RNA-seq analysis of single bovine blastocysts

Background: Use of RNA-Seq presents unique benefits in terms of gene expression analysis because of its wide dynamic range and ability to identify functional sequence variants. This technology provides the opportunity to assay the developing embryo, but the paucity of biological material available from individual embryos has made this a challenging prospect. Results: We report here the first application of RNA-Seq for the analysis of individual blastocyst gene expression, SNP detection, and characterization of allele specific expression (ASE). RNA was extracted from single bovine blastocysts (n = 5), amplified, and analyzed using high-throughput sequencing. Approximately 38 million sequencing reads were generated per embryo and 9,489 known bovine genes were found to be expressed, with a high correlation of expression levels between samples (r > 0.97). Transcriptomic data was analyzed to identify SNP in expressed genes, and individual SNP were examined to characterize allele specific expression. Expressed biallelic SNP variants with allelic imbalances were observed in 473 SNP, where one allele represented between 65-95% of a variant’s transcripts. Conclusions: This study represents the first application of RNA-seq technology in single bovine embryos allowing a representation of the embryonic transcriptome and the analysis of transcript sequence variation to describe specific allele expression.EEA BalcarceFil: Chitwood, James L. University of California Davis. Department of Animal Science; Estados UnidosFil: Rincon, Gonzalo. University of California Davis. Department of Animal Science; Estados UnidosFil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Medrano, Juan F. University of California Davis. Department of Animal Science; Estados UnidosFil: Ross, Pablo J. University of California Davis. Department of Animal Science; Estados Unido

El rol de las biotécnicas de reproducción asistida en la transmisión del virus de la diarrea viral bovina

Las biotécnicas de reproducción asistida han adquirido importancia comercial en los últimos años. Asociado a estas técnicas surge el interrogante sobre su rol en la posible transmisión de agentes infecciosos vía semen y ovocitos utilizados como material de partida. La elevada prevalencia y capacidad de causar infecciones reproductivas del virus de la Diarrea Viral Bovina (vDVB) lo ha convertido en un problema potencial en la producción in vitro de embriones. La presente revisión aborda los antecedentes previos sobre la temática y muestra resultados propios sobre las vías de infección del virus de la Diarrea Viral Bovina (vDVB) asociadas a las técnicas de producción in vitro (PIV) y su impacto en la reproducción bovina. Los resultados obtenidos demuestran alteraciones en el desarrollo folicular ovárico asociadas a infecciones persistentes por vDVB. Estas alteraciones fueron reflejadas en la baja eficiencia obtenida en la fecundación in vitro (FIV) como resultado de la interacción temprana entre el virus y la línea germinal del ovario. Por otra parte, se determinó que el vDVB-no citopático (vDVB-ncp) puede atravesar la zona pelúcida de ovocitos bovinos infectados al inicio de la etapa de maduración in vitro (MIV). Este hallazgo pone en evidencia la importancia de controles sanitarios en los sistemas de producción in vitro basados en la comprensión de los riesgos de transmisión del virus a partir de semen y ovocitos. Asimismo, la asociación del vDVB con gametas fue demostrada cuando la FIV se realizó tanto con ovocitos como con semen de animales persistentemente infectados (PI), donde se observó una disminución en las tasas de división y desarrollo de embriones. La información presentada en este artículo de revisión aporta al conocimiento sobre las implicancias de infecciones por vDVB en los sistemas de PIV y su efecto en el desarrollo embrionario, como así también al impacto de la transmisión de la infección en el ganado bovino mediante técnicas de reproducción asistida.Biotechnical of assisted reproduction have become commercially important in recent years. Associated with these techniques the question about his possible role in the transmission of infectious agents via semen and oocytes used as starting material arises. The high prevalence and ability to cause reproductive infections of the bovine viral diarrhea virus (BVDV) has become a potential problem in the in vitro production embryos. This update addresses the previous data on the subject and shows own results on the ways of infection with bovine viral diarrhea (BVDV) associated with in vitro production techniques and their impact on the reproduction bovine. The results demonstrate changes in the ovarian follicular development associated with persistent BVDV infection. These changes were reflected in the low efficiency obtained in the in vitro fertilization as a result of early interaction between the virus and ovarian germ line. Moreover, it was determined that noncytopathic BVDV can cross the zona pellucida of bovine oocytes infected at the beginning of the stage of in vitro maturation. This finding highlights the importance of to understand fully the risks of transmission of virus via semen and oocytes and appropriate quality assurances are used in in vitro production embryos systems (IVP). Likewise, the association of BVDV was demonstrated with gametes when IVF was performed with both types from animals infected persistent, where a decrease was observed in cleavage rates and embryo development. The information obtained in this review article contributes to knowledge about the implications of BVDV infections in IVP systems and its effect on embryonic development, as well as the impact of the transmission of infection in cattle by breeding techniques assisted.EEA BalcarceFil: Gonzalez Altamiranda, Erika Analia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Odeon, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Combined epidermal growth factor and hyaluronic acid supplementation of in vitro maturation medium and its impact on bovine oocyte proteome and competence

The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus–oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus–oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 μg/mL HA, and 100-μM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P < 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P < 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P < 0.05) with respect to EGF-HA–matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive zygotes at Day 7 developed into the different stages with similar rates (∼4%) independently of the medium condition. Modifications of IVM medium composition markedly affected protein profile of bovine oocytes in a differential manner. The proteomic approach revealed the presence of 68 spots in both treatments, 41 exclusively found in the FSH-FBS group and 64 exclusive for the EGF-HA group. Taken together, these results indicate that combined EGF-HA supplementation of in vitro maturation medium could be used to improve oocyte meiotic competence and ensure a better timing to develop into the blastocyst stage.EEA BalcarceFil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Buschiazzo, Jorgelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentina. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca; ArgentinaFil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Alberio, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentin

Interaction of bovine viral diarrhea virus with bovine cumulus–oocyte complex during IVM: Detection in permissive cells

Structural changes in the zona pellucida (ZP) of bovine oocytes seem to modulate their interaction with various viral agents, facilitating the viral infection in in vitro production systems. To evaluate the susceptibility of bovine oocytes to noncytopathogenic bovine viral diarrhea virus (ncp-BVDV), cumulus–oocyte complexes were exposed to 107 ​tissue culture-infective doses (TCID50)/mL of an ncp-BVDV strain during IVM (in vitro maturation). After that, cumulus cells and the ZP were removed by hyaluronidase and pronase treatment, respectively, and the percentages of oocytes with polar body were analyzed as a sign of nuclear maturation. After passage through cell culture, the virus was isolated from granulosa cells, ZP-free mature oocytes, and ZP-intact mature oocytes. These results were confirmed by reverse transcription–polymerase chain reaction. After consecutive washes, the virus remained associated with ZP-free oocytes, maintaining its replication and infectivity in permissive cells. Based on these findings, it is concluded that the classical viral isolation procedure has a predictive value to detect BVDV associated with ZP-free oocytes and that it was novelty demonstrated that both washing and trypsin treatment of oocytes were ineffective to remove BVDV infection.EEA BalcarceFil: Gonzalez Altamiranda, Erika Analia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

Changes in the aerobic vaginal flora after treatment with different intravaginal devices in ewes

The objective of this study was to characterize the vaginal bacterial flora and subsequent pregnancy rates after AI in sheep subjected to 4 different estrous synchronization regimes – the use of two intravaginal devices (silicone or polyurethane sponges), and two different treatment periods (7 or 14 days). Twenty-one multiparous Texel ewes were randomly allocated to 4 treatment groups during the breeding season. In the ewes from Group I (n = 6) and Group II (n = 5), a polyurethane sponge containing 60 mg MAP was inserted in the vagina for a period of 7 or 14 days, respectively. In the ewes of Group III (n = 5) and Group IV (n = 5), an intravaginal progesterone releasing insert (IVP4) containing 160 mg of progesterone in an inert silicone device, was inserted for 7 or 14 days, respectively. At device withdrawal, each ewe was treated with 200 IU eCG i.m. Standard bacteriological procedures were performed on vaginal mucus samples obtained before the introduction of the devices, at device withdrawal and on the day of AI in all groups. Estrus was recorded with the aid of vasectomized rams every 12 h, and AI was performed 52–54 h after device withdrawal, using fresh semen. The intervals between device withdrawal and estrus were: Group I: 56.4 ± 21.5 h; Group II: 42.0 ± 33.9 h; Group III: 51.6 ± 21.5 h; Group IV: 37.2 ± 10.7 h, while the pregnancy rates were: Group I: 83.3%; Group II: 60.0%; Group III: 60.0%; Group IV: 60.0%. The pregnancy rates and the interval between device withdrawal and the occurrence of estrus did not differ between treatments. The predominant bacterial flora population at device insertion was mostly gram positive (G+) (90%) bacteria. The strains most frequently found were Bacilllus sp., Staphylococcus sp. and Corynebacterium sp. Of the 19 isolates made at device removal, 79% were gram negative, with the Escherichia sp. being the most frequently isolated. At the time of AI and regardless of the device used, the 14-day treatment group presented an initial gram positive bacterial flora, while the 7-day groups presented gram negative flora (82%). It could be concluded that the use of intravaginal devices, regardless of their composition (silicone or polyurethane), may generate changes in the normal vaginal bacterial flora of the vaginal mucus. These changes did not reflect on the subsequent fertility. The use of intravaginal devices should however include the adoption of strict hygiene procedures, to minimize the growth of bacterial flora.EEA BalcarceFil: Manes, Jorgelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentina.Fil: Fiorentino, María Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Laboratorio de Bacteriología; ArgentinaFil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Alberio, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Sánchez, Esteban O. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentina.Fil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Laboratorio de Bacteriología; Argentin

Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART)

Improve intra‐uterine insemination in rabbits using ultra‐high temperature skim milk as extender to keep semen at room temperature

Two experiments were carried out to examine in vitro quality and in vivo fertility of rabbit semen diluted in ultra‐high temperature (UHT) skim milk. In the first experiment, pooled ejaculates of 10 adult rabbits were divided in three aliquots. Each aliquot was diluted in saline solution, TrisC or UHTm extender and kept at room temperature for 24 h. Sperm quality assessment was performed during all the incubation periods. In the second experiment, 27 adult rabbit does were inseminated with semen incubated for 5 h. Embryo recovery was performed 96 h after insemination. Results showed that treatments diluted in UHTm registered the highest values of spermatozoon with total motility, intact and functional plasma membrane and greater number of embryos recovered in rabbit does. We conclude that UHT skim milk would be a good extender for improved intra‐uterine insemination in rabbits and to keep sperm cells for several hours at room temperature.EEA BalcarceFil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Ledesma, Alba. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Manes, Jorgelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentina.Fil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Cano, Adriana Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentina.Fil: Luciano, Cecilia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Paraná. Unidad Cunícola; ArgentinaFil: Alberio, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentin

<i>In vivo</i> embryo production.

<p><i>In vivo</i> embryo production.</p