%). Surveillance systems included abortion surveillance (34 countries), routine herd screening (28 countries), movement testing (14 countries), routine testing at bull stations (9 countries),and screening tests at slaughterhouses (4 countries). The review highlighted variability in technical conditions and screening tests among countries. These results are discussed with regard to the European Union regulations, disease risks and epidemiological situations, with the aim of improving surveillance efficacy and efficiency.

La brucellose bovine est une zoonose majeure qui entraîne chez les bovins de graves troubles de la reproduction ainsi qu'une baisse importante de la production. La surveillance et le contrôle de cette maladie font l'objet d'une réglementation spécifique au niveau européen, à travers des dispositions conditionnant l'obtention et le maintien du statut officiel indemne, lequel facilite l'accès aux marchés d'exportation. Les normes européennes permettent d'harmoniser la surveillance et le diagnostic de la brucellose tout en laissant aux pays une certaine marge de manoeuvre quant au choix des mesures appliquées pour atteindre les objectifs souhaités. En se basant sur les résultats d'une enquête conduite auprès des experts des Laboratoires de référence nationaux pour le diagnostic de la brucellose dans chaque pays, les auteurs font le point sur les systèmes de surveillance de la brucellose bovine appliqués actuellement en Europe en fonction du statut des pays au regard de la brucellose. Il a été demandé aux experts de présenter une synthèse des données de surveillance et de décrire le dispositif technique de suivi et les tests de dépistage effectués en cas d'avortement, ainsi que la surveillance sérologique conduite à l'échelle des troupeaux, les contrôles exercés sur les mouvements d'animaux et toute autre composante de la surveillance en 2015. Au total, 34 des 37 pays interrogés (92 %)ont répondu à l'enquête. Les systèmes de surveillance en place portaient sur les points suivants : surveillance des avortements (34 pays), dépistage systématique à l'échelle des troupeaux (28 pays), tests sur les animaux déplacés (14 pays), dépistage systématique dans les stations de monte et les taurelleries (neuf pays) et dépistage à l'abattoir (quatre pays). Les résultats de l'enquête mettent en relief la diversité du contexte technique et des tests de dépistage d'un pays à l'autre. Les auteurs analysent ces résultats au regard de la réglementation de l'Union européenne, des risques sanitaires spécifiques et des diverses situations épidémiologiques, dans le but d'améliorer l'efficacité et l'efficience de la surveillance.

La brucelosis bovina es una importante zoonosis, causante de graves trastornos reproductivos y de cuantiosas pérdidas en el sector de la producción pecuaria. En los reglamentos europeos, que rigen las actividades de vigilancia y control, se establecen también requisitos específicos para obtener y mantener el estatutode «oficialmente libre de la enfermedad¼, que facilita el acceso a los mercados de exportación. Estas normas europeas permiten armonizar las actividades devigilancia y diagnóstico de la brucelosis y ofrecer a la vez cierta flexibilidad alos países en la elección de las medidas necesarias para cumplir los objetivos fijados. Los autores pasan revista a los sistemas de vigilancia de la brucellosis bovina actualmente implantados en todo el continente europeo, en funcióndel estatuto del país con respecto a la enfermedad, utilizando para ello los datosde un cuestionario dirigido a los especialistas en diagnóstico de la brucelosis decada laboratorio nacional de referencia. A cada uno se le pedía una síntesis delos datos de vigilancia y una descripción (incluidas las condiciones técnicas) delas pruebas de detección realizadas con fines de vigilancia de abortos, análisis serológico de rebaños, controles en caso de movimiento de animales y demás componentes de la vigilancia que se hubieran aplicado en 2015. Se obtuvieron así datos de 34 países sobre un total de 37 (un 92%). Los sistemas de vigilancia descritos incluían la vigilancia de abortos (34 países), la realización sistemática de pruebas de detección en rebaños (28 países), en corrales de toros (nueve países) y en mataderos (cuatro países) y la realización de pruebas antes y/o después del movimiento de animales (14 países). El estudio puso de manifiesto la heterogeneidad reinante entre los países en cuanto a las pruebas de detección practicadas y a sus condiciones técnicas. Los autores examinan esos resultados en relación con las normas de la Unión Europea, los riesgos de enfermedad y las situaciones epidemiológicas, con el objetivo de conferir más eficacia y eficiencia a las labores de vigilancia.

