SOF Medium Research Articles

139 BOVINE PREIMPLANTATION EMBRYOS SECRETE EXTRACELLULAR VESICLES, WHICH MAY INDICATE EMBRYO COMPETENCE

Pre-implantation embryos secrete extracellular vesicles (EV) most likely to communicate with the surroundings. The objective of this study was to determine the distribution (size and concentration) of EV secreted by bovine pre-implantation embryos with different developmental competence. The IVF bovine embryos were produced from oocytes recovered from slaughterhouse ovaries. Presumptive zygotes were in vitro cultured (IVC) in groups in 4-well plates (30 zygotes per 500-µL well) using SOFaa medium at 39°C under 5% CO2, 5% O2, and 90% N2 until the morula stage (Day 5 post IVF). Morulae were cultured individually in 96 well at 39°C under until blastulation time (Day 6.5–7.5) in EV-free SOF medium. Culture medium was collected only from embryos that developed to the blastocyst stage that were classified in a group of early (Day 6.5) or late (Day 7.5) blastulation. Blastocysts were kept in culture until Day 11 to assess embryo developmental competence, considering embryo size (>350 µm) and total cell count (>500 blastomeres). For EV analysis, 4 groups were organised a posteriori: G1: Day 6.5-competent; G2: Day 6.5-not competent; G3: Day 7.5-competent; G4: Day 7.5-not competent. The EV in culture media were analysed using a nanoparticle tracking analysis (Nanosight NS300). Statistical analysis was performed using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Early blastulation rate (Day 6.5) was 40.3% (112/278), whereas late blastulation rate (Day 7.5) was 20.5% (57/278), showing a significant difference (P < 0.0001). Embryos derived from Day 6.5 blastocysts have a higher probability (39.3%: 44/112) of posthatching development [until Day 11; Day 7.5, 10.5% (6/57); P = 0.0001]. At Day 11, competent embryos (G1) derived from Day 6.5 blastocysts have a higher diameter and total cell number (447 µm; 688 cells) than those derived from Day 7.5 blastocysts (G3; 405 µm, 598 cells; P < 0.05 for both parameters). It was possible to detect EV from collected medium of individual embryos independent of their competence. Neither the EV size nor the EV concentration was statistically different between Day 6.5 and Day 7.5 blastocysts (without considering their further competence; 2.9 × 108, 147 nm; and 3.0 × 108, 149 nm, respectively). However, independent of the day of blastulation, competent embryos had a significantly lower concentration of EV (2.7 × 108 v. 3.3 × 108; P = 0.03). Moreover, competent embryos from early and late blastocysts (G1 and G3) tend to produce a lower amount of EV (G1: 2.8 × 108; G2: 3 × 108; G3: 2.6 × 108; G4: 3.5 × 108; P = 0.05). Furthermore, EV concentration was statistically different between G3 and G4 (P = 0.002). No differences in EV size were observed among groups (G1: 145 nm; G2: 148 nm; G3: 146 nm; G4: 151 nm). Our results provide an initial approach to study the EV secreted by individual pre-implantation embryos to assess their competence. From these results, we can conclude that blastulation time affects the future development of bovine embryos and a model based on blastulation time and EV secretion could be a simple noninvasive tool to improve embryo selection.

45 SURVIVAL OF HOLSTEIN IN VITRO-PRODUCED EMBRYOS CULTURED IN NOVEL SYNTHETIC OVIDUCTAL FLUID MEDIA (SCF1) AND DEHYDRATED PRIOR TO CRYOPRESERVATION

In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 M sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 M ethylene glycol and 0.5 M sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4′6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µM step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 M sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P < 0.01). A 20-min equilibration time decreased apoptosis (P < 0.01) and tended to increase post-thaw survival (P < 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1.Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

Correlation between the cryosurvival, cell number and diameter in bovine in vitro produced embryos

The selection of quality embryos is a prerequisite of cryopreservation process. Present study was conducted to examine the correlation between the diameter and cryotolerance, on the cell number of the cryopreserved embryos. The blastocyst stage embryos were collected at culture days 7–9, evaluated morphologically under a microscope and divided according to the diameter into three groups: Group 1; (larger than 150 μm), Group 2; (diameter of 100–150 μm), Group 3; (smaller than 100 μm). Blastocysts were vitrified-thawed using the classical vitrification method and then cultured in SOF medium drops at 24 h. Blastocysts were considered viable if they re-expanded or hatched from the zona pellucidae. Finally re-expanded blastocysts from the Group 1 and Group 2 to determine the differential count of cells in the ICM and TE. The re-expansion ability of blastocysts 100–150 μm in diameter (69.56%) was significantly higher than other groups (52.17 and 47.36%). The value of the correlation coefficient between the re-expansion rate and cell number of blastocysts in the group 2 (r = 0.784) tended to be higher than that in the group 1 (r = 0.512) and group 3 (r = 0.491) (p < 0.05). For ICM/total cell ratio yield group 2 embryos showed higher rate (0.28), compared to the other groups (0.19 and 0.16). In conclusion, the present study demonstrates that the correlation between diameter of embryos and their cryosurvival based on re-expansion ability and cell allocation.

Effect of Culture Media on Mare Oocytes Maturation

432 mare oocytes were collected from ovaries of slaughtered animals and cultured in three mediums supplimented with hormons (FSH, LH) and nutritive substances and were cultivated for 24 hours in three media, the highest cultivation rate in three media was obatined in TCM-199 medium 53.33% (80 oocytes), followed by SOF medium, maturation rate was 39.65% (58 oocytes), but the less maturation rate (34.54%) was observed in the medium composed of TCM+ 5% SOF + 5% FCS + 1% glutamine + 1% A.A + 500 I¼l antibiotic). Oocytes were selected and classified in two quality categories (mature, degenerated).

Comparison of Culture Media for in vitro Maturation of Oocytes of Indigenous Zebu Cows in Bangladesh

The objectives of the present study were to select an effective basic medium including its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The ovaries of cows were collected from slaughter house and the follicular fluid was aspirated from 2 to 8 mm diameter follicles. The COCs with more than 3 cumulus cell layers and homogenous cytoplasm were selected for maturation. The oocytes were matured in media for 24 hrs at 39℃ with 5% CO2 in humidified air. The maturation of oocytes was evaluated by examining the presence of first polar body under microscope. An efficient basic medium was determined after culturing COCs in either TCM 199 or SOF medium in Experiment 1. An efficient hormone supplementation was determined after culturing COCs in either FSH or gonadotrophin supplemented TCM 199 in Experiment 2. An efficient protein supplementation was determined after culturing COCs in either FBS or Oestrous cow serum (OCS) supplemented TCM 199 in Experiment 3. The oocyte recovery rate per ovary was 3.35. The overall rate of IVM was 74.6%. The maturation rate was 75.5±3.9 and 62.2±20.2% in TCM and SOF medium, respectively (P>0.05). The maturation rate of oocytes was significantly higher (76.6±13.2%) in FSH supplemented medium than gonadotrphin supplemented counterpart (69.7±10.8%) (P 0.05). In conclusions, both TCM 199 and SOF supplemented with either FBS or OCS, and FSH may be used as medium for IVM of indigenous zebu oocytes in Bangladesh.

18 EMBRYO AGGREGATION IN PIG IMPROVES CLONING EFFICIENCY AND EMBRYO QUALITY

In this study, we analysed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst size and cell number, DNA fragmentation levels by TUNEL assay, and the relative expression of genes associated with pluripotency, apoptosis, trophoblast markers, and DNA methylation in the porcine. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% polyvinyl alcohol) by a DC pulse of 1.2 kVcm for 80 μs. Then, embryos were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of zona-free embryos was achieved in a well of wells system in 100 μL of SOF medium. Two experimental groups were used, one control group with a single reconstructed embryo per microwell (1×) and the other group placing 3 reconstructed embryo per microwell (3x aggregation group). Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality. Our results showed that aggregation of 3× embryos increased blastocyst formation rate and blastocyst size of pig cloned embryos (Fisher’s test P &lt; 0.05 and Student’s t-test P &lt; 0.05, respectively). The DNA fragmentation levels in 3× aggregated cloned blastocysts were significantly decreased compared to 1x blastocyst (Student’s t-test P &lt; 0.05). Levels of Oct4, Klf4, Igf2, Bax, and Dnmt1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially nondetectable (Student’s t-test P &lt; 0.05). Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

92 INSULIN-LIKE GROWTH FACTOR-1 PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS PRODUCED IN VITRO FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

The objective of this study was to evaluate the protective effect of insulin-like growth factor (IGF-1) on blastocyst development and cryotolerance of bovine embryos in in vitro culture (IVC) under oxidative stress induced by menadione (MD). Cumulus-oocyte complexes (n = 1421) were matured in TCM-199 with bicarbonate, hormones, and 10% FCS for 22 h. After fertilization, the presumptive zygotes were cultured up to 7 days in SOF medium with 2.5% FCS and 0.5% BSA (control), and also supplemented with 100 μM IGF-1 (IGF). At Day 6, MD was included in the culture medium (0 μM, control; or 5.0 μM, MD) during 24 h. Cultures were conducted at 38.5°C in 5% CO2 in air. The cleavage and blastocysts rates were evaluated, respectively, at Days 3 and 7 (IVF = Day 0). At Day 7, a sample of the blastocysts was stained with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the intracellular ROS levels or was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA). Stained embryos were immediately evaluated under an epifluorescence microscope (excitation 495/550 nm and emission 404/590 nm, respectively, for ROS and TUNEL), and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. Other blastocysts were vitrified (Ingámed®, Maringá-PR, Brazil), and after warming, they were cultured for 24 h to evaluate the re-expansion rates. The results were compared by ANOVA followed by Student’s t-test (mean ± s.e.M) and re-expansion rates by chi-square test (P &lt; 0.05). The cleavage rates did not differ (P &gt; 0.05) among groups (77.1 ± 1.9% to 82.75 ± 2.2%). The blastocyst rates were similar between control (35.4 ± 2.0%) and IGF (34.5 ± 3.7%), and both were higher (P &lt; 0.05) than MD (21.3 ± 2.7%); the IGF+MD group (28.3 ± 1.6%) was similar (P &gt; 0.05) to all groups. The intracellular levels of ROS were higher (P &lt; 0.05) for the MD group (21.7 ± 0.7) than for control (17.0 ± 1.6), and both were similar (P &gt; 0.05) to the IGF (19.2 ± 0.6) and IGF+MD (18.0 ± 1.0) groups. The highest rates of apoptosis were found in the MD group (22.3% ± 2.3) and the smallest in IGF (9.1% ± 0.7), and both differed (P &lt; 0.05) from control (12.8% ± 1.0), and IGF+MD (15.6% ± 1.6). The re-expansion rates were similar between control (77.4%) and IGF (69.2%), and both were higher (P &lt; 0.05) than MD (49.1%); however, the IGF+MD group (57.6%) was similar (P &gt; 0.05) to IGF and MD groups. In conclusion, the supplementation with IGF-1 during IVC reversed the detrimental effects of MD on embryonic levels of ROS and apoptosis, as well as improved the embryo development and cryotolerance of blastocysts under oxidative stress. Financial support was provided by FAPESP (#2012/10083–8 and #2013/07382–6).

173 IN VITRO EMBRYO PRODUCTION FROM OOCYTES FERTILISED WITH UNSORTED OR X-SORTED SPERM AND ISSUED FROM SUBFERTILE HIGH GENETIC MERIT COWS SUBMITTED OR NOT TO A 48-h COASTING PERIOD FOLLOWING FSH STIMULATION

High genetic merit cows may be sidelined from breeding schemes because of reproductive disorders. In vitro production of embryos (IVP) issued from ovum pickup (OPU) can be an alternative to bypass infertility problems as experienced in humans and thus accelerate genetic progress (Duszewska et al. 2012 J. Anim. Feed Sci. 21, 217–233). The aim of this work was to evaluate if IVP from high genetic merit subfertile cows could benefit to the breeding scheme under commercial conditions, at the Biotechnology MIDATEST Station located in Denguin, Southwest, France. Holstein cows (n = 16) from 3.5 to 13 years old with different reproductive pathologic problems (repeated breeding, failure in in vivo embryo production, embryo mortality, permanent cysts, oviduct infection) were used in an OPU-IVP program. Donor cows were stimulated with decreasing doses pFSH twice daily during 3 days (Stimufol®, total dose: 350 µg of pFSH). The OPU was performed 48 h after the last FSH injection in the “coasting” group v. 12 h after the last FSH injection for “no-coasting” group and in vitro matured using a standard IVM protocol. Oocytes were fertilised with frozen-thawed unsorted or X-sorted sperm in TALP medium from different bulls (n = 42) without any previous IVP testing. Presumptive zygotes were cultured in SOF medium (Minitub®) plus 1% cow serum up to Day 7 at 38.5°C in 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity. The OPU-IVP was repeated 1 to 6 times (3.75 ± 2.6) for each donor cow. Grade 1 blastocysts and expanded blastocysts (according to IETS classification) were recorded on Days 6.5 and 7. Embryo production was analysed with ANOVA and blastocyst yield was analysed by chi-square. The results in both coasting or no-coasting groups and the effect of fertilization using unsorted or X-sorted sperm are presented in Table 1. The embryonic development rate was significantly higher when using unsorted semen to fertilize the oocytes compared to X-sorted sperm (P &lt; 0.05). On the other hand the coasting period had no significant effect neither on the number of collected oocytes nor on the embryonic development rates. In conclusion, our work confirmed the efficacy of OPU-IVP techniques to produce grade 1 embryos using X-sorted or unsorted sperm in subfertile high genetic merit cows. Table 1.Oocyte collection and in vitro embryo production, in donor cows submitted or not to a 48-h coasting period and effect of fertilization with unsorted v. X-sorted sperm

88 ENRICHMENT OF CULTURE MEDIUM WITH CROCETIN IMPROVES IN VITRO EMBRYO DEVELOPMENT IN CATTLE

The high incidence of developmental failure of bovine in vitro-produced embryos is due to suboptimal culture conditions that induce oxidative stress. Indeed, increased oxidative stress is one of the main factors affecting in vitro mammalian embryo development, decreasing the viability of IVP embryos. It is known that saffron has a powerful antioxidant capacity, mainly due to its active components crocin and crocetin. The aim of this study was to evaluate whether enriching the in vitro culture medium with crocetin improves in vitro embryo production efficiency in cattle. The range of concentrations of crocetin was chosen after a preliminary dose response trial (322 total presumptive zygotes were cultured with 0, 1, 10, and 50 μM, over 2 replicates) that showed beneficial and deleterious effects, respectively, with the lowest and highest concentration compared with the control (36.6 ± 5.6, 57.4 ± 4.5, 46.4 ± 4.4, and 6.8 ± 3.7% blastocyst rates, respectively, with 0, 1, 10, and 50 μM; P &lt; 0.01). Therefore, the range of concentrations to test was reduced. Abattoir-derived bovine oocytes (n = 832, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 0 (control; n = 208), 1 μM (n = 208), 2.5 μM (n = 208), and 5 μM (n = 208), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson and Nelson 2010, Manual of the IETS, 86–105). The percentages of total transferable embryos and grade 1 and 2 blastocysts were recorded. As the chronology of development is a reliable parameter to assess quality, the percentage of fast-developing embryos (i.e. hatched and expanded blastocysts) was also compared among groups. Differences among groups were analysed by ANOVA, and Tukey method was used as a post-hoc test. Data are presented as means ± s.d. The supplementation of crocetin during culture did not affect cleavage rate (74.9 ± 6.3, 76.4 ± 8.4, 81.4 ± 4.3, and 76.4 ± 8.4%, respectively, with 0, 1, 2.5, and 5 μM). However, post-fertilization embryo development improved with 1 µM crocetin compared with the control, both in terms of total embryo output (43.8 ± 4.4, 61.1 ± 5.2, 50.4 ± 6.7, and 53.3 ± 7.3%, respectively, with 0, 1, 2.5, and 5 μM; P &lt; 0.01) and grade 1 and 2 blastocysts (41.0 ± 3.6, 54.3 ± 5.4, 46.2 ± 6.7, and 49.4 ± 6.5%, respectively, with 0, 1, 2.5, and 5 μM; P &lt; 0.05), whereas no differences were observed among the other groups. Moreover, the percentage of fast developing embryos increased with 1 µM (P &lt; 0.05) crocetin compared with the control, with no other differences recorded among groups (17.7 ± 5.8, 34.7 ± 5.7, 24.9 ± 5.1, and 28.7 ± 7.8%, respectively, with 0, 1, 2.5, and 5 μM). In conclusion, these results demonstrated a beneficial effect of low concentrations of crocetin (1 μM) during culture both on blastocyst yield and quality, as indicated by the improved chronology of embryo development.

217 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO DEVELOPMENT IN BOVINE USING HIGH CYSTEAMINE CONCENTRATION DURING IN VITRO MATURATION AND SPERM CO-CULTURE WITH CUMULUS-OOCYTE COMPLEXES

In bovine, the intracytoplasmic sperm injection (ICSI) technique remains inefficient probably because of low levels of male sperm decondensation. In species with frequent fertilization failure, high cysteamine (Cys) concentration during in vitro maturation (IVM) has been used to improve IVF. Cysteamine, a precursor of glutathione, plays a critical role on sperm decondensation. The aim of this work was to improve ICSI efficiency in bovine by (1) increasing endogenous glutathione levels from oocytes using high Cys during IVM; and (2) incubating sperm with cumulus-oocyte complexes (COC) before ICSI, to mimic the physiological capacitation process. In experiment 1, we tested the effect of high Cys concentrations during IVM over the development of IVF embryos. In experiment 2, we performed ICSI after IVM with 1 mM Cys, based on IVF results. The COC were collected from slaughtered cow ovaries and IVM for 21 h with 10, 1, and 0.5 mM Cys v. 0.1 mM Cys (standard condition). Then, IVF was performed using 16 × 106 sperm mL–1 for 5 h on BO medium. For ICSI, COC were IVM with 1 mM Cys (ICSI 1 mM groups), and sperm used for injection was previously incubated with COC for 3 h (Inc. groups), as was done for IVF. Sham and diploid parthenogenetic (PA Diplo) controls were also included. Metaphase II oocytes were selected for ICSI, and injected oocytes were activated by a 4-min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h (except for PA Diplo) and treated with 2 mM DMAP for 3 h. For ICSI control groups, COC were matured using 0.1 mM Cys. All embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post-IVF/ICSI, respectively. The total cell numbers of blastocysts were counted at Day 7, after Hoechst 33342 staining. Results are shown in Table 1. In conclusion, an increase of 5- to 10-fold of Cys concentration during IVM was not detrimental for development to blastocyst after IVF. The use of 1 mM Cys during IVM combined with the use of sperm co-cultured wit IVM COC before sperm injection is a good strategy to improve in vitro development of bovine ICSI embryos. Table 1.Effect of 1 mM cysteamine (Cys) during IVM over the development of IVF bovine embryos (top part) and effect of 1 mM Cys during IVM over embryo development of ICSI embryos, using sperm previously incubated (Inc.) with COC (bottom part)

188 HAPLOID ACTIVATION OF BOVINE OOCYTES WITH IONOMYCIN AND SINGLE OR COMBINED ACTIVATING AGENTS

Haploid activation of bovine oocytes is important for reproductive technologies such as intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT). Nevertheless, it is still a highly inefficient procedure. The aim of this work was to combine different activation drugs, known to have different targets along the activation cascade, to find a more effective activation protocol. Cumulus-oocyte complexes (COC) were aspirated from slaughtered ovaries and in vitro-matured (IVM) for 22 h. Oocytes were activated with 5 µM ionomycin (IO) for 4 min and then randomly allocated into 1 of the following treatments: 50 µM roscovitine (ROSC), 10 µg mL–1 cycloheximide (CHX), ROSC and 10 µM PD0325901 (ROSC/PD), or CHX and PD (CHX/PD) for 5 h; 15 µM dehydroleucodine (DHL) or DHL and ROSC (DHL/ROSC) for 3 h; DHL and CHX for 3 h followed by 2 h with CHX; 5-min exposure to 7% ethanol 4 h post-IO (ET); or ET followed by ROSC (ET-ROSC). Controls were IO followed by 3 h of exposure to 1.9 mM 6-DMAP with or without a previous 3-h culture in TCM-199 (3 h in DMAP and DMAP, respectively). Embryos were cultured in SOF medium. Pronuclear formation (PN) and second polar body extrusion (2PB) were assessed by 5 µg mL–1 propidium iodide oocyte staining, 17 h after IO. Activation was defined as the presence of at least 1 PN, and 2PB extrusion rate was calculated regardless of the nuclear stage. Data were analysed by Fisher’s Test (P &lt; 0.05). Activation (Table 1) was similar in all groups, with the exception of ROSC/PD and ET-ROSC that were the highest and DHL that was the lowest. Although ROSC or CHX seemed to improve 2PB rate when combined with DHL, cleavage decreased significantly, suggesting DHL itself, or its combination with these drugs, negatively affects embryo development. Group ET showed activation rates comparable to other treatments, but it was not reflected on cleavage, suggesting that ET induces PN formation but it might be inefficient to trigger embryo development. Nevertheless, this observation was not made for ET-ROSC, as it showed a higher cleavage rate than ET and ROSC alone. The mitogen-activated protein kinase (MAPK) inhibitor PD showed different effects when combined with ROSC or CHX, despite that they both act on the mammary fat pad (MPF). In ROSC/PD, a slight improvement was observed on activation and cleavage rates compared with ROSC. Group CHX/PD resulted in a slightly higher 2PB percentage, but a lower activation percentage that derived in a significantly lower cleavage than CHX. In conclusion, ROSC and CHX were the most effective single treatments for haploid activation. Moreover, some combined treatments, namely DHL/ROSC and DHL/CHX, proved to be as effective or better at 2PB extrusion rate, which is the defining feature in haploid activation. Table 1.Activation, second polar body extrusion (2PB) and cleavage of bovine oocytes activated with ionomycin followed by single or combined activating agents1

