¿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

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

Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4) Strains Isolated from In Vitro Bovine Embryos production in Argentina

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as “starting material” for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.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: Manrique, Julieta Marina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perez, Sandra. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Odeón, Anselmo Carlos. 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: Jones, Leandro Roberto. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Verna, Andrea Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

Vaginal mucus from ewes treated with progestogen sponges affects quality of ram spermatozoa

The use of intravaginal sponges (IS) to synchronize estrous onset in ewes provokes vaginitis, an increase in the vaginal bacterial load, and growth of bacterial species that are not present during spontaneous estrous behavior. The objective of the study was to compare the functional sperm parameters after incubating it with mucus collected from the vagina of ewes during spontaneous estrus or estrous synchronized with IS. Pooled spermatozoa were co-incubated with: (1) vaginal mucus collected from ewes in spontaneous estrus; (2) vaginal mucus collected from ewes in estrus pretreated with progestogen-impregnated IS; (3) synthetic mucus; and (4) medium without mucus as a control group. Sperm samples were evaluated after incubating it for 30 and 90 minutes. The number of colony-forming units (CFUs/mL), pH, and osmolality were greater in the mucus collected from ewes treated with IS than from those untreated (P ¼ 0.046; P < 0.0001, and P < 0.0001, respectively). The percentage of sperm with progressive motility was lower after incubation with vaginal mucus collected from estrous ewes treated with IS than in the other three treatments both, 30 and 90 minutes after incubation (P ¼ 0.0009 and P < 0.0001, respectively). The sample incubated for 30 minutes with mucus from ewes treated with IS had a lower percentage of sperm with intact plasma membrane than all the other treatments (P < 0.0001). The percentage of sperm with functional membrane was significantly lower in the sample incubated for 30 minutes with vaginal mucus from ewes treated with IS than in the other three treatments (P < 0.0001). After 90 minutes, the percentage was still lower than that in the sample collected from ewes during their spontaneous estrus (P ¼ 0.0005). The lowest percentages of sperm with acrosome damage were observed in sperm incubated with mucus collected from sheep in spontaneous estrus for 30 and 90 minutes (P < 0.0001 and P ¼ 0.008, respectively). The percentage of apoptotic spermatozoa was greater in samples incubated during 30 minutes with vaginal mucus collected from ewes treated with IS than in the other three groups (P ¼ 0.0005). The functionality and the viability of ram sperm is negatively affected by the cervical mucus of ewes pretreated with progestagen-impregnated IS used in estrous synchronization treatments. This may partially explain the decrease in conception rate obtained with treatments with IS.EEA BalcarceFil: Manes, Jorgelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentina. Universidad de la República. Facultad de Veterinaria. Departamento de Fisiología; UruguayFil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Fiorentino, María Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Laboratorio de Bacteriología; ArgentinaFil: Ungerfeld, Rodolfo. Universidad de la República. Facultad de Veterinaria. Departamento de Fisiología; Urugua

Exogenous progesterone during in vitro fertilization improves developmental competence of partially cumulus-denuded bovine oocytes

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a four-hour capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases.Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis,which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P<0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 µM). Cleavage rate significantly increased at 1 µM P4, which translated to increased total embryo production after 7 days of in vitro culture (P<0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 µM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.Fil: Suqueli García, María Florencia. 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; ArgentinaFil: Gabbanelli, Nadia. 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; ArgentinaFil: Rios, Glenda Laura. 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; 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; 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

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

Effect of gaseous atmosphere on different supplementation medium for In Vitro canine oocyte maturation

The aim of the present research was to verify the influence of two different atmosphere gases (A: 5%CO2, B: 5%CO2, 5%O2 and 90%N2) on three different maturation media on in vitro maturation (IVM) rates for oocytes from anoestrous and diestrous bitches that were cultured in vitro for 48h. Oocytes harvested from bitches were selected and allocated into three maturation media groups, cultured in atmosphere A or B. Group 1, oestrous bitch serum; Group 2, bovine serum albumin (BSA), glutamine, cisteamine; Group 3, BSA, estrogen (E2), epidermal growth factor (EGF). Progression of nuclear maturation was evaluated under fluorescent microscopy after staining with Hoechst 33342 solution. The results demonstrated that different maturation media was not influenced by the atmosphere in any case. Group 1 showed the lowest maturation rates in both cases: 4.3±1.3 vs 7.3±2.1 and 7.9±2.5 for groups 1, 2 and 3 respectively in atmosphere A (p≤ 0.5), 0 vs 7.2± 2.9 and 9.3± 4.0 in atmosphere B (p= 0.33). Maturation rates were similar among treatments 2 and 3 in both atmospheres, were slightly higher for treatment 3. Moreover, group 1 obtained the highest degeneration rates: 65.5±7.3 vs 39.63±7.1, 28.19±6.4 (p= 0.018) and 71.43±12.2 vs 27.71±10, 25.33± 10.20 (p= 0.010) for mediums 1, 2, and 3 in atmospheres A and B respectively. Our findings indicate that atmosphere B seems to be more favorable habitat for canine oocytes and independently of gassed, maturation with not controlled supplementation, with estrous bitch serum, is not a good option.EEA BalcarceFil: González, Noelia. Universidad de Zaragoza. Facultad de Veterinaria. Reproduction and Obstetric Unit; EspañaFil: Gil, Lydia. Universidad de Zaragoza. Facultad de Veterinaria. Reproduction and Obstetric Unit; EspañaFil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: Canizo, Jésica Romina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; ArgentinaFil: De Blas, José Ignacio. Universidad de Zaragoza. Facultad de Veterinaria. Infectious Diseases Unit; EspañaFil: Alberio, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentin

Effect of Seminal Plasma on Post‐Thaw Quality and Functionality of Corriedale Ram Sperm Obtained by Electroejaculation and Artificial Vagina

We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post‐thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.EEA BalcarceFil: 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; ArgentinaFil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Cesari, Andreina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentina.Fil: Alberio, Ricardo. 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; Argentin