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An abstract of the study "Development Stage of Bovine Blastocysts and the Addition of Leukemia Inhibitory Factor (LIF) and Basic Fibroblast Growth Factor (bFGF) During Culture in the Establishment of Embryonic Stem Cells-Like Colonies," by M. D. Guastali and colleagues is presented.

An abstract of the study "Global Gene Expression Patterns of Fresh and Vitrified In Vitro-Produced Bovine Blastocysts," by M. J. Sudano et al, is presented.

The increased storage of lipid granules in in vitro-produced (IVP) bovine embryos seems to be related to the presence and concentration of fetal calf serum (FCS) during culture. The presence of high concentration of lipids on embryos reduces their viability after cryopreservation, which has been one of the main obstacles for the success of vitrification of IVP bovine embryos (Moore et al.2007 Theriogenology 68, 1316-1325). The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma-Aldrich, St. Louis, MO, USA), which raises the levels of intracellular cAMP (Seamon et al.1981 Proc. Natl. Acad. Sci. USA, 78, 3363-3367). Nelore oocytes were matured in TCM-199 + 10% FCS, FSH, and LH in 5% CO2in air atmosphere, at 38.5°C. After 24 h of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions. Presumptive zygotes were cultured in 2 concentrations of FCS: Control 0% (SOFaa + 5 mg mL-1BSA; basic medium, BM), and Control 2.5% (BM supplemented with 2.5% FCS). On Day 6 of culture embryos were divided into 2 additional treatments: Forskolin 0% (BM + 10 μM forskolin; and Forskolin 2.5% (BM supplemented with 2.5% FCS and 10 μM forskolin). All embryos were cultured in a 5% CO2, 5%O2, and 90% N2atmosphere at 38.5°C for 7 days, when blastocyst formation rate was evaluated. Embryo viability was also checked by staining the embryos with Hoechst 33342 and propidium iodide. Data were analyzed by ANOVA followed by Tukey''s test, using a 5% significance level. No statistical differences were observed among treatments on cleavage rates, evaluated on Day 3 of culture, or on blastocyst formation rates. Although no statistical differences was observed between treatments on percentage of viable cells, embryos cultured with 0% FCS, independently of the presence of forskolin, presented significantly more damaged cells than embryos cultured with 2.5% FCS (P< 0.05). The results indicate that the presence of FCS is important to reduce degeneration of blastomeres during culture. Moreover, the presence of forskolin on Day 6 of culture did not influence embryo development, indicating that this drug could be a good alternative to reduce embryo lipid content in bovine IVP embryos produced in presence of FCS. Table 1.Effect of fetal calf serum and forskolin on embryo cultureAcknowledgments: FAPESP 07/53505-1. [ABSTRACT FROM AUTHOR]

An abstract of the study "Evaluation of Apoptosis in In Vitro-Produced Bovine Embryos Matured With Forskolin," by D. M. Paschoal and colleagues is presented.

An abstract of the study "Effect of Meiotic Arrest Using Butyrolactone I and Roscovitine on In Vitro Production of Bovine Embryos," by R. R. D. Maziero and colleagues is presented.

An abstract of the study "Comparison of the Expression of Sox2 and Stat3 in Bovine Blastocyst and Hatched Blastocyst," by T. S. Rascado et al. is presented.

An abstract of the study "An Easy-to-Perform Method to Assess Viability of Feline Freeze-Dried Sperm," by L. C. O. Magalhães et al, is presented.

Several factors exert considerable influence on oocyte production and quality, such as donor age, nutrition, season and others. However, very few studies have been done to verify the effects of breed on the quantity or quality of oocytes, or both. In Brazil, the Nellore breed has been developed as a result of many years of selective breeding from 6 major ancestor bulls imported from India in the 1960s. These animals have characterised their respective lineages, as evidenced by the well-defined genetic traits. This study was carried out to investigate the influence of the genetic lineage of oocyte donors on the number and quality of oocytes obtained through ultrasound-guided follicular aspiration in Nellore cows derived from 2 lineages of bulls [Karvadi (K) and Taj Mahal (T)]. Both maternal (Km and Tm) and paternal (Kp and Tp) lineages were investigated. Oocyte aspirations were repeatedly performed with an aspiration interval of 15 days without the superovulation procedure in 56 donor females over a period of 2 years. At the beginning of the experiment, the animals were on average 78 ± 12 months old with a body condition score of 3.6 ± 0.08 (on a 5-point scale). Recovered cumulus-oocyte complexes were counted, morphologically examined and classified into 7 categories (grades I to VII) according to the number of layers of the cumulus-oocyte and the appearance of the cytoplasm. Data were analysed using ANOVA and Tukey's test after transformation into log(x + 1). Values are reported as means ± s.e.m. and differences (P > 0.05) were taken as significant. The mean number of oocytes retrieved from donors of lineage Tp-Tm was higher (n = 9; 28.23 ± 1.92; P < 0.05) than that obtained for lineages Kp-Tm (n = 11; 21.34 ± 1.32), Kp-Km (n = 15; 21.28 ± 1.73) and Tp-Km (n = 21; 16.72 ± 1.31). There was no significant difference in the mean number of recovered oocytes between donors of lineages Kp-Tm and Kp-Km; however, lineage Tp-Km yielded a lower number of oocytes. Donors of lineage Kp yielded more (P < 0.05) oocytes of grade III than lineage Tp (10.11 ± 0.66 vs 8.79 ± 0.58, respectively). These lineages produced a higher mean number of oocytes graded as I, II and III (3.14 ± 0.21, 4.93 ± 0.33 and 10.11 ± 0.66 vs 3.19 ± 0.21, 5.59 ± 0.44 and 8.79 ± 0.58, respectively) than any other categories. However, more oocytes (P < 0.05) of grades I, II and III were obtained for lineage Tm (3.64 ± 0.25, 5.9 ± 0.42 and 11.67 ± 0.67) than for lineage Km (2.85 ± 0.19, 4.92 ± 0.40 and 7.73 ± 0.56, respectively), with similar results for oocytes of grades IV, V, VI and VII. In conclusion, the genetic lineage of the oocyte donor influenced the production and quality of oocytes obtained through ultrasound-guided follicular aspiration in Nellore cows. Selection of donor cows based on their oocyte morphology and availability can further improve IFV programs. [ABSTRACT FROM AUTHOR]