Schmallenberg virus: experimental infection in goats and bucks.

BACKGROUND: Schmallenberg virus (SBV) is an emerging Orthobunyavirus of ruminant livestock species currently circulating in Europe. SBV causes a subclinical or mild disease in adult animals but vertical transmission to pregnant dams may lead to severe malformations in the offspring. Data on the onset of clinical signs, viremia and seroconversion in experimentally infected adult animals are available for cattle and sheep but are still lacking for goats. For a better understanding of the pathogenesis of SBV infection in adult ruminants, we carried out experimental infections in adult goats. Our specific objectives were: (i) to record clinical signs, viremia and seroconversion; (ii) to monitor viral excretion in the semen of infected bucks; (iii) to determine in which tissues SBV replication took place and virus-induced lesions developed. RESULTS: Four goats and two bucks were inoculated with SBV. Virus inoculation was followed by a short viremic phase lasting 3 to 4 days and a seroconversion occurring between days 7 and 14 pi in all animals. The inoculated goats did not display any clinical signs, gross lesions or histological lesions. Viral genomic RNA was found in one ovary but could not be detected in other organs. SBV RNA was not found in the semen samples collected from two inoculated bucks. CONCLUSIONS: In the four goats and two bucks, the kinetics of viremia and seroconversion appeared similar to those previously described for sheep and cattle. Our limited set of data provides no evidence of viral excretion in buck semen.

Dietary propylene glycol and in vitro embryo production after ovum pick-up in heifers with different anti-Müllerian hormone profiles.

Rapid genetic improvement in cattle requires the production of high numbers of embryos of excellent quality. Increasing circulating insulin and/or glucose concentrations improves ovarian follicular growth, which may improve the response to superovulation. The measurement of anti-Müllerian hormone (AMH) can help predict an animal's response to superovulation treatment. The aim of the present study was to investigate whether increasing circulating insulin concentrations, through propylene glycol (PG) drenches, could improve in vitro embryo production in oestrus-synchronised superovulated heifers with different AMH profiles. Holstein heifers were grouped according to pre-experimental AMH concentrations as low (L) or high (H). The PG drench increased circulating insulin and glucose concentrations and reduced ß-hydroxybutyrate and urea concentrations compared with the control group. AMH was a good predictor of follicle and oocyte numbers at ovum pick-up (OPU), and of oocyte and embryo quality (AMH H>AMH L). PG in the AMH H group increased the number of follicles and blastocyst quality above that in the control group, but did not improve these parameters in the AMH L group. These results indicate that short-term oral PG supplementation modifies an animal's metabolic milieu and is effective in improving in vitro embryo production, after superovulation-OPU, more markedly in heifers with high rather than low AMH concentrations.

Sire effect on early and late embryonic death in French Holstein cattle.

We investigated the effect of maternal sire on early pregnancy failure (between D0, day of insemination and D90) in their progeny during the first and second lactations (n=3508) in the Holstein breed. The estimated breeding value (EBV) for cow fertility of 12 bulls (reliability⩾0.95) was used to create the following three groups: low, medium and high EBV (EBV from -0.7 to 1 expressed as genetic standard deviation relative to the mean of the breed). In their daughters (93 to 516 per bull), progesterone measurement was carried out on the day of artificial insemination (AI; D0) to check whether the cows were in the follicular phase and on D18 to 25 to assess non-fertilisation-early embryonic mortality (NF-EEM). Late embryonic mortality (LEM) and early foetal death (FD) were determined by ultrasonography on D45 and D90 and by the return to oestrus after the first AI. Frequencies of NF-EEM, LEM, FD and pregnancy were 33.3%, 11.7%, 1.4% and 48.5% and incidences were 35.1, 19.0, 2.7 and 51.1, respectively. Sire EBV was significantly related to the incidences of pregnancy failure between D0 and D90, fertilisation failure-early embryonic mortality (FF-EEM) and LEM but not to the incidence of FD between D45 and D90 of pregnancy. The relative risk (RR) of FF-EEM was significantly higher (RR=1.2; P<0.05) for the progeny group of low EBV bulls when compared with high EBV bulls. The same effect was observed when comparing LEM of the progeny groups from the low EBV bulls to those from moderate and high EBV bulls (RR, respectively, of 1.3 and 1.4; P<005). The incidence of FF-EEM was significantly higher when cows were inseminated before 80 days postpartum compared with later, and for the extreme values of the difference between milk fat and protein content measured during the first 3 months of lactation. FF-EEM was also significantly related to the year of observation. The incidence of LEM was higher for the highest producing cows and was influenced by interaction between milk yield×lactation rank and milk yield×milk protein content. In conclusion, this study showed large differences in early pregnancy failure between progeny groups and highlights the interest of accurate characterisation of embryonic death in order to identify potential candidate genes for female fertility.

