bluuebunny/crossref_metadata_2025 huggingface dataset:
271 EFFECT OF BULL ON IN VITRO FERTILIZATION OF BOVINE OOCYTES
Authors: Garcia O. S. , Ferro R. S. , Bezerra J. M. D. , Sales M. S.
Published: August 2010

The main objective of this work was to evaluate the effect of the bull on IVF of bovine oocytes. These oocytes were obtained from ovaries collected in the slaughterhouse and selected for their maturation process in TCM-199 culture medium with 20% estrus cow serum, 10 IU of eCG, and 1 mg mL-1 gentamicin. The oocytes were cultivated for 20 to 24 hours in 39.0°C culture incubator and moist environment with 5% CO2. For the oocyte insemination, frozen/thawed semen of 8 bulls was used by means of swim-up capacity procedure according to Parrhit (1989) and then motility, sperm concentration, and strengh were evaluated. In all cases, 25 to 30 oocytes, previously selected according to cumulus oophorus density were artificially inseminated with 1 × 106 spermatozoids mL-1. The total of selected oocytes by bull for this work was: 5028 (1), 1247 (2), 2888 (3), 650 (4), 1529 (5), 699 (6), 984 (7), and 1250 (8). The results were evaluated according to the average of oocytes matured in vitro and to embryonic division 48 hours after AI. In order to evaluate the effects by bull, the statistical examination of chi-square proportion comparison was used. The average values and standard deviation (SD) motility were 57.0% ± 13.7; 53.2% ± 11.3; 51.2% ± 8.20; 40.9% ± 5.0; 48.1% ± 4.92; 33.5% ± 4.42; 53.5% ± 11.1 and 40.0% ± 8.9 for bulls 1 to 8, respectively. There was a highly significant difference (P < 0.001) in relation to the percentage of division: 46, 42, 34, 18, 30, 31, 30, and 17% among bulls 1 to 8, respectively, in addition to variability among these bulls. The results obtained confirm the effect of the bull on conception rate. The conclusion is that frozen/thawed semen with 50% motility can increase the percentage of embryonic division.
278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO
Authors: Imai K. , Somfai T. , Ohtake M. , Inaba Y. , Aikawa Y. , Sugimura S. , Yoshioka H. , Kobayashi S. , Konishi K.
Published: August 2010

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU.
This work was supported by the Research and Development Program for New Bio-industry Initiatives.
280 INDISTINGUISHABLE TRANSCRIPTIONAL PROFILES BETWEEN IN VIVO- AND IN VITRO-PRODUCED BOVINE FETUSES
Authors: Jiang L. , Marjani S. L. , Bertolini M. , Lewin H. A. , Anderson G. B. , Yang X. , Tian X. C.
Published: August 2010

