¿Por qué y para qué producir embriones bovinos in vitro?

Nuevo récord mundial de embriones bovinos producidos in vitro promueve un giro hacia el uso de la criopreservación asociada al incremento sostenido en la producción de estos embriones.EEA BalcarceFil: Ríos, Glenda. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Buschiazzo, Jorgelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina

Cholesterol and desmosterol incorporation into ram sperm membrane before cryopreservation: Effects on membrane biophysical properties and sperm quality

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-β-cyclodextrin (MβCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MβCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MβCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MβCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MβCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.Fil: Carro, Maria de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; ArgentinaFil: Peñalva, Daniel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Hozbor, Federico Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; ArgentinaFil: Buschiazzo, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; Argentin

Follicular waves and status of oocyte maturation: Detection of caspases as indicator of apoptosis in immature oocytes

Objetivos. Establecer la relación entre el número de folículos ≤14mm, los ovocitos VG (estadio de vesícula germinal) y la duración de la estimulación ovárica controlada (EOC). Determinar el estatus apoptótico de los ovocitos VG mediante la identificación de caspasas activas. Establecer la relación entre las oleadas foliculares, los ovocitos VG y su viabilidad según la prueba de caspasas. Diseño. Estudio transversal de cohortes, prospectivo. Materiales y métodos. Se incluyeron 27 pacientes en tratamiento de fertilidad de alta complejidad, de las cuales se obtuvieron 61 ovocitos VG. Análisis estadístico: ANOVA, regresión lineal y test Chi2 . Resultados. Un aumento del número de folículos ≤14mm se relacionó con un aumento de ovocitos VG recuperados. Cuando la duración de la estimulación ovárica fue ≤9 o ≥11 días, la recuperación de ovocitos VG fue mayor con respecto a 10 días. El incremento en el número de folículos de la 2ª oleada fue directamente proporcional al número de ovocitos VG. Se recuperó un número mayor de ovocitos VG de los folículos de la 2ª oleada folicular. El número de ovocitos negativos y positivos para caspasa no presentó diferencia significativa, sin embargo, se observó una tendencia mayor de ovocitos VG negativos para caspasas en los folículos de la 2ª oleada. Conclusión. La duración de la EOC influye significativamente en la recuperación de ovocitos VG, mostrando que EOC cortas o prolongadas incrementan la cantidad de ovocitos inmaduros recuperados, provenientes de diferentes oleadas. Teniendo en cuenta la necesidad de obtener una mayor cantidad de ovocitos viables, tanto de la 1ª como de la 2ª oleada, deberíamos planificar estrategias. Identificar las oleadas foliculares durante la EOC podría modificar la conducta y los resultados reproductivos.Objectives. To establish the relation between thenumber of follicles ≤14mm, the number of GV oocytes(stage of germinal vesicle) and the durationof COS. To determine the apoptotic status of GVoocytes, by identifying active caspases. To establishthe relation between follicular waves, GV oocytesand their viability according to the caspase test. Design.Cross sectional study of cohorts, prospective.Materials and methods. 61 GV oocytes obtainedfrom 27 patients included in high complexity fertilitytreatment. Statistical analysis: ANOVA, linearregression with Poisson or Binomial distribution.Chi2 test. Results. An increase in the number offollicles ≤14mm was related to an increase in therecovery of GV oocytes. When the duration of theovarian stimulation was ≤9 or ≥11 days, the recoveryof GV oocytes was higher with respect to 10days. A greater number of GV oocytes were recoveredfrom the follicles of the 2nd follicular wave. Agreater number of VG oocytes were recovered fromthe follicles of the 2nd follicular wave. The numberof negative and positive caspase oocytes did notpresent a significant difference, however, a greatertendency of negative VG oocytes was observed forcaspases in the follicles of the 2nd wave. Conclusion.The duration of the COS significantly influencedthe recovery of GV oocytes, showing that short COSor prolongation increases the number of immatureoocytes recovered coming from different wave.Taking into account the need to obtain a greaternumber of viable oocytes, both from the 1st and the2nd wave, we should plan strategies. Identifying thefollicular waves during the EOC could modify thebehavior and the reproductive results.Fil: Manchado, Carmen. Centro de Reproducción y Genética Humana.; ArgentinaFil: Casco, Agustina. Centro de Reproducción y Genética Humana.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Casanova, Pía. Centro de Reproducción y Genética Humana.; ArgentinaFil: Buschiazzo, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; ArgentinaFil: Pené, Alicia. Centro de Reproducción y Genética Humana.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentin

Desmosterol Incorporation Into Ram Sperm Membrane Before Cryopreservation Improves in vitro and in vivo Fertility