177 PERFORMANCE OF Gyr PREPUBERTAL HEIFERS IN IN VITRO EMBRYO PRODUCTION

Bovine in vitro embryo production is highly relevant for dairy systems in Brazil, and Gyr dams are commonly used as oocyte donors. The aim of this study was to evaluate the use of prepubertal Gyr heifers as oocyte donors, an alternative to anticipate reproduction of those animals. For that, 11 Gyr [4 prepubertal (PP) donors and 7 adult cows © donors] were used in ovum pickup (OPU) sessions. The PP cows presented an average of 282.5 kg and 26.75 months, and had never displayed oestrous. Non-lactating cows presenting an average of 492 kg and 136 months were selected for C. Five replicates were performed, totaling 27 OPU sessions (C-17, PP-10) and 2–3 sessions per animal. Follicular wave was synchronised by aspiration of follicles larger than 8 mm 96 h before OPU. Cumulus-oocyte complexes (COC) were classified accordingly to their quality in viable (G1, G2, and G3) or non-viable (G4). Viable oocytes were matured and fertilized, and the presumptive zygotes were cultured in SOF medium at 38.5°C and 5% CO2 in air. Cleavage rate was assessed 48 to 72 h post-insemination (hpi) and blastocyst rate at 168 hpi. Mean number of structures was analysed by t-test, and percentage of viable, G1, G2, G3, G4, cleavage, and blastocyst rates were compared among groups by Fisher’s exact test (GraphPadInstat, La Jolla, CA, USA; P = 0.05). Results are followed by standard error values. All procedures were approved by a local ethics committee. We found that despite higher (P &lt; 0.05) numbers for both viable oocytes (PP: 15 ± 2.6; C: 6.11 ± 0.76) and total oocytes (PP: 23.70 ± 2.83; C: 8.82 ± 1.19) in the PP group, the rate of viable oocytes was similar (P &gt; 0.05) among PP and C groups (PP: 61.5 ± 6.51%, C: 66.79 ± 3.79%). Mean numbers of G1, G2, G3, and G4 oocytes were higher (P &lt; 0.05) in the PP group (G1 = 7.1 ± 1.18; G2 = 4.9 ± 1.74; G3 = 3.9 ± 1.09; G4 = 7.8 ± 1.38) than in the C group (G1 = 2.70 ± 0.740; G2 = 2.47 ± 0.44; G3 = 1.11 ± 0.31; G4 = 2.52 ± 0.39). However, the proportion was similar (P &gt; 0.05) among PP and C groups (PP: G1 = 29.5 ± 4.21%; G2 = 19.5 ± 2.85%; G3 = 15.9 ± 13.5%; G4 = 35.1 ± 6.33%; and C: G1 = 27.24 ± 4.44%; G2 = 29.60 ± 5.08%; G3 = 12.34 ± 3.01%, G4 = 30.79 ± 4.93%). Cleavage rate (PP: 91.3 ± 17.94%; C: 74.09 ± 4.65%), mean blastocyst number per OPU session (PP: 3.3 ± 1.29; C: 1.76 ± 0.28), and blastocyst rate (PP: 19.74 ± 7.40%; C: 27.03% ± 4.07%) were similar (P &gt; 0.05) among groups. We conclude that prepubertal heifers presented increased numbers of viable oocytes per OPU session, but blastocyst yield was similar to adult cows. This data suggests that prepubertal Gyr heifers can be used as oocyte donors. Support from FAPERJ and Embrapa is acknowledged.

246 INFLUENCE OF BREED AND SEASON ON IN VITRO EMBRYO PRODUCTION

A retrospective analysis of in vitro production (IVP) data was done to determine the influence of breed and season on the production of viable oocytes and embryos. Cumulus‐oocyte complexes (COC) were obtained from 1946 ultrasound-guided follicle aspiration (ovum pickup) sessions performed at random stages of the oestrous cycle without superstimulation in Bos taurus and Bos indicus donors in commercial IVP in Argentina. Frozen-thawed conventional semen was used in beef cattle and conventional (n = 139) and sexed-selected (n = 481) semen in dairy cattle. The COC were classified, matured in B-199 medium, fertilized in IVF-SOF medium (Day 0), and cultured in SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, and saturated humidity for 7 days. The number of viable COC and transferable embryos in each breed and season were compared by ANOVA and means were compared by Fisher’s Least Significant Difference test. Proportions were first transformed by arcsin and then analysed by ANOVA. To simplify the interpretation of the results, breeds were grouped as follows: dairy Bos taurus (Holstein, n = 620), beef Bos taurus (Angus and Bonsmara, n = 229), Bos taurus × Bos indicus (Brangus and Braford, n = 1045), and Bos indicus (Brahman, n = 52). There was no interaction between breed and season for any of the end points analysed (P &gt; 0.1). Mean (± standard error of the mean) numbers of viable COC and transferable embryos were higher (P &lt; 0.01) in Bos indicus × Bos taurus (19.3 ± 0.4 and 5.3 ± 0.2, respectively) and Bos indicus (15.8 ± 1.4 and 6.8 ± 0.9, respectively) than in beef (11.6 ± 0.5 and 3.0 ± 0.2, respectively) and dairy (8.0 ± 0.2 and 1.6 ± 0.1, respectively) Bos taurus donors. Cleavage rates were higher (P &lt; 0.01) in Bos indicus (72%) than in the other breeds (57% for Bos indicus × Bos taurus and dairy Bos taurus and 54% for beef). Transferable embryo rates were higher (P &lt; 0.01) in Bos indicus (41%) and Bos indicus × Bos taurus (30%) than in beef Bos taurus (26%). Dairy Bos taurus had the lowest (P &lt; 0.01) embryo rates of all breeds (21%). In dairy Bos taurus, cleavage rates, the number of embryos produced, and transferable embryo production rates were higher (P &lt; 0.01) when conventional semen was used (62%, 2.8 ± 0.15, and 27%, respectively) compared to sexed-selected semen (55%, 1.3 ± 0.1, and 19%, respectively). With regards to season, the number of viable COC was highest (P &lt; 0.01) in the spring (14.3 ± 0.5), lowest in the summer (11.3 ± 1.0), and intermediate in the fall (12.2 ± 1.2) and winter (13.7 ± 1.2), which did not differ. Although not affected significantly by season, the number of embryos produced was numerically lower in the summer (2.8 ± 0.4) than in the spring (4.2 ± 0.2), winter (4.5 ± 0.5), or fall (4.6 ± 0.5). In conclusion, in vitro embryo production was directly influenced by breed and season. Bos indicus influenced cattle and the spring season were preferable for commercial IVP programs that did not include superstimulation.

Corrigendum to: 131 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED 4-DAY-OLD CATTLE EMBRYOS: INFLUENCE OF THE OOCYTE MATURATION MEDIA

Chromosomal abnormalities were related to embryo developmental failures in cattle, being highly increased in IVF-produced compared with multiple-ovulation embryo transfer-produced embryos. The origin of this difference remains unclear, but they were related to the suboptimal environmental culture laboratory conditions. We studied the influence of 3 different maturation media in the appearance of chromosomal abnormalities in early in vitro-produced embryos. A total of 562 oocytes classified as Class A were collected by follicular aspiration in slaughterhouse ovaries and matured by 20 h in 3 different culture media supplemented with 10% FCS and antibiotics as follows: TCM-199 (T, 193 oocytes); DME (D, 178 oocytes); and RPMI-1640 (R, 191 oocytes). Matured oocytes were fertilized by 16 h with 1 × 106 spz mL–1 in IVF-TALP media and cultured by 72 h in SOF media. Later, 48 h after fertilization, 562 presumptive embryos were evaluated showing a normal cleavage rate of 64.7, 55.2, and 54.9% in T, D, and R groups, respectively. After culture, only 181 early embryos (90 hpi) showing normal development were correctly karyotyped following our standard laboratory techniques. Individual blastomeres were stained with Giemsa and assessed by direct observation at ×1250 magnification in a brightfield microscope. The percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Only embryos showing almost 2 different blastomeres correctly karyotyped were included in this study. Cleavage rates were statistically higher (P &lt; 0.05) in the oocytes matured in T media in comparison with the oocytes matured in D or R media. The percentage of diploid embryos were almost equals in the 3 groups evaluated (Table 1). There was some variation when each kind of chromosomal abnormality was assessed individually, but no statistical differences were observed. These results are in consonance with our previous studies in which we demonstrated the maturation time and morphological quality are the 2 main oocyte-derived factors influencing the ploidy of early embryos. It was also demonstrated that chromosomal complements were affected, in a much lesser extent, by the maturation media supplementation. However, in the present study, the maturation media did not statistically affected embryo ploidy. However, the higher percentage of cleaved embryos using TCM-199 observed agree with fact that this maturation media is one of the most widely used in IVF procedures in cattle. Based on our data, we suggest that oocyte maturation, a well established technique in cattle, could be performed using different maturation media without expecting major differences in the embryo ploidy, and therefore, the differences observed in cleavage rate must be originated in other physiological factors. Table 1.Results

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS

Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P &lt; 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P &lt; 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (&gt;200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.

124 CRYOPRESERVED BOVINE OVIDUCTAL EPITHELIAL CELLS SURVIVAL, GROWTH, AND ABILITY TO SUPPORT EARLY EMBRYO DEVELOPMENT

Previous experiments demonstrated that co-culture of bovine embryos with bovine oviducal epithelial cells (BOEC) improved blastocyst rate and quality (Cordova et al. 2014). However, the use of primary cell support for improving embryo development in vitro may introduce a higher variability of the results between different BOEC batches used, as well as sanitary risks. The use of well-controlled large batches of frozen BOEC may help to solve these problems. Therefore, the aim of the present study was to characterise the survival and functionality of frozen-thawed BOEC. Bovine oviducts attached to ovaries showing recent ovulation were collected at a local slaughterhouse during 4 replicates (3 oviducts per replicate). Epithelial cells were expelled by gentle squeezing and washed 3 times. Half of the cell pellet was diluted 100-fold in culture medium (TCM199 + 10% FCS) for culture of fresh cells. The other half was diluted 10-fold in cell freezing medium (TCM199 + 20% FCS + 10% dimethyl sulfoxide), allowed to equilibrate in this medium for 10 min, and frozen at –80°C in a container filled with isopropyl alcohol. After 4 h, the tubes were transferred into LN for at least 1 h. The tubes were then thawed (5 min in 37°C water bath), diluted 1 : 1 in cell culture medium, and centrifuged for 10 min at 100 × g. The pellet was then diluted 100× in cell culture medium. Fresh or frozen-thawed cells were seeded in 4-well NUNC plates for 7 days at 38.8°C in a humidified atmosphere with 5% CO2 in air. The medium was renewed every 48 h, and the viability of cells was assessed by calcein-AM and ethidium homodimer labelling. After 7 days of culture, the medium was replaced by SOF medium + 5% FCS, and bovine in vitro-produced zygotes were added the day after and co-cultured for 8 days at 38.8°C in a humidified atmosphere with 5% CO2 in air to evaluate embryo development. Half of the medium was renewed every 48 h. Frozen-thawed cells showed the same viability than fresh ones at Days 0 and 7 of culture and reached confluence at the same time (Day 7). Development results are shown in Table 1. Frozen and fresh cells support early embryo development at the same rate. In conclusion, the present study showed that BOEC frozen on the day of collection are equivalent to fresh BOEC in regards to their survival and proliferation and their ability to support early embryo development. At collection, the cells may face stresses that are just as considerable as freezing/thawing (temperature shock, scrapping, change of environment). This may explain why they are not affected by freezing than at collection. The differentiation status of these cells is now under analysis by immunocytochemistry. Table 1.Cleavage rate and blastocyst rate in 3 different types of culture systems

227 SUPPLEMENTATION WITH CYSTEINE, CYSTEAMINE, AND CATALASE DURING IN VITRO MATURATION PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

Menadione (MD), a naphthoquinone used as a vitamin K source in animal feed, can generate reactive oxygen species (ROS) and cause apoptosis. Hence, we examined whether MD reduces development of pre-implantation bovine embryos by inducing ROS production and apoptosis and tested the hypothesis that the anti-developmental actions of MD would be reduced by supplementation of the in vitro maturation (IVM) medium with antioxidants (a mixture of 0.6 mM cysteine + 100 μM cysteamine + 100 UI mL–1 catalase). Cumulus-oocyte complexes (COC; n = 3334; 50 per well in 11 replications) were matured in IVM medium (TCM-199 with bicarbonate, hormones, and 10% FCS) for 22 h. After fertilization, presumptive zygotes were cultured in synthetic oviduct fluid medium (SOF) with 2.5% FCS and 0.5% BSA up to 7 days (Day 0 = IVF). On Day 6, MD was included in the SOF medium (0 μM: Control; or 5.0 μM: MD5) during 24 h. All cultures were conducted at 38.5°C in 5% CO2 in air. In a second experiment, COC were matured in TCM-199 supplemented with or without the mixture of antioxidants (Antiox; only during IVM) and with or without MD at Day 6 in a 2 × 2 factorial design (Control, Control/Antiox, MD5, MD5/Antiox). The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively. A sample of the blastocysts (n = 1112) was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA) to detect the apoptotic cells or with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the ROS levels. Stained embryos were evaluated under an epifluorescence microscope and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. The means (±s.e.m.) were compared by ANOVA followed by Tukey's test (P &lt; 0.05). In the first experiment, and the cleavage rates were similar (P &gt; 0.05) for Control (80.5% ± 0.9) and MD5 groups (78.9% ± 1.2), but the blastocyst rates were lower (P &lt; 0.05) in the MD5 group (25.9% ± 2.1) when compared with Control (36.4% ± 1.5). The rate of apoptotic cells in blastocysts was higher (P &lt; 0.05) in the MD5 group (19.2% ± 0.7) than in Control (10.0% ± 0.5), as well as the fluorescence intensity for ROS quantification (0.7 ± 0.1 v. 1.1 ± 0.1, respectively, for Control and MD5 groups). In an effort to rescue the impaired embryonic development, a mixture of antioxidants was added to the IVM medium and the results were transitional in response in which MD5/Antiox (28.3% ± 3.5) was intermediate (P &gt; 0.05) between the Controls [Control (38.2% ± 2.5) and Control/Antiox (34.7% ± 1.9) groups] and the MD5 (21.4% ± 2.9) group. Principal effects evaluated were the presence of MD (without: 35.9% ± 2.3 v. with: 24.8% ± 2.3; P &lt; 0.05) or Antiox (without: 28.4% ± 2.3 v. with: 32.3% ± 2.3; P &gt; 0.05). In conclusion, addition of MD to embryonic culture reduces the blastocyst development and increases intracellular levels of ROS and the occurrence of apoptosis. Anti-developmental actions of MD on embryonic development can be partially blocked by treating oocytes with a mixture of antioxidants during IVM.Financial support was provided by FAPESP (2012/10083-8 and 2013/07382-6).