During the past several decades, in vitro fertilization (IVF) has been increasingly used in animal production and human infertility treatment. In vitro production (IVP) has been shown to cause reduced developmental competence, aberrant gene expression, and developmental abnormalities. Our objective was to determine how in vitro procedures influence global gene expression during fetal development. To this end, we analyzed the gene expression profiles of liver and placentome tissue samples (n = 18) from IVP and in vivo-derived fetuses at Days 90 and 180 of gestation (n = 5 IVP and n = 4 in vivo-derived pregnancies for each day of gestation). Standard in vitro maturation and fertilization protocols were employed. Putative zygotes were co-cultured with bovine oviductal epithelial cells to the blastocyst stage. In vivo embryos were collected 7 days after AI by nonsurgical uterine flushing. Blastocyst-stage IVP and in vivo embryos were transferred to synchronized recipients and monitored until collection at Day 90 or 180. The pregnancy rate at Day 90 was 12% and 27% for IVP and in vivo pregnancies, respectively (Bertolini et al. 2004 Reproduction 128, 341-354). To conduct expression profiling, total RNA from each tissue sample and a standard reference was indirectly labeled with Cy3 and Cy5, respectively, and hybridized in duplicate to custom, bovine 13 K oligonucleotide microarrays. After Loess normalization, a two-way (origin and day) ANOVA model (GeneSpring 7.3.1) was used to identify differentially expressed genes in each tissue. The P-values were adjusted for multiple comparisons using a 5% false discovery rate (FDR). The expression of 11 candidate genes was confirmed independently by quantitative RT-PCR. Surprisingly, in both the liver and placentome tissues, no differential gene expression was detected between the IVP and in vivo fetuses at Day 90 and 180. This was observed even when the FDR was relaxed to 10% and 20%. A total of 879 genes (523 genes ≥ 1.5-fold) were differentially expressed during liver development from 90 to 180 days of gestation. Conversely, no differential gene expression was detected in the placentomes during this developmental period. Our findings show that during early and mid gestation, surviving IVP fetuses had normal patterns of gene expression. It is possible that embryos with less severe perturbations may survive with their gene expression normalized as development proceeds. Additionally, initial changes in gene expression caused by IVP may affect subsequent development, but do not necessarily persist throughout gestation.
Present addresses: L. Jiang, Columbia University, New York, NY, USA; S. L. Marjani, Yale University, New Haven, CT, USA; M. Bertolini, University of Fortaleza, CE, Brazil. This work was supported by USDA grants to X.Y, H.A.L., and X.C T.
282 NOVEL SEQUENTIAL MEDIA FOR THE PRODUCTION OF CENTRAL AMERICA'S FIRST IN VITRO-PRODUCED SENEHOL CALVES
Authors: Koyner P. , Pino J. E. , Lasso M. , Rodriguez F.
Published: August 2010

Panama, like many countries in Central America, has a tropical climate with high temperature and humidity throughout the year. These conditions negatively affect the fertility and productivity of dairy cattle; therefore, it is necessary to develop and apply technologies to improve those parameters in heat-stressed cattle. One such technology is in vitro embryo production (IVP). The objectives of this study were to a) introduce, for the first time in Panama, IVP procedures for bovine embryos and b) evaluate the efficacy of novel sequential media (SM) on in vitro embryo development and pregnancy rates. Oocytes collected from slaughterhouse ovaries of Holstein cows were matured and fertilized (with Senepol semen) in vitro using standard procedures. The resultant zygotes were cultured in an atmosphere of 6% CO2, 5% O2, and 89% N2 at 38.5°C in either control medium plus BSA (3 mg mL-1; mSOF, Tanaka et al. 1996 JICA Manual) for 144 h post-insemination (hpi; n = 1,072) or a semi-defined medium for 96 hpi followed by a second semi-defined medium for 48 h additional culture (n = 1,081; see Table 1). The experiment was replicated 5 times between January 2007 and May 2009, and data were analyzed using chi-square. Zygotes cultured in SM resulted in more >4-cell embryos at 48 hpi than controls (93 v. 81%; P < 0.01), more >8-cell embryos at 96 hpi than controls (79 v. 65%; P < 0.01), and more blastocysts at 144 hpi than controls (45 v. 37%; P < 0.05). A subset of quality grade 1 blastocysts produced in SM (n = 35) or mSOF (n = 30) were transferred nonsurgically to synchronized recipients. Pregnancy rates at 60 days were similar between SM and mSOF embryos (62 v. 45%, respectively). From the first 14 embryo transfers, 7 calves have been born (4 from SM and 3 from mSOF). These results demonstrate that the SM used in this study, which contained antioxidants and growth factors, supported enhanced in vitro embryo development. Additional transfers are needed to determine if the use of SM will also result in a statistically higher pregnancy rate, which would be economically important from a commercial point of view. This represents the first report of IVP calves in Central America.

Table 1.Composition of novel sequential media
292 EVALUATION OF THREE PROTEIN SOURCES FOR THE IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Ordoñez-Leon E. A. , Cancino G. , Hernandez-Ceron J. , Medrano J. A. , Ducolomb Y. C. , Romo S.
Published: August 2010

Bovine embryo development in vitro can be affected by many factors, including protein source, which can cause embryo development failure. The use of in vitro culture media supplemented with serum-free compounds could allow a better understanding of embryo requirements during the preimplantation stages by eliminating a highly variable and undefined compound such as serum. The objective of this study was to evaluate the effect of 3 different protein supplements used during IVM, IVF, and IVC on embryo production. Ovaries were collected from slaughtered cows and then aspirated to obtain oocytes for in vitro embryo production procedures. A total of 2056 oocytes were used, from which 685 were processed with maturation medium supplemented with 10% serum replacement (SR) (Gibco Knockout Serum Replacement, Invitrogen, Carlsbad, CA, USA), a defined serum-free formulation (TCM-199 + SR), fertilization medium with SR (TALP + SR), and culture medium with SR (SOF + SR). These were compared with 675 and 696 oocytes processed with the same IVM, IVF, and IVC media, but supplemented with 10% FCS or 10% heat-inactivated estrous cow serum (ECS), respectively. Data obtained from the variables studied were processed by analysis of variance and means were compared by Tukey’s test. The percentages of embryos produced with FCS (52.4%) and ECS (52.7%) were significantly higher compared with the percentage obtained with SR (41.5%) (P < 0.05). The percentages of morulae were similar in the groups supplemented with FCS (36.5%) and SR (36.7%), but significantly higher than the percentage in the ECS group (26.9%) (P < 0.05). For blastocysts, the percentages of embryos developed with FCS (35.2%) and ECS (35.6%) were significantly higher than that obtained with SR (29.2%) (P < 0.05). When evaluating expanded blastocysts, the percentage obtained in the FCS (45.9%) group was significantly higher than that in the ECS group (33.2%), and this was significantly higher than that obtained in SR (21%), with all these differences being significant (P < 0.05). It is concluded that it is possible to produce bovine embryos in vitro using FCS, ECS, or SR as supplements in IVM, IVF, and IVC media. Significant differences were found in different embryo stages, with the highest proportion of embryos developing with the addition of FCS, whereas supplementation with SR only improved the production of morulae.
We thank Consejo Nacional de Ciencia y Tecnologia (CONACYT-Mexico) for the graduate student’s scholarship.
294 USE OF FORSKOLIN TO PRODUCE IN VITRO BOVINE EMBRYOS UNDER TWO CONCENTRATIONS OF FETAL CALF SERUM
Authors: Paschoal D. M. , Sudano M. J. , Magalhães L. C. O. , Crocomo L. F. , Landim-Alvarenga F. C.
Published: August 2010

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.
297 ASSESSMENT OF THE TRANSPORTATION OF BOVINE OOCYTES EXPERIMENTALLY EXPOSED TO BOVINE HERPESVIRUS TYPE 1 (BoHV-1) DURING THE PERIOD OF MATURATION IN IN VITRO OVIDUCTS
Authors: Piccolomini M. M. , Batista M. L. , Góes A. C. , Pavão D. L. , Alves M. F. , Dangelo M.
Published: August 2010