Prediction of pregnancy viability in bovine in vitro-produced embryos and recipient plasma with Fourier transform infrared spectroscopy.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared (FTIR) metabolomics to predict pregnancy outcome. Individually cultured, in vitro-produced (IVP) blastocysts were transferred to recipients as fresh and vitrified-warmed. Spent CM and plasma samples were evaluated using FTIR. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the receiver operator characteristic curve (AUC). Within all IVP fresh embryos (birth rate=52%), high AUC were obtained at birth, especially with expanded blastocysts (CM: 0.80±0.053; plasma: 0.89±0.034). The AUC of vitrified IVP embryos (birth rate = 31%) were 0.607±0.038 (CM, expanded blastocysts) and 0.672±0.023 (plasma, all stages). Recipient plasma generally predicted pregnancy outcome better than did embryo CM. Embryos and recipients with improved pregnancy viability were identified, which could increase the economic benefit to the breeding industry.

Development rate and gene expression of IVP bovine embryos cocultured with bovine oviduct epithelial cells at early or late stage of preimplantation development.

The use of somatic cells for coculture with embryos has been amply investigated to study embryo maternal interactions. The use of bovine oviduct epithelial cells (BOEC) has been shown to improve the blastocyst rate and quality, affecting their gene expression profile. In this study, we evaluated different timings of BOEC coculture for the development of in-vitro-produced embryos and their effects on blastocysts rate and mRNA abundance of some genes that are important for embryo development. Our results confirmed the positive effects of BOEC on early development of bovine embryos. The presence of the cells during the first four days or during the last four days of development was enough to produce the full BOEC effect. When the presence of BOEC was restricted to the four first days, the kinetics of blastocyst development was accelerated, with significantly more blastocysts at Days 6 and 7 than when the cells were present all along the culture or only during the last four days. Older cells used at early stage were not active anymore. Using young cells at late stage did not improve the cell effect, compared with the older ones. Therefore, the lower effect of BOEC at late stage, compared with early period, may not be explained by cell aging. In addition, the presence of BOEC, at early or late stages, induced changes in the embryos expression profile of genes known to be related to embryo quality, suggesting reduced apoptosis and increased capacity to struggle against oxidative stress after coculture. In conclusion, we confirmed the effect of BOEC on the rate and quality of bovine IVP embryos development. We found for the first time that the presence of BOEC during the four first days of the 8-days development is enough to produce these effects. These first four days represent the period of the presence of the embryos in the oviduct in vivo, highlighting the physiological relevance of this in vitro model of coculture. In addition, we found that the presence of BOEC at early stages of development induced modification of transcription profile in the blastocyst, four days later, suggesting an epigenetic regulation induced by BOEC in growing embryos.

Energy and lipid metabolism gene expression of D18 embryos in dairy cows is related to dam physiological status.

We analyzed the change in gene expression related to dam physiological status in day (D)18 embryos from growing heifers (GH), early lactating cows (ELC), and late lactating cows (LLC). Dam energy metabolism was characterized by measurement of circulating concentrations of insulin, glucose, IGF-1, nonesterified fatty acids, ß-hydroxybutyrate, and urea before embryo flush. The metabolic parameters were related to differential gene expression in the extraembryonic tissues by correlation analysis. Embryo development estimated by measuring the length of the conceptuses and the proportion of expected D18 gastrulating stages was not different between the three groups of females. However, embryo metabolism was greatly affected by dam physiological status when we compared GH with ELC and GH with LLC but to a lesser extent when ELC was compared with LLC. Genes involved in glucose, pyruvate, and acetate utilization were upregulated in GH vs. ELC conceptuses (e.g., SLC2A1, PC, ACSS2, ACSS3). This was also true for the pentose pathway ( PGD, TKT), which is involved in synthesis of ribose precursors of RNA and DNA. The pathways involved in lipid synthesis were also upregulated in GH vs. ELC. Despite similar morphological development, the molecular characteristics of the heifers' embryos were consistently different from those of the cows. Most of these differences were strongly related to metabolic/hormone patterns before insemination and during conceptus free-life. Many biosynthetic pathways appeared to be more active in heifer embryos than in cow embryos, and consequently they seemed to be healthier, and this may be more conducive to continue development.

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.

[Use of an in vitro model in bovine to evidence a functional and molecular dialogue between preimplantation embryo and oviduct epithelial cells]. / Mise en évidence d'un dialogue moléculaire et fonctionnel entre l'embryon préimplantatoire bovin et l'oviducte grâce à un modèle in vitro.

Beyond being a pipe between ovary and uterus, the oviduct is an active player in different aspects of early reproductive processes, in particular in the transport of embryos to the site of implantation and the regulation of its early development. Different studies evidenced a communication between oviduct and early embryo at the molecular and functional levels. Since the study of these interactions is difficult in vivo, different in vitro systems have been developed to mimic the maternal milieu during early development. These systems allowed to confirm the action of the cells on the quality of early development (blastocyst rate and viability). In turn, the embryos are producing signals that are able to modify and adapt the activity of maternal cells.

Breast-cancer anti-estrogen resistance 4 (BCAR4) encodes a novel maternal-effect protein in bovine and is expressed in the oocyte of humans and other non-rodent mammals.

STUDY QUESTION: Does BCAR4 have a role in mammalian embryo development? SUMMARY ANSWER: Expression, localization and functional data support that BCAR4 is a maternal-effect protein in non-rodent mammals. WHAT IS KNOWN ALREADY: BCAR4 was previously identified as an oocyte-specific gene in cattle, and as a marker of certain breast tumors in humans. STUDY DESIGN, SIZE, DURATION: Human oocytes were obtained from patients undergoing IVF, but had failed to mature after ovarian stimulation. Dog oocytes were obtained from ovariectomized bitches. Pig, horse and bovine ovaries were obtained from commercial slaughterhouses for extraction of immature oocyte-cumulus complexes. In vivo matured bovine matured oocytes were obtained after ovulation induction and ovulation inducing treatment of Montbeliard heifers. MATERIALS, SETTING AND METHODS: Expression at the RNA level was analyzed by reverse transcription coupled to polymerase chain reaction. Western blot and immunolabeling coupled to confocal or electronic microscopy were used to analyze bovine protein expression and intracellular localization. For the functional approach, short-interfering RNA were microinjected into mature bovine oocytes, followed by IVF; cleavage and embryo development were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: The BCAR4 gene is conserved in mammalian species from various orders and has been lost in rodents after divergence with lagomorphs. The transcript is expressed in the oocytes of humans and domestic species. We bring the first experimental evidence of the BCAR4 protein in mammals. In cattle, the protein is not detected in immature oocytes but starts to be synthesized during maturation, increases in the zygote and persists until the morula stage. The protein is detected throughout the cytoplasm in mature oocytes, concentrates in and around the pronuclei in the zygote, and appears to shuttle in and out of the nuclei starting in the 2-cell embryo; BCAR4 is also present at the junctions between blastomeres from 2-cell to morula. In our functional approach, targeting the BCAR4 transcript by small-interfering RNA significantly compromised development to the morula or/and blastocyst stages (P < 0.05, logistic regression). LIMITATIONS, REASONS FOR CAUTION: As indicated above, protein expression and function were investigated in cattle and mostly in vitro matured oocytes were used. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a novel candidate gene whose mutation or deregulation may underlie certain cases of unexplained female infertility.

Can video cameras replace visual estrus detection in dairy cows?

