Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150 mu g D-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment I (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos. (C) 2009 Elsevier B.V. All rights reserved.

Factors affecting fertilisation and early embryo quality in single- and superovulated dairy cattle

Data on fertilisation and embryo quality in dairy cattle are presented and the main factors responsible for the low fertility of single-ovulating lactating cows and embryo yield in superovulated dairy cattle are highlighted. During the past 50 years, the fertility in high-producing lactating dairy cattle has decreased as milk production increased. Recent data show conception rates to first service to be approximately 32% in lactating cows, whereas in heifers it has remained above 50%. Fertilisation does not seem to be the principal factor responsible for the low fertility in single-ovulating cows, because it has remained above 80%. Conversely, early embryonic development is impaired in high-producing dairy cows, as observed by most embryonic losses occurring during the first week after fertilisation. However, in superovulated dairy cattle, although fertilisation failure is more pronounced, averaging approximately 45%, the percentage of fertilised embryos viable at 1 week is quite high (>70%). Among the multifactorial causes of low fertility in lactating dairy cows, high feed intake associated with low concentrations of circulating steroids may contribute substantially to reduced embryo quality. Fertilisation failure in superovulated cattle may be a consequence of inappropriate gamete transport due to hormonal imbalances.

Global poly(A) mRNA expression profile measured in individual bovine oocytes and cleavage embryos

The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus-oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p < 0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (I arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p < 0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Xenooplasmic Transfer between Buffalo and Bovine Enables Development of Homoplasmic Offspring

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.

Bovine oocyte vitrification: Effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24 h of INK respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 It of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-I or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification. (C) 2007 Elsevier B.V. All rights reserved.

Effects of flunixin meglumine, recombinant bovine somatotropin and/or human chorionic gonadotropin on pregnancy rates in Nelore cows

The objective was to compare pharmacological strategies aiming to inhibit prostaglandin F2 alpha (PGF(2 alpha)) synthesis (flunixin meglumine; FM), stimulate growth of the conceptus (recombinant bovine somatotropin; bST) and progesterone (P(4)) synthesis (human chorionic gonadotropin; hCG), as well as their combinations, regarding their ability to improve pregnancy rates in beef cattle. Lactating Nelore cows (N = 975), 35 to 70 days postpartum, were synchronized and inseminated by timed artificial insemination (TAT) on Day 0. On Day 7, cattle were allocated into eight groups and received one of the following treatments: saline (S) on Days 7 and 16 (Group Control); S on Day 7 and FM on Day 16 (Group FM); bST on Day 7 and S on Day 16 (Group bST); bST on Day 7 and FM on Day 16 (Group bST + FM); hCG on Day 7 and S on Day 16 (Group hCG); hCG on Day 7 and FM on Day 16 (Group hCG + FM); bST and hCG on Day 7 and S on Day 16 (Group bST + hCG), or bST and hCG on Day 7 and FM on Day 16 (Group bST + hCG + FM). The aforementioned treatments were administered at the following doses: 2.2 mg/kg FM (Banamine (R); Intervet Schering-Plough, Cotia, SP, Brazil), 500 mg bST (Boostin (R); Intervet Schering-Plough), and 2500 IU hCG (Chorulon (R); Intervet Schering-Plough). Pregnancy diagnosis was performed 40 days after TAI by transrectal ultrasonography. Pregnancy rates were not significantly different among treatments. However, there was a main effect of hCG treatment to increase pregnancy rates (63.0 vs. 55.4%; P = 0.001). Concentrations of P(4) did not differ significantly among groups on Day 7 or on Day 16. However, consistent with the higher pregnancy rates, hCG increased P(4) concentrations on Day 16 (10.6 vs. 9.6 ng/mL, respectively; P = 0.05). We concluded that hCG treatment 7 days after TAI improved pregnancy rates of lactating Nelore cows, possibly via a mechanism leading to induction of higher P(4) concentrations, or by reducing the luteolytic stimulus during maternal recognition of pregnancy. (C) 2011 Elsevier Inc. All rights reserved.