Pregnancy rates in ewes are markedly low after cervical insemination with frozen-thawed sperm. Sensitivity of ram sperm to freeze-thawing is related to the lipid composition of the membrane, particularly to its low sterol content. Recently, we proved that sterol content of ram sperm can be increased by treatment with methyl-β-cyclodextrin-sterol complexes and we provided mechanistic based evidence on the differential behavior of cholesterol and desmosterol in the ram sperm membrane. In the present study, we evaluated the role of increasing cholesterol and desmosterol content of ram sperm before cryopreservation, on the extent and distribution of sterols, cryocapacitation status, acrosome integrity, DNA damage associated with apoptosis and fertility competence in vitro and in vivo of post-thawed sperm. After freeze-thawing, similar levels of sterol content were evidenced in control sperm cells and in those pre-incubated with either cholesterol or desmosterol. Still, moderately higher levels of sterols were registered in treated sperm compared to the control, indicating no physiological excess of sterols after thawing or sterol losses that exceed the control. Live cell imaging of fluorescent cholesterol evidenced the presence of sperm sub-populations differentially affected by freeze-thawing. Similar unimodal frequency profiles were observed between sterol-enriched groups, while the control exhibited a sub-population of sperm compatible with low sterol content. Tyrosine phosphorylation was significantly lower when ram sperm incorporated cholesterol compared to the control. No difference in this capacitation parameter was found between the latter and desmosterol-enriched sperm. The percentage of sperm with damaged acrosomes post-thawing, assessed by a fluorescent lectin, was reduced in sperm that incorporated sterols before freezing, irrespective of the sterol class. These results suggest that sterols exert a stabilizing effect on the acrosome. No differences were found in levels of apoptotic DNA fragmentation among experimental groups. As to fertility trials, desmosterol-enriched sperm gave rise to higher rates of in vitro activated oocytes by heterologous fertilization and to significantly lower pregnancy loss in vivo. Our research provides new insights on sterol incorporation into ram sperm prior to cryopreservation, in particular on the additional benefit of incorporating desmosterol as a strategy to improve fertility outcome.Fil: Carro, Maria de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; ArgentinaFil: Ramírez Vasquez, Rafael Ramón Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; ArgentinaFil: Peñalva, Daniel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Buschiazzo, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Hozbor, Federico Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; Argentin

Effect of meiotic maturation on yolk platelet lipids from Bufo arenarum oocytes

Progesterone induces the resumption of meiosis in Bufo arenarum full-grown arrested oocytes through a nongenomic mechanism called meiotic maturation. Growing evidence indicates that lipids are involved in the maturation process. They are mainly located in yolk platelets, the principal organelles of amphibian oocytes. The aim of the present study was to analyze the effect of progesterone-induced maturation on lipids from B. arenarum yolk platelets. Ovarian oocytes, manually obtained, were incubated with progesterone to induce maturation. Yolk platelets were isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derivatized by methanolysis, and were identified and quantified in a gas–liquid chromatograph. Phospholipid content decreased in progesterone-treated oocytes, mainly as a result of a decrease at the level of phosphatidylcholine (PC). The turnover of this lipid is considered crucial for the completion of meiosis. Sphingomyelin also underwent a decrease that could be related to the important role of ceramide as an inducer of germinal vesicle breakdown. Maturation effect on fatty acid composition registered significant changes in PC whose saturated fatty acids increased. A net increase in arachidonic acid was observed in phosphatidylserine after progesterone treatment. The contents of total triacylglycerols and diacylglycerols were not significantly modified by hormone effect while free fatty acids underwent a significant increase as a result of polyunsaturated fatty acids increase. Altogether, our results demonstrate that yolk platelet lipids are involved in the resumption of the meiotic cell cycle, thus suggesting that these organelles participate in a dynamic role during amphibian developmentFil: Buschiazzo, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Alonso, Telma Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

1502-15 MATEMÁTICA Cónicas

Material de estudio para alumnos de 5º año - Área Matemática

1503-14 MATEMATICA Conicas

Material de estudio para alumnos de 4º año Construcciones, Área Matemática

Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol homeostasis within the COC. Modulation of membrane cholesterol by MβCD improved survival of bovine oocytes and preserved integrity of GM1-related rafts after vitrification

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

Linoleic acid stimulates neutral lipid accumulation in lipid droplets of maturing bovine oocytes

Linoleic acid (LA) is a polyunsaturated fatty acid present in high concentrations in bovine follicular fluid; when added to maturation culture media, it affects oocyte competence (depending on the type and concentration of LA used). To date, little is known about the effective level of incorporation of LA and there is apparently no information regarding its esterification into various lipid fractions of the oocyte and its effect on neutral lipid storage. Therefore, the objective was to assess the uptake and subcellular lipid distribution of LA by analyzing incorporation of radiolabeled LA into oocyte polar and neutral lipid classes. The effects of various concentrations of LA on the nuclear status and cytoplasmic lipid content of bovine oocytes matured in vitro was also analyzed, with particular emphasis on intermediate concentrations of LA. Neutral lipids stored in lipid droplets were quantified with a fluorescence approach. Linoleic acid at 9 and 43 μM did not affect the nuclear status of oocytes matured in vitro, and 100 μM LA inhibited germinal vesicle breakdown, resulting in a higher percentage of oocytes arrested at the germinal state (43.5 vs. 3.0 in controls; P < 0.05). Bovine oocytes actively incorporated LA from the maturation medium (83.4 pmol LA per 100 oocytes at 22 hours of incubation; P < 0.05) and metabolized it mainly into major lipid classes, e.g., triacylglycerols and phospholipids (61.1% and 29.3%, respectively). Supplementation of the maturation medium with LA increased triacylglycerol accumulation in cytoplasmic lipid droplets at all concentrations assayed (P < 0.05). In conclusion, LA added to a defined maturation medium at concentrations that did not alter the nuclear status of bovine oocytes matured in vitro (9 and 43 μM) improved their quality by increasing the content of neutral lipids stored in lipid droplets. By directing the free fatty acid (LA) to triacylglycerol synthesis pathways and increasing the degree of unsaturation of membrane phospholipids, the oocyte was protected from lipotoxic effects (with an expectation of improved cryotolerance).EEA BalcarceFil: Carro, María De Las Mercedes. 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: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Oresti, Gerardo Martin. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca; ArgentinaFil: Alberio, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentin