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Forskolin® (F-6886) is being used to induce lipolysis and increase cryotolerance, to be an activator of adenylate cyclase, and elevating the cyclic adenosine monophosphate (cAMP) levels. The objective of this experiment was to induce the chemical lipolysis of embryos to improve vitrification and the hypothesis would be that Forskolin decrease the amount of lipid droplets, improve the production of blastocysts, and increase the survival rate after vitrification and warming. Eight random effect were performed which oocytes (N = 1172) were matured in TCM 199® supplemented with 10% of fetal bovine serum (FBS), under 5% CO2 atmosphere, at a temperature of 38.5°C and absolute humidity for 24 h. Semen was selected by Percoll gradient with a final concentration of the 2 × 106 sperm mL–1. Presumptive zygotes were cultured in SOFaa and 2.5% of FBS and were kept in an incubator with 5% CO2, 5% O2 and 90% N2 at 38.5°C and absolute humidity until Day 6, when Forskolin was added and remained until Day 7; control (group without Forskolin); F 2.5 µM (group with 2.5 µM Forskolin); F 5 µM (group with 5 µM Forskolin). On Day 7 (Day 0 = IVF) the rate of blastocyst formation was observed then they were vitrified. Apoptosis was analysed using the TUNEL technique, and the lipid content analysis was performed with Sudan Black B® (S-0395). To estimate the lipid content of embryos, 1 photo at a blastocyst group was performed and submitted to the program ImageJ 1.14 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The embryos were limited to obtain the area (μm2), and gray intensity mean (arbitrary units), and gray intensity per area was calculated (arbitrary units/μm2). Data were analysed by ANOVA with PROC GLM of SAS (SAS Institute, Cary, NC, USA). Sources of variation in the model including treatment and replicas were regarded as fixed and random effects, respectively. Data are presented as mean and standard least-squares error. For all analyzes was adopted the significance level of 5%. There was no difference in blastocyst rate: control (37.0 ± 4.0%), F 2.5 μM (38.6 ± 4.0%), F 5 μM (40.7 ± 4.0%). There were difference in lipids content between all groups: control (136.8 ± 2.2ab); F 2.5 μM (128.5 ± 2.2b), F 5 μM (135.6 ± 2.3c; P

Made available in DSpace on 2016-04-01T18:44:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2016-04-01T18:49:46Z : No. of bitstreams: 1 ISSN0378-4320-2010-121-01-186-187.pdf: 85775 bytes, checksum: ac2d45d3d4ee8bd33ac1c9b26f7c4e4f (MD5) Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil

Made available in DSpace on 2016-04-01T18:44:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2016-04-01T18:49:47Z : No. of bitstreams: 1 ISSN0378-4320-2010-121-01-178-179.pdf: 96576 bytes, checksum: 8fb40716f73b8e12f798baf47cfcb93c (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil FAPESP: 2008/57504-2

Made available in DSpace on 2016-04-01T18:44:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2016-04-01T18:49:51Z : No. of bitstreams: 1 ISSN0378-4320-2010-121-01-168-170.pdf: 593173 bytes, checksum: e693ecec394342ab838a2765096e715a (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, DDepartamento de Clínica Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, DDepartamento de Clínica Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. It was suggested that the inhibition of spontaneous nuclear IVM might allow for more time to accumulate the molecules important for embryonic development. The objective of this work was to evaluate blocking oocyte meiosis with the addition of forskolin. Slaughterhouse-derived bovine Zebu ovaries were collected and carried to the laboratory. Oocytes (n = 584) with at least 3 intact layers of cumulus cells and homogeneous cytoplasm were selected for IVM. The oocytes were transferred to drops of TCM 199 plus 10% FCS and hormones. The oocytes remained in IVM medium in 3 different concentrations of forskolin (6886), 0.1, 0.05, 0.025 mM, and a control group (withouth forskolin), for 6 h. Then they were maturated for an additional 18 h in forskolin-free medium. The first period above was an attempt to block (Block) and the second to resume (Res) the oocyte meiosis. The oocytes were incubated in a humidified atmosphere with 5% CO2 at 38.5°C in an air incubator. The oocytes were assessed for the stage of nuclear maturation, to see if they were in M II. Then oocytes were in vitro fertilized (IVF) with frozen Nelore bull semen (Bos taurus indicus). Presumptive zygotes (20–30/group) were cultured in SOFaa (synthetic oviducal fluid) supplemented with 5 mg mL–1 of BSA; the embryos were kept in an incubator with 5% CO2, 5% O2, and 90% N2 at 38.5°C and absolute humidity. On Day 7 (Day 0 = IVF) the blastocyst, the number of viable cells, and apoptosis rate (terminal deoxynucleotide transferase uridine nick-end labelling) were observed. Data were analysed with ANOVA using SAS PROC GLM (SAS Inst. Inc., Cary, NC, USA). Sources of variation in the model, including treatment and replication, were respectively considered fixed and random effects. If ANOVA was significant, the contrasts of means were performed using the least-squares difference. Data are presented as the mean and the standard error of least-squares. For all analyses, we used a significance level of 5%. No differences were observed for the stage of nuclear maturation of the oocyte (N = 336; control: 67.7 ± 8.3; F 0.025 mM, Block/Res: 67.7 ± 8.9; F 0.05 mM, Block/Res: 65.9 ± 9.8; F 0.1 mM, Block/Res: 50.2 ± 8.9), the blastocyst rate (N = 584; Control: 36.7 ± 3.7; F0.025 mM, Block/Res: 32.6 ± 3.7; F0.05 mM, Block/Res: 29.2 ± 3.7; F0.1 mM, Block/Res: 25.1 ± 3.7), and total number of intact cells (N = 10–15 embryos/group; Control:140.1 ± 13.0; F0.025 mM, Block/Res: 129.9 ± 13.0; F0.05 mM, Block/Res: 139.0 ± 13.0; F0.1 mM, Block/Res: 104.4 ± 13.0; P > 0.05). However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (N = 10–15 embryos/group): Control: 12.1 ± 2.5a; F 0.025 mM, Block/Res: 12.9 ± 2.5a; F0.05 mM, Block/Res: 13.5 ± 2.5a; F 0.1 mM, Block/Res: 30.2 ± 2.5b (P

The cryopreservation of sperm from different segments of the epididymis is the ultimate opportunity to preserve the gene pool of animals of great breeding value. Despite the fact that samples from the caput epididymis are immotile, their DNA integrity could justify their use in ICSI procedures. Thus, the objective of the present study was to evaluate the DNA integrity and sperm motility of stallion sperm collected from different regions of the epididymis. Equine epididymides were obtained from 10 stallions using retrograde flush with 40mL of Botu-Semen per caudae epididymis. Sperm from the caudae epididymis were obtained with a slicing technique in association with a float-up method. The samples were divided into 3 groups: Botu-Semen (control), Fert-Talp and Sperm-Talp, and diluted 1:1 with these media. The samples were centrifuged (1000 x g/10 min) to concentrate the sperm, the supernatant was removed, and the pellets were resuspended in Botu-Crio . Samples were loaded into 0.5mL straws and cooled at 5 C for 20 min. They were then frozen in nitrogen vapor (6 cm above the level of liquid nitrogen) for an additional 20 min, plunged into liquid nitrogen and stored. Sperm chromatin structure assay was performed using a semen aliquot diluted in 200 mL of buffer solution (0.186g disodium EDTA, 0.790 g Tris-HC1, 4.380 g NaC1 in 500 mL deionized water, pH 7.4). This was added with 400 mL of detergent/acid solution. Thirty seconds later, 1.2 mL acridine orange solution was added (Evenson, In: Sorsa M, Norppa H (ed), Monitoring of

Even though FCS provides energy substrates, amino acids, vitamins, growth factors, and heavy-metal chelators, its supplementation has been associated with several embryo abnormalities such as mitochondrial degeneration, metabolic deviations, excessive lipid accumulation, and decreased embryo survival after cryopreservation. The aim of the present study was to evaluate the effect of high FCS concentration in the gene expression pattern of in vitro-produced bovine embryos. Slaughterhouse ovaries were used to obtain oocytes (N = 360), which were matured and fertilized in vitro (Day 0). Presumptive zygotes were divided in 2 culture media: with low (SOFaa with 0.5% BSA and 2.5% FCS) or high (SOFaa with 0.5% BSA and 10% FCS) FCS concentration. Cleavage was evaluated on Day 3. Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2, and 90% N2). The produced blastocysts were placed in PBS solution and washed five times. A single blastocyst was frozen in a minimal volume of PBS and stored at –80°C until RNA extraction. Total RNA extraction was performed using the PicoPure RNA isolation Kit (Applied Biosystems®, Foster City, CA, USA). Extracted RNA was evaluated through 2100-Bioanalyzer (Agilent Technologies®, Palo Alto, CA, USA) and DNAse treated (Qiagen®, Valencia, CA, USA). RiboAmp RNA Amplification Kit (Applied Biosystems®) was used to amplify the RNA (T7 RNA polymerase-catalysed amplification reaction). The aRNA output was evaluated through NanoDrop ND-1000 (NanoDrop Technologies®, Wilmington, DE, USA). A biotin-labelled cRNA and fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization (N = 3 per group) using GeneChip Bovine Genome Array (Affymetrix®). Following hybridization, probe arrays were washed, stained, and scanned. Microarray data analysis was performed in the software FlexArray 1.6.1.1. Genes with a fold change of at least 1.5 and a probability of P 0.05) between low and high FCS concentrations, respectively. A total of 40 genes were differentially expressed between low and high FCS concentration. A total of 28 genes were annotated, with 37 genes up-regulated and 3 genes down-regulated by high FCS concentration. The associated network functions of gene expression, RNA damage and repair, and post-transcriptional modification; and cell-to-cell signalling and interaction were generated by Ingenuity Pathway Analysis® (Redwood City, CA, USA). Differentially expressed genes involved in carbohydrate metabolism (GAPVD1, MGAT4A), lipid metabolism (ELOVL5), cellular assembly and organisation (EZR, LRP2), and cell death and survival (DRT8) were identified. In conclusion, high FCS supplementation was associated with different expression profiles of genes regulating carbohydrate and lipid metabolism, cellular assembly and organisation, and cell death and survival. The authors acknowledge support from FAPESP and LNBio-CNPEM.

Harvesting and freezing epididymal sperm is a technology that enables the preservation of the gene pool from animals that had died either unexpectedly or because of colic conditions. This technique may also be employed in animals that have to be euthanized because of traumatic injuries. Therefore, the objective of the present study was to improve the process of freezing epididymal sperm using a freezing extender without the prior centrifugation of the samples. Twelve stallions aging between 3 and 6 years and of different breeds were used (Quarter Horse, Mangalarga, and Brazilian Jumping Horse). Stallions were castrated, and the cauda epididymides were isolated from the testis. The connective tissue was carefully dissected, and the cauda epididymides were straightened. A 200-µL pipette tip was attached to a 20-mL syringe, and the cauda epididymides were flushed using 40 mL of either (A) BotuSemen® (Nidacon, Mölndal, Sweden) or (B) BotuCrio® (Nidacon). They were then immediately processed at room temperature (25°C). Samples flushed with B were randomly subjected to either of the 2 procedures: B1) directly loaded into 0.5-mL straws or B2) centrifuged at 600g for 10 min, the supernatant was discarded, and the pellet was resuspended with B and loaded into 0.5-mL straws. The straws were kept at 5°C for 20 minutes followed by another 20 min at 6 cm above liquid nitrogen before immersion. After thawing at 46°C for 20 s the samples were analyzed by computer-assisted semen analysis (HTM – IVOS 12) and plasma membrane integrity was assessed using fluorescent probes (carboxyfluorescein diacetate and propidium iodide). Data were analyzed by ANOVA followed by the Tukey test (P

The maintenance of oocytes in the germinal vesicle stage for a few hours could result in more competent oocytes for use in biotechnology. Related to this, forskolin (Sigma-Aldrich, St. Louis, MO, USA) is an efficient inhibitor of nuclear maturation because of its ability to increase the levels of intracellular cyclic adenosine monophosphate. This study aimed to show whether the use of forskolin would be able to inhibit maturation in bovine oocytes, producing a higher rate of in vitro embryos. Nellore oocytes from a slaughterhouse (n = 960) were matured in TCM-199 with Earle’s salt + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium with forskolin at 3 different concentrations, 0. 1 mM (n = 240), 0.05 mM (n = 240), and 0.025 mM (n = 240), whereas untreated oocytes acted as controls (n = 240). The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h (Day 0) of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions as described above. Semen was selected through Percoll gradient, and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocysts were evaluated. Apoptosis in blastocysts was accessed through TUNEL reaction. Data were analysed by ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate (n = 297): control (n = 88): 36.7% ± 3.7; 0.1 mM forskolin (n = 61): 25.1% ± 3.7; 0.05 mM forskolin (n = 70): 29.2% ± 3.7; 0.025 mM forskolin (n = 78): 32.6% ± 3.7 (P > 0.05). However, when we analysed the apoptosis rates, differences were found among groups: control: 6.0% ± 6.3a; 0.1 mM forskolin: 33.4% ± 6.3b; 0.05 mM forskolin: 27.2% ± 6.3ab; 0.025 mM forskolin: 10.0% ± 6.3ab (P

Alterations to a sperm cell that are similar to apoptosis reduce the sperm cell longevity in the female reproductive tract. The aim of the present study was to evaluate the effect of sperm motility factors [caffeine, heparin, and penicillamine, hypotaurine, and epinephrine (PHE)] on phosphatidylserine translocation in equine epididymal semen. Equine epididymal samples were obtained from 10 stallions using retrograde flush with 40 mL of BotuSemen® (BS) per epididymal cauda. The samples were divided into 3 groups: BS (control), Fert-TALP (Fert), and Sperm-TALP (SP), and diluted 1 : 1 with these media. Samples containing 100 × 106 sperm cells were loaded into straws, and then incubated at room temperature (25°C) for 15 min. The samples were centrifuged (1000g for 10 min), the supernatant was removed and the pellets were resuspended in BotuCrio®. Samples were loaded into 0.5-mL straws and cooled at 5°C for 20 min. Then, they were frozen in nitrogen vapor (6 cm above the level of LN) for an additional 20 min and finally plunged into nitrogen and stored. The straws were thawed at 46°C/20 min. Sperm motility was evaluated by computer-assisted semen analysis (CASA; HTM-IVOS 12). To evaluate the pancreatic secretory trypsin inhibitor (PSTI), the Annexin V-FITC Apoptosis Detection Kit (6710KK; Pharmingen, San Jose, CA, USA) was used according to the manufacturer’s recommendations. The samples were diluted in Annexin V buffer solution to a concentration of 2 × 106 spermatozoa mL–1 and then 5 mL of Annexin V-FITC, 5 mL of propidium iodide (PI, 50 mg mL–1), and 2 mL of Hoechst 33342 dye (H342, 40 mg mL–1) were added. The samples were then homogenized and incubated for 15 min. Then, 400 µL of Annexin V buffer solution was added to obtain a final concentration of 1 × 106 spermatozoa mL–1. The samples were evaluated. Flow cytometry was carried out at BD LSR II (Becton Dickinson, Mountain View, CA, USA). Mean and standard deviation were calculated, and then the normality test was evaluated by the Kolmogorov-Smirnov test. An analysis of variance and Tukey test with a P