Short-term urea feeding decreases in vitro hatching of bovine blastocysts

Cows fed high-protein diets may have impaired reproductive performance. Although the pathogenesis has not been completely elucidated, it appears that not only the uterus, but also the follicle and oocyte, are affected by excessive plasma urea nitrogen (PUN) concentrations. Thus, the objective was to determine the effects of short-term urea feeding on the competence of bovine oocytes. Forty crossbred heifers (Bos indicus vs Bos taurus) were allocated to two groups, namely CONTROL (maintenance diet) and UREA (maintenance diet supplemented with 75 g of urea/day), following a cross-over design. Heifers received their respective diets for 6 d (without adaptation). On the sixth day, blood samples were harvested both before and 3 h after feeding, and cumulus oocyte complexes (COCs) were collected by ovum pick-up. Although PUN concentrations were higher in UREA than CONTROL heifers (31.31 mg/dL +/- 1.13 vs 22.12 mg/dL +/- 0.86; mean +/- SEM), neither the number of COCs recovered (8.8 +/- 1.0 vs 9.2 +/- 0.8, UREA vs CONTROL, respectively) nor their quality (based on morphology) differed significantly between groups. Next, oocytes were fertilized and cultured in vitro to assess developmental rates. There was an absence of significant differences between groups for rates of cleavage (Day 3) or blastocyst formation (Days 6, 7 and 9), but the hatched blastocyst rate on Day 11 after fertilization was lower (P < 0.05) in the UREA than the CONTROL groups (64.3 vs 83.5%). Therefore, we inferred that the effects of urea were only manifest later in development. In conclusion, high PUN concentrations decreased oocyte competence in heifers, reinforcing the hypothesis that poor reproductive performance in cows with high PUN was due, at least in part, to a deleterious effect on oocytes. (C) 2011 Elsevier Inc. All rights reserved.

Cell cycle and apoptosis in normal and cloned bovine near-term placentae

The bovine maternal epithelium is composed of cuboidal cells interspersed with low columnar cells having centrally located nuclei. Bovine trophoblast is composed of two cell types: mononuclear trophoblastic and giant trophoblastic cells that can have two or more nuclei. Number of apoptotic cells and proliferative cells are variable in both cell populations. This study compared tissue growth and apoptosis by flow cytometry in the cell population found at distinct placental regions (central region of placentomes, <= 1-cm microplacentomes and the interplacentomal region) between normal and cloned near-term bovine pregnancies. After a morphological comparison between regions and groups (controls vs. clones), a lesser proportion of diploid to tetraploid cells was observed in the central region of placentomes and in microplacentomes from cloned-derived pregnancies. In addition, cloned animals had a fewer apoptotic cells in the central region of the placentome and in interplacentomal region and a greater proliferative capacity in all regions (cells in G(2)/M) near term as opposed to control animals. These results may reveal the existence of a relationship between such changes in the proportions of uterine and trophoblastic epithelial cells at the end of pregnancy and normal placental function. This could be related to faulty placentation in early pregnancy, placental insufficiency during pregnancy or lack of placental and/or fetal maturation in late pregnancy, which may contribute to someof the abnormalities after in vitro embryo manipulations, such as poor preparation and initiation of parturition, prolonged gestation and lesser post-natal survival in some cloned animals. (C) 2008 Elsevier B.V. All rights reserved.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Proliferation of chick embryo neuroblasts grown in the presence of horse serum requires exogenous transferrin.

We have previously shown that neuroblasts from cerebral hemispheres of 6-day-old chick embryos are able to proliferate when grown in the presence of fetal calf serum. We report here that in the presence of horse serum alone the proliferative rate of neuroblasts is strongly reduced. A high proliferative rate is restored upon the addition of bovine transferrin and to a lesser extent with added FeSO4 or hemin. These findings suggest that the transferrin of horse serum cannot be used by chick neuroblasts in vitro, while bovine transferrin exogenously added is active in promoting cell proliferation. We propose that the stimulatory activity of the fetal calf serum is due to bovine transferrin, since when this serum is fractionated by gel filtration, the fractions that stimulate the proliferation of neuroblasts grown in the presence of horse serum are located in the molecular weight area of transferrin, and they do contain transferrin as seen by immunoblotting with a specific anti-transferrin antibody.