A 6-mo experiment was conducted in a dairy herd to evaluate a video system for estrus detection. From October 2007 to April 2008, 35 dairy cows of three breeds that ranged in age from 2 to 6 yr were included in the study. Four daylight cameras were set up in two free stalls with straw litter and connected to a computer equipped with specific software to detect movement. This system allowed the continuous observation of the cows as well as video storage. An observation method related to the functionality of the video management software ("Camera-Icons" method) was used to detect the standing mount position and was compared to direct visual observation (direct visual method). Both methods were based on the visualization of standing mount position. A group of profile photos consisting of the full face, left side, right side, and back of each cow was used to identify animals on the videos. Milk progesterone profiles allowed the determination of ovulatory periods (reference method), and a total of 84 ovulatory periods were used. Data obtained by direct visual estrus detection were used as a control. Excluding the first postpartum ovulatory periods, the "Camera-Icons" method allowed the detection of 80% of the ovulatory periods versus 68.6% with the direct visual method (control) (P = 0.07). Consequently, the "Camera-Icons" method gave at least similar results to the direct visual method. When combining the two methods, the detection rate was 88.6%, which was significantly higher than the detection rate allowed by the direct visual method (P < 0.0005). Eight to 32 min (mean 20 min) were used daily to analyze stored images. When compared with the 40 min (four periods of 10 min) dedicated to the direct visual method, we conclude that the video survey system not only saved time but also can replace direct visual estrus detection.

Reduction of body-weight gain enhances in vitro embryo production in overfed superovulated dairy heifers.

The aim of our study was to test whether a reduction in dietary intake could improve in vitro embryo production in superovulated overfed dairy heifers. Cumulus-oocyte complexes of 16 Prim' Holstein heifers (14 +/- 1 months old) were collected by ovum pick-up (OPU), every 2 weeks following superovulation treatment with 250 microg FSH, before being matured and fertilized in vitro. Embryos were cultured in Synthetic Oviduct Fluid medium for 7 days. Heifers were fed with hay, soybean meal, barley, minerals and vitamins. From OPU 1 to 4 (period 1), all heifers received individually for 8 weeks a diet formulated for a 1000 g/day live-weight gain. From OPU 5 to 8 (period 2), the heifers were allocated to one of two diets (1000 or 600 g/day) for 8 weeks. Heifers' growth rates were monitored and plasma concentrations of metabolites, metabolic and reproductive hormones were measured each week. Mean live-weight gain observed during period 1 was 950 +/- 80 g/day (n = 16). In period 2 it was 730 +/- 70 (n = 8) and 1300 +/- 70 g/day (n = 8) for restricted and overfed groups respectively. When comparing period 1 and period 2 within groups, significant differences were found. In the restricted group, a higher blastocyst rate, greater proportions of grade 1-3 and grade 1 embryos, associated with higher estradiol at OPU and lower glucose and beta-hydroxybutyrate, were observed in period 2 compared with period 1. Moreover, after 6 weeks of dietary restriction (OPU 7), numbers of day 7 total embryos, blastocysts and grade 1-3 embryos had significantly increased. On the contrary, in the overfed group, we observed more <8 mm follicles 2 days before superovulation treatment, higher insulin and IGF-I and lower nonesterified fatty acids in period 2 compared with period 1 (no significant difference between periods for embryo production). After 6 weeks of 1300 g/day live-weight gain (OPU 7), embryo production began to decrease. Whatever the group, oocyte collection did not differ between period 1 and 2. These data suggest that following a period of overfeeding, a short-term dietary intake restriction (6 weeks in our study) may improve blastocyst production and embryo quality when they are low. However, nutritional recommendations aiming to optimize both follicular growth and embryonic development may be different.

Genetic and environmental factors influencing first service conception rate and late embryonic/foetal mortality in low fertility dairy herds.

The objective of this study was to identify factors affecting variation in conception rate to first artificial inseminations (AI) (CR: number of pregnant cows on D80-100/inseminated cows) and the incidence of embryonic/foetal loss (LEM) between 21 and 80 days of pregnancy (number of cows non-pregnant on D80-100/pregnant on D21) in 44 low fertility dairy herds of the west-central region of France. Reproductive status was assessed using progesterone milk concentration on D0 = Day of AI and D21-24, plasma PSPB concentration on D30-35, rectal palpation on D80-100 and observed return to oestrous. The final data set contained 1285 Prim'Holstein cows, 5.0% (64/1285) were inseminated in the luteal phase (progesterone > or = 3 ng/ml on D0), 61.3% (787/1285) were pregnant on D21-24 (progesterone < 3 ng/ml on D0 and > or = 5 ng/ml on D21-24), 15.4% lost their embryo/foetus between D21-24 and D80-100 (198/1285) and 45.8% (589/1285) were pregnant on D80-100. The incidence of late embryonic/foetal loss (LEM) was 25.2% (198/787). Multivariate logistic regression models including the random herd effect were used to analyse the relationship between AI centre, AI sire, cow's sire, parity, interval between calving and AI, milk production, milk protein content, body condition score (BCS) on D0, season of calving, season of AI, estimated genetic index on CR and LEM incidence. CR was significantly related to parity (p < 0.05), milk production after calving (p < 0.05) and estimated genetic value (p < 0.01). A significant difference in CR was observed for calving to AI interval > or = 70 days versus > or = 90 days, but the overall effect of the interval was not significant (p = 0.11). LEM incidence was affected by period of AI (p < 0.05), milk production (p < 0.05) and BCS (p < 0.05), but was not related to estimated genetic index. In conclusion, in these low fertility herds, the incidence of LEM was high and 25% of the cows lost their embryo after 21 days of pregnancy. LEM was affected by specific factors (season, BCS), which were not related to CR. The absence of a relationship between estimated genetic index and LEM in spite of its effect on CR indicates that estimated genetic merit has a greater effect on early embryonic loss or fertilisation failure than on later stages of embryo development.