The high concentration of lipids on embryos reduces their viability after cryopreservation. The use of drugs to modify their metabolism has been used to produce embryos with greater resistance to cryopreservation. The present experiment aimed to induce cytoplasmic lipolysis in in vitroproduced (IVP) bovine embryos using forskolin (Sigma-Aldrich, St. Louis), which raises the levels of intracellular cAMP. Nelore cow cumulus–oocyte complexes (COC) were matured in TCM 199 and fertilized with frozen Nelore bull semen. Presumptive zygotes were cultured in SOFaa supplemented with BSA in the presence of 2.5% FCS. Embryos were kept in a humidified atmosphere with 5% CO2, 5% O2, and 90% N2at 38.5°C. On Day 6, embryos were divided into 2 groups: 2.5% FCS (2.5% FCS without forskolin) and 2.5% FCS+F (2.5% FCS plus 10μM forskolin). Embryo cleavage was recorded on Day 3 (IVF: D0), and blastocyst production on Day 7. Embryo viability was estimated by the index of total number of cells per embryos observed after the staining with propidium iodite and Hoechst 33342. In vitroproduced embryos were compared with embryos obtained in vivofrom Nelore cows. Embryos were vitrified using the protocol developed by Campos-Chillòn et al.(2006). After thawing, the re-expansion rate and cell number were again estimated (after 12h). For statistical analysis, percentage cleaved and percentage blastocyst, percentage re-expansion, and total number of cells were transformed using the arcsine transformation (√y/100) and analysed using ANOVA followed by Tukey’s test. The level of significance adopted was 5%. No statistical differences were observed between IVP embryos concerning cleavage (2.5% FCS: 87.48±9.52 and 2.5% FCS+F: 85.13±7.57) and blastocyst formation rates (2.5% FCS: 46.8±13.28 and 2.5% FCS+F: 46.31±11.39). Also, no differences were detected in total number of cells per embryos (2.5% FCS: 162.4±43.3; 2.5% FCS+F: 147.6±35.3 and in vivo: 143.5±11.5) when IVP and in vivoproduced embryos were compared. After vitrification the re-expansion rate was similar between IVP and in vivoproduced embryos (2.5% FCS: 75.07±9.81; 2.5% FCS+F: 81.09±10.90 and in vivo: 86.40±18.62). But the total cell number of IVP embryos was significantly lower than the in vivoproduced embryos [2.5% FCS: 42.6±17.2a(P<0.001); 2.5% FCS+F: 59.9±46.2a(P<0.05) and in vivo: 124.2±12.9b]. If one side the viability cell between IVP and in vivoproduced embryos is the same, after vitrification in vivoproduced embryos showed greater resistance after the vitrification. The addition of forskolin did not interfere in embryo production or quality, but this drug was not efficient in increasing embryo tolerance after vitrification. FAPESP 07/53505-1. [ABSTRACT FROM AUTHOR]

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitroproduced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N=4320) were matured and fertilized in vitro(Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3μM PES from Day 4 (after 96h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N=60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivocontrol (n=15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means±SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7±1.7, 82.3±1.6, 86.3±1.4, 87.0±1.5, P>0.05) and augmented blastocyst production (30.5±2.5a, 41.8±2.4b, 40.5±2.6b, 47.2±2.8b, P0.05) cleavage (87.0±1.3 and 84.4±1.3), blastocyst production (42.0±2.8 and 43.0±2.0), and apoptosis in blastocysts (20.7±2.0band 18.9±2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivocontrol group presented the lowest apoptosis rate (6.3±1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality. São Paulo Research Foundation – FAPESP. [ABSTRACT FROM AUTHOR]

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm), BL-I (50 μm) and association of drugs (ROS 6.25 μm and BL-I 25 μm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL-I and ROS+BL-I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post-thawing embryos. Embryos from ROS+BL-I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process., (© 2016 Blackwell Verlag GmbH.)