6 EQUINE SPERM INDUCES PRONUCLEAR FORMATION BY INTRACYTOPLASMIC SPERM INJECTION IN BOVINE, SWINE, AND FELINE OOCYTES INDEPENDENTLY OF CHEMICAL ACTIVATION ASSISTANCE

Interspecific intracytoplasmic sperm injection (ICSI) is a valuable tool to study early events of fertilization in species for which oocyte availability is reduced. Equine in vitro fertilization remains unsuccessful and ICSI is the technique of choice for the in vitro production of high-value embryos. Therefore, the objective of this study was to evaluate the rate of pronuclear (PN) formation after ICSI with stallion sperm in bovine, swine and feline oocytes with or without chemical activation assistance. Ovaries from cows and pigs were collected at abattoirs whereas gonads from female domestic cats were obtained from ovariectomized animals at veterinary sterilization centers. Cumulus-oocyte complexes were matured in TCM-199 supplemented following standard protocols for each species. ICSI was performed in 100-μL drops of TALP-HEPES, using frozen-thawed semen from one stallion. Spermatozoa were held separate in 3-μL droplets of 7% (vol/vol) polyvinylpyrrolidone, where one of them was immobilized by swiping the injection pipette across its tail, and then injected into the matured oocyte. After ICSI, some oocytes were chemically activated with 5 μM ionomycin for 4 min (cow and cat) or with an electric pulse (sow) followed by 3 h in culture medium to allow extrusion of the second polar body and then exposure to 1.9 mM 6-DMAP solution for 3 h. Embryos were cultured in SOF medium. After 17 h of culture, embryos were stained with propidium iodide to identify the percentage of oocytes activated and with PN. Haploid and diploid parthenogenetic controls were included. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of the partenogenetic control groups were assessed. There were no statistical differences (chi-squared analysis) in PN formation between the activated and nonactivated groups within species. When the activated group was compared between the different species, no differences were observed. However, for the nonactivated group, significant differences were observed between species. The feline oocyte showed the higher percentage of PN and activation, whereas the bovine oocyte exhibited the lower rate of PN formation (cat: 22/27, 81.48%; swine: 19/39, 71.64%; cow:18/63, 43.07%). Our results suggest that the feline oocyte can be used as model to study fertilization events associated with the stallion sperm due to the higher efficiency in supporting PN formation. Our results indicate that the equine sperm is capable of inducing PN formation in these 3 species without further chemical activation assistance.

273 EFFECT OF IONOMYCIN ASSOCIATED WITH ROSCOVITINE, DEHYDROLEUCODINE, CYCLOHEXIMIDE, OR ETHANOL ON HAPLOID ACTIVATION OF BOVINE OOCYTES

Haploid activation of bovine oocytes after ICSI is a routine procedure. However, embryos frequently contain an abnormal chromosome set as a result of the drugs employed. We compared the efficiency of ionomycin (Io) followed by roscovitine (ROSC), cycloheximide (CHX), ethanol, and dehydroleucodine (DhL) to induce haploid parthenogenetic activation in bovine. Pronuclear (PN) formation, second polar body (2PB) extrusion, embryo development, and ploidy of blastocysts were evaluated. To this aim, COC were aspirated from slaughtered ovaries and IVM for 22 h. Oocytes were activated with 5 µM of Io for 4 min and then randomly allocated into 1 of the following treatments: 25 or 50 µM ROSC or 10 µg mL–1 of CHX for 5 h; 15 or 30 µM DhL for 3 h; or 5 min of exposure to 7% ethanol 4 h post-Io. Controls were Io followed by (1) 3 h in TCM-199 and 3 h in 1.9 mM 6-DMAP (Io-3h-DMAP) and (2) 3 h of exposure to 1.9 mM 6-DMAP (Io-DMAP). Oocytes were cultured in SOF medium. The PN formation and 2PB extrusion were assessed by 5 µg mL–1 of propidium iodide oocyte staining, 17 h after Io. Cleavage, morulae, and blastocyst stages were evaluated at Days 2, 5, and 8 of in vitro development, respectively. Chromosome number of blastocysts was evaluated by Giemsa staining. Data were analysed by Fisher's test (P &lt; 0.05). Rates of 2PB extrusion were 75, 61.1, 60, 56.3, 54.6, and 42.9% for 15 µM DhL (n = 23), 50 µM ROSC (n = 22), Io-3h-DMAP (n = 9), CHX (n = 17), 25 µM ROSC (n = 22), and ethanol (n = 22), respectively, with no differences between groups. A PN was observed in over 81% of the oocytes activated with ethanol, 25 µM ROSC, CHX, 50 µM ROSC, and 15 µM DhL. Lower percentages of 2PB extrusion and PN formation were observed for 30 µM DhL (n = 22; 6.3 and 0%, respectively). The highest cleavage rates were 83.2% for 25 µM ROSC (n = 185), not differing from 78% in Io-DMAP (n = 159). Cleavage rates for 50 µM ROSC (n = 185), CHX (n = 143), and ethanol (n = 74; 80.5, 80.4 and 67.6%, respectively) were not different from Io-3h-DMAP (n = 78; 71.8%) and Io-DMAP. Cleavage rates for 15 µM DhL (n = 70) and 30 µM DhL (n = 93) were the lowest (48.6 and 25.8%). Blastocyst rates were the highest for CHX and 50 µM ROSC, not differing from Io-3h-DMAP (21.7 and 10.8 v. 18%). Very few or no blastocysts were obtained with ethanol, 25 µM ROSC, 30 µM DhL, and 15 µM DhL (4.1, 3.8, 1.1, and 0%, respectively), although ethanol was not different from Io-3h-DMAP. Chromosome number analysis showed that ethanol (n = 2) and CHX (n = 2) resulted in a higher percentage of haploid embryos (50% each), followed by 50 µM ROSC (n = 8), 25 µM ROSC (n = 3), and Io-3h-DMAP (n = 8; 37.5, 33.3% and 12.5%, respectively), although they were not different. Remaining embryos were diploid, aneuployd, or mixoployd. In conclusion, DhL and ROSC proved to be as effective as CHX or ethanol regarding 2PB extrusion and resulting ploidy, defining features when activating oocytes in ART, suggesting they could be efficiently used in bovine to assist ICSI.