In vitro-produced Bos taurus indicus (zebu) and Bos taurus indicus × Bos taurus taurus (cross-bred) embryos behave differently when vitrified. The present experiment aimed to examine the effect of vitrification on embryos produced in the presence or absence of FCS. Cumulus-oocyte complexes (COC) were matured in TCM-199 and fertilized in human tubal fluid medium with frozen Nelore bull semen. On Day 1 (Day 0 = IVF), presumptive zygotes were cultured with SOFaa + BSA in the presence of FCS (Group 2.5%) or in the absence of FCS (Group 0%) until Day 7. The cleavage was analysed on Day 3 and the blastocyst rate on Day 7. Blastocysts were vitrified and, after warming (Campos-Chillòn et al. 2006) the viability was evaluated. Data were analysed with ANOVA, using the general linear model (GLM) of SAS (SAS Inst Inc., Cary, NC, USA). Sources of variation in the model included FCS concentration and first-order interactions; all factors were considered fixed effects. The arcsine transformation (√y/100) was applied to percentage data. If the ANOVA was significant, means were separated using the Tukey test. There was no difference in cleavage (for zebu embryos: Group 0%: 87.2 ± 6.8; Group 2.5%: 87.4 ± 9.5; for cross-bred embryos: Group 0%: 79.6 ± 11.9; Group 2.5%: 73.1 ± 13.7; P > 0.05). On the other hand, zebu embryos cultured in the presence of FCS reached blastocysts at a higher rate than cross-bred embryos in the absence of FCS (for zebu embryos: Group 0%: 33.3 ± 12.4ab; Group 2.5%: 46.8 ± 13.2a; for cross-bred embryos: Group 0%: 21.8 ± 8.3b; Group 2.5%: 33.6 ± 10.1ab; P 0.05) and cell number per embryo (zebu embryos: Group 0%: 65.1 ± 34.7; Group 2.5%: 42.6 ± 17.2; cross-bred embryos: Group 0%: 64.3 ± 44.2; Group 2.5%: 52.0 ± 31.5; P > 0.05) between species groups and within species were seen. However for zebu embryos, Group 0% showed a lower damaged cell rate than Group 2.5%. The same effect was not observed in the cross-bred embryos (zebu embryos: Group 0%: 20.3 ± 22.7c; Group 2.5%: 63.3 ± 27.0d; cross-bred embryos: Group 0%: 25.4 ± 24.3cd; Group 2.5%: 45.8 ± 34.6cd; P

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 vitro produced 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 0.3 μM PES from Day 4 (after 96 h 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 vivo control (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, P 0.05) 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 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control 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.

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 vitro produced (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% N2 at 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 10 μM 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 vitro produced embryos were compared with embryos obtained in vivo from 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 12 h). 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 vivo produced embryos were compared. After vitrification the re-expansion rate was similar between IVP and in vivo produced 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 vivo produced embryos [2.5% FCS: 42.6 ± 17.2a (P

In order to enhance the use of biotechnology in females it is necessary to have good knowledge of the ovarian events, and thus enable the manipulation of the estrus cycle to concentrate ovulations in an established period of time. In this experiment, we evaluated the ovary response and corpus luteum (CL) parameters in a short-duration protocol associated or not to lecirelin (L)(GnRH) and/or estradiol benzoate (EB). Twenty-four cyclic Santa InÊs ewes were divided into 4 groups: Control animals (G1; n = 6) received 45 μg of d-cloprostenol (Prolise®, Tecnopec, Sao Paulo, Brazil) on Day 0. Intravaginal sponges containing medroxyprogesterone acetate (MAP60®, Tecnopec) were inserted on Day 3. Then on Day 7 sponges were removed and 400 IU of eCG and 45 μg of d-cloprostenol were administered. G2 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1; G3 (n = 6) animals underwent the same protocol as G1 but with administration of 25 μg of L (Gestran plus®, Tecnopec) 30 h after sponge withdrawal; G4 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1 and 25 μg of L 30 h after sponge withdrawal. Ewe ovaries were assessed via transrectal ultrasound (US), and CL were measured 4 days after ovulation. On the same day blood samples were taken to measure plasma progesterone levels via radioimmunoassay. Twenty-three out of 24 animals ovulated. One ewe from G4 that did not ovulate was taken out of the experiment. The average size of the largest pre-ovulatory follicle was 7.3 mm (G1), 7.0 mm (G2), 6.8 mm (G3), and 7.4 mm (G4) (P > 0.05). There were 1.8, 1.2, 2.0, and 1.4 ovulated follicles per animal in G1, G2, G3, and G4, respectively. The results for estrus start and duration after sponge withdrawal were G1: 32/38.6; G2: 37/69.3; G3: 29.3/37.3; G4: 44/68 h, respectively. Ovulation time span was G1: 65.3; G2: 88; G3: 53.3; G4: 82.4 h after sponge withdrawal. There was a high correlation of ovary mensuration and progesterone level (r = 0.64; P < 0.0001) on the fourth day after ovulation. Progesterone levels after ovulation were 2.37 ng mL-1 (G1); 1.5 ng mL-1 (G2); 3.22 ng mL-1 (G3) and 1.99 ng mL-1 (G4), being higher in G3 than in G2 (P < 0.05). It was seen that the EB is prejudicial to the CL function. Despite the fact that there was no significant difference of progesterone levels within G1, G3, and G4, animals in G3 displayed higher levels of progesterone; hence, there is a need for further studies using a larger number of animals and fertility test. CNPq e Tecnopec.

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% CO2 in 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-1 BSA; 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% N2 atmosphere 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 culture Acknowledgments: FAPESP 07/53505-1.

The ultrarapid freezing technique was developed as an alternative to slow conventional freezing to avoid formation of ice crystals (Tucker MJ and Liebermann J 2007, Vitrification in Assisted Reproduction, 87-92). The recent use of ethylene glycol tetraacetic acid (EGTA) instead of glycerol as a cryoprotectant for sperm is a result of efforts to reduce osmotic and cytotoxic effects. Accordingly, the objective of the present study was to compare the cryoprotective effects of EGTA v. glycerol on bovine sperm frozen using an ultrarapid method. A pool of epididymal sperm collected from 5 bulls was maintained in Botusui extender (Biotech, Botucatu, Brazil) containing 5% BSA, either EGTA (5%) or glycerol (5%), and 0 or 2 mg mL of polyvinyl alcohol (PVA). All samples were loaded into 0.25-mL French straws and either placed into a freezing machine (TK 4000 compacta, TK equipamentos para reproducao, Uberaba, Brazil) and cooled at a rate of 50°C/min, or exposed to liquid nitrogen (LN2) vapor for 5 to 15 min before plunging into LN2. After overnight storage in LN2, straws were thawed and examined using CASA (HTM IVOS 12, Hamilton Thorne Research, Beverly, MA, USA) for total motility (TM), progressive motility (PM), path velocity (VAP), progressive velocity (VSL), and track speed (VCL). Acridine Orange (AO) was used to check DNA integrity of sperm cells (Chirinea VH et al. 2006 Ciencia Animal Brasileira 7, 407-415). Our results indicate that, despite the potentially damaging effects of the extreme temperature changes, epididymal sperm showed 95% of DNA integrity and, therefore, should be able to participate in the fertilization process. Addition of PVA had a negative effect on sperm motility characteristics. CASA revealed that glycerol was a more suitable cryoprotectant for ultrarapid freezing of bull epididymal spermatozoa than was EGTA (see Table 1). Table 1. CAPES.