A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

Effect of supplementation of green tea polyphenols on the developmental competence of bovine oocytes in vitro

The objective of the present study was to examine the effect of green tea polyphenols (GTPs) supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5ºC for 24 h) and fertilized (38.5ºC for 15-18 h) and embryos were cultured (38.5ºC for 192 h) in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99% catechin derivatives, with the major components being 50% (-)-epigallocatechin gallate, 22% (-)-epicatechin gallate, 18% (-)-epigallocatechin, and 10% (-)-epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 µM) of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4%, P < 0.05). However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 µM) were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 µM) significantly reduced the rate of blastocyst formation (P < 0.05). Supplementation of the culture medium with 15 µM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 µM) significantly reduced embryo development (P < 0.05). In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 µM) during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.

The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3

Morphological and functional characterization of placenta during gestation in bovine clones derived by somatic nuclear transfer

La technique de clonage par transfert nucléaire de cellules somatiques (SCNT) présente une page importante dans les annales scientifiques, mais son application pratique demeure incertaine dû à son faible taux de succès. Les anomalies placentaires et de développement fœtal se traduisent par des pertes importantes de gestation et des mortalités néonatales. Dans un premier temps, la présente étude a caractérisé les changements morphologiques des membranes fœtales durant la gestation clonée en les comparant à des gestations contrôles obtenues à partir de l’insémination artificielle. Les différentes anomalies morphologiques des placentomes telles que l’œdème chorioallantoique, la présence de zones hyperéchoiques et irrégulières dans la membrane amniotique et la présence de cellules inflammatoires dégénérées compromettent le développement fœtal normal de la gestation clonée. L’examen ultrasonographique représente une technique diagnostique importante pour faire le suivi d’une gestation et de caractériser les changements placentaires dans le cadre d’évaluation globale du bien-être fœtal. Le profil hormonal de trois stéroïdes (progestérone (P4), estrone sulfate (E1S), et œstradiol (E2)) et de la protéine B spécifique de gestation (PSPB) dans le sérum des vaches porteuses de clones SCNT a été déterminé et associé aux anomalies de gestations clonées. Une diminution de la P4 sérique au jour 80, une élévation du niveau de la concentration de la PSPB au jour 150, et une augmentation de la concentration d’E2 sérique durant le deuxième et troisième tiers de la gestation clonée coïncident avec les anomalies de gestation déjà reportées. Ces changements du profil hormonal associés aux anomalies phénotypiques du placenta compromettent le déroulement normal de la gestation clonée et gênent le développement et le bien-être fœtal. Sur la base des observations faites sur le placenta de gestation clonée, le mécanisme moléculaire pouvant expliquer la disparition de l’épithélium du placenta (l’interface entre le tissue maternel et le placenta) a été étudié. L’étude a identifié des changements dans l’expression de deux protéines d’adhérence (E-cadhérin et β-catenin) de cellules épithéliales pouvant être associées aux anomalies du placenta chez les gestations clonées. Le tissu de cotylédons provenant de gestations clonées et contrôles a été analysé par Western blot, RT-PCR quantitatif, et par immunohistochimie. Les résultats présentaient une diminution significative (p<0.05) de l’expression des dites protéines dans les cellules trophoblastiques chez les gestations clonées. Le RT-PCR quantitatif démontrait que les gènes CCND1, CLDN1 et MSX1 ciblés par la voie de signalisation de la Wnt/β-catenin étaient significativement sous exprimés. La diminution de l’expression des protéines E-cadherin et β-catenin avec une réduction de l’activation de la protéine β-catenin durant le période d’attachement de l’embryon peut potentiellement expliquer l’absence totale ou partielle de l’attachement des membranes fœtales au tissu maternel et éventuellement, l’insuffisance placentaire caractéristique des gestations clonées chez la vache. La caractérisation morphologique et fonctionnelle du placenta durant les gestations clonées à haut risque est essentielle pour évaluer le statut de la gestation. Les résultats de la présente étude permettront de prédire le développement et le bien-être fœtal de façon critique à travers un protocole standardisé et permettre des interventions médicales pour améliorer le taux de succès des gestations clonées chez les bovins.