[Insemination: history and state of the art in animals]. / L'insémination: historique, état des lieux chez l'animal.

Insemination has been used since 1950 in the bovine and since the seventies in other domestic species. The use of insemination varies between the different species in terms of activity and method. The methods depend on the anatomical and physiological characteristics of each species, which lead to different sites of semen deposition and different minimal numbers of inseminated spermatozoa to obtain efficient fertility results. The use of frozen semen influences also the methods of insemination. Intra-uterine insemination is principally used when only small quantities of semen are available and/or to reduce the number of inseminated spermatozoa. Recent results in different species indicate that the use of intra-uterine insemination could be developed in the next years.

Influence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle.

One of the major sources of success in embryo transfer is timing of AI relative to the LH surge and ovulation. The aim of this study was to compare the embryo production following superovulation during a PGF2alpha (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle). CIDR-B (CIDR-B ND, Virbac, Carros, France) was inserted on Day 11 of a previously synchronized cycle and left for 5 days. A total dose of 350 microg FSH was administered (eight injections i.m. for 4 days; first on Day 13, decreasing doses) and PGFalpha analog (750 microg i.m.: Uniandine ND, Schering-Plough, Levallois-Perret, France) injected at the time of third FSH injection. Artificial inseminations were performed 12 and 24 h after standing estrus (Day 0). Embryos were collected on Day 7. Luteinizing hormone was measured by EIA (Reprokit Sanofi, Libourne, France) from blood samples collected every 3 h for 36 h, starting 24 h after PGF2alpha (control cycle) or 12 h after CIDR-B removal (CIDR-B cycle). The effects of treatment group and interval between the LH peak and AI (two classes, < 10 and > or = 10 h) on embryo production and quality were analyzed by ANOVA. No effect of treatment was observed on embryo production variables. The intervals between the end of treatment and onset of estrus and between end of treatment and LH surge were greater in heifers treated during a control than a CIDR-B cycle, respectively (45.5 +/- 1.4 versus 31.9 +/- 0.7; 42.0 +/- 1.6 versus 31.0 +/- 1.5; P < 0.05), but maximal LH and estradiol concentrations, at the preovulatory surge were similar in control and CIDR-B synchronized heifers. The numbers of viable and Grade I embryos were significantly increased (P < 0.01) when animals had an interval from LH peak to first AI > or = 10 h (7.2 +/- 0.9 and 3.5 +/- 0.6) when compared to shorter intervals (4.2 +/- 1.1 and 2.0 +/- 0.7) whereas total number of embryos was unchanged (11.8 +/- 1.4 versus 10.3 +/- 1.8). It is concluded that late occurrence of LH peaks in relation to estrous behavior is associated with a lower embryo quality when first AIs are performed systematically 12 h after standing estrus. Further studies are needed to know if results may be improved when making AI at a later time after standing estrus or if LH assays are useful to better monitor AI time.

Risks of transmissible diseases in relation to embryo transfer.

Realizing the potential of Embryo transfer (ET) for rapid, cheap and widespread dissemination of genetic material, the risk of transmission disease through the embryos must be considered. The aim of this paper is to evaluate theses risks at each step of production, storage and transfer. The pathogen agent may potentially originate from the donor male (semen) or the donor female (oocytes, embryos) and finally from the environmental conditions. As the differences between in vivo and in vitro derived embryos have been well described, evaluation of the potential risks should be assessed separately for in vivo and in vitro produced embryos. Even if this paper insist on the diseases or diseases agents that are more questionable, it clearly appears that ET remains the more safety way to transfer gene, provided prevention measures are properly handled (use of donor that are specific pathogen free, washing of embryos, additional treatment...) and furthermore it can be easily seen as the best way to prevent some disease transmissions (TSEs, leukosis, foot-and-mouth disease...).

Acetyl- and pseudo-cholinesterase activities of plasma, erythrocytes, and whole blood in male beagle dogs using Ellman's assay.

Organophosphate and carbamate ester insecticides, main causes of pesticide poisoning, inhibit cholinesterase (ChE) enzymes. The aim of this study was to measure and compare baseline values for pseudocholinesterase and acetylcholinesterase (AChE) enzyme activities of different blood fractions in the dog to aid in diagnosis of anticholinesterase poisoning. After collecting blood samples from 23 6-24-mo-old male beagle dogs, Ellman's colorimetric assay was run on plasma, red blood cells (RBC), and whole blood fractions prepared in triplicate. The procedure described in a commercially available kit was applied to plasma and RBC. Hemolyzed whole blood fractions (final dilution 1:8) avoided the time-consuming and laborious separation of plasma and RBC. In addition to the kit substrate acetylthiocholine (ASCh), we used butyrylthiocholine (BSCh) as substrate. Whatever the substrate, ChE activity was lower in RBC than in other blood preparations. It was higher when using ASCh rather than BSCh as substrate (mean IU/L+/-SD): 563+/-144 and 303+/-45 respectively, in contrast to plasma (1640+/-310 and 2510+/-450). Whole blood enzyme activity did not differ significantly according to substrate: ASCh, 1590+/-190; BSCh, 1620+/-250) with a 2 to 3% within-day coefficient of variation. Enzyme activity was significantly lower in dogs <1-y old. This study confirms the low ChE activity in dog RBC compared to other species and other blood fractions. It shows that using whole blood instead of separating RBC from plasma minimizes the variability of ChE activity in the hemoglobin-rich fraction.

Influence of the type of energy supply on lh secretion, follicular growth and response to estrus synchronization treatment in feed-restricted suckler beef cows.

The present experiment aimed to compare the efficiency of supplementation (+17.5 MJ Net Energy/d starting 47 +/- 4 days after calving) with concentrate (CS, maize grain, n = 10) or with forage (FS, maize silage, n = 10) in estrus-synchronized (Norgestomet implant 10 days inserted 60 +/- 4 days postpartum + PMSG at implant removal) beef cows previously restricted (47 MJ Net Energy/d, 785 g CP/d, 70% of requirements). The type of diet had no significant effect on basal LH concentrations (CS: 0.18 +/- 0.12 vs FS: 0.11+/- 0.02 ng/mL), LH pulse frequency (CS : 0.7 +/- 0.3 vs FS: 0.8 +/- 0.2 pulse/10 h), LH pulse amplitude (CS: 0.55 +/- 0.50 vs FS : 0.62 +/- 0.50 ng/mL) or estradiol (E2) concentrations (CS: 3.3 +/- 0.8 vs FS: 4.6+ /- 0.8 pg/mL) 13 days after the beginning of energy supplementation. No differences between CS and FS cows were observed for the number of small, medium and large follicles nor on the size of the largest follicle from 11 days before implant insertion to implant removal (IR). After IR, an LH surge was observed in 2 of the CS and 4 of the FS cows. The type of energy supplementation had no significant effect on LH (CS: 0.16 +/- 0.06 ng/mL vs FS 0.48 +/- 0.06 ng/mL; P > 0.05) or on estradiol concentrations (CS : 7.8 +/- 0.2 vs FS : 8.9 +/- 0.2 pg/mL, P > 0.10) measured hourly from 29 to 49 h after IR. Cows that ovulated after IR tended to have higher E2 concentrations than cows that did not ovulate (9.4 +/- 0.2 vs 6.3 +/- 0.2 pg/mL, P = 0.08). Similar ovulation and pregnancy rates were observed in CS and FS cows (CS: 6/10 vs FS: 7/10 and CS: 6/10 vs FS: 5/10 respectively, P > 0.05). To conclude, energy supplementation with forage was as effective as energy supplementation with concentrate to influence follicular growth, ovulation and pregnancy percentage after estrus synchronization treatment in diet-restricted beef cows.