The necessity of greater elucidation about oocyte pathogen interactions made us examine the interaction of bovine herpesvirus type 1 (BoHV-1), Los Angeles strain, with oocytes matured in vitro to assess the potential hazard of transmission of such infectious agent through IVF. The process of capacitation and transportation of oocytes through the oviduct depends on the quality of the female gamete. Keeping in mind that the BoHV-1 causes morphophysiological modification in oocytes, the aim of this study was to evaluate, in an experimental model of in vitro oviduct, whether the pathogen interferes in the transportation of oocytes. This was determined using the percentage of oocyte recovery and assessment of the alteration in the morphology of the oviduct lumen. Oviducts and oocytes were collected from bovine ovaries derived from slaughterhouse cows. The oviducts were dissected, washed, and placed in Petri dishes (100 × 20 mm). On the previous portions of the infundibulum, in vitro-matured control oocytes or oocytes exposed to the virus (105.5 TCID50 mL-1) were introduced (20 oocytes per group) and then immersed in 100 mL of TCM-199 medium. Each dish was incubated at 38°C, 5% CO2, and 95% humidity for 24 h. Then, we introduced in the infundibulum, through intramammary infusion probe, 5 mL from a physiologic solution containing 1% of bovine fetal serum. Each oviduct effluent was collected separately and evaluated through a stereoscope for the recovery and counting of oocytes. Immediately after the recovery, both oviduct groups were sectioned longitudinally and observed through an optical microscope (100 ×) for a morphological evaluation of the luminar area. For the control group, the percentage of recovery was 14.9% (65/437), whereas in the infected group, the oocyte recovery was 23.4% (100/428). The statistical analysis was made according to Student’s t-test (P < 0.05), and presented a significant difference in the final results. The previous results show that the group of oocytes that was exposed to the virus presented a higher percentage of recovery compared with the control group. The oviducts that received the exposed oocytes presented areas where it seemed there has been cytopathic effect represented by dark and lumpy sections, in which there were round and degenerating cells mainly located in the oviducts’ peripheral area. The morphophysiological modifications in contaminated oocytes and oviducts probably complicate their transportation and also interferes in the process of their liberation. It is relevant to elucidate the importance of the studies above because of their participation in the recovery of these gametic cells in bovine oviducts, as well as the transmission during IVF processes.
Vitrocel/Embriolife.
299 OVUM PICK-UP USE IN NELORE COWS SUPERSTIMULATED WITH FSH OR eCG
Authors: Ribeiro L. V. P. , Rigolon L. P. , Cavaliere F. L. B. , Martinez A. C. , Martins R. R. , Ribeiro M. G. , De Abreu C. O.
Published: August 2010

The success of reproductive technologies, such as embryo transfer and IVF, greatly depend on the response to superovulation. Using hormonal therapy, it is possible to increase the reproductive rates of beef and dairy cattle for individual and herd treatment. The objective of the present study was to assess the effect of stimulating treatment with FSH or eCG on the oocytes production in Nelore (Bos indicus) donors. The experiment was carried out at Biotec (Cesumar farm, Maringá, Paraná State, Brazil). Forty-two cows that originated from the same genetic group, between 4 and 9 years old were used, with an average weight of 420 kg. The cows received an intramuscular injection of estradiol benzoate (EB, 2 mg) and an ear implant of Norgestomet® (3 mg). Then, they were divided in 3 groups: control group (n = 14); group 1 = eCG treatment (n = 14); and group 2 = FSH treatment (n = 14). In group 2, cows received on 120 IU of FSH on Days 5 and 6, divided in 4 doses that were administrated at 12-h intervals. On Day 7, all implants were removed and ovum pick-up (OPU) was performed. The results were statistically analyzed by Proc GLM (version 8.0, SAS Institute, Cary, NC, USA) and the mean differences were analyzed by Tukey test. Of 42 OPU sessions, a total of 627 oocytes were recovered, of which 502 were viable and 125 were degenerated, but there were no significant differences (P > 0.05) between the groups, as shown in Table 1. We conclude that the gonadotrophic stimulations with FSH or with eCG used in this study did not increase the overall quality of the viable oocytes. More research is needed to determine the optimal ovarian stimulation protocol in Nelore cows.

Table 1.Number of oocytes recovered after OPU session in cows that were superstimulated with FSH or eCG1
30 OVULATION AND PREGNANCY RATES IN CYCLING NELLORE HEIFERS AFTER FIXED-TIME ARTIFICIAL INSEMINATION PROTOCOLS USING DIFFERENT CONCENTRATIONS OF PROGESTERONE, ASSOCIATED OR NOT WITH eCG
Authors: Pegorer M. F. , Ereno R. L. , Satrapa R. A. , Barros C. M.
Published: August 2010

The objective of the present study was to evaluate the influence of different P4 concentrations, associated or not with eCG, in the ovulation and pregnancy rates of Nellore heifers submitted to hormonal protocols for FTAI. In experiment (exp.) 1, heifers (n = 15) presynchronized with a hormonal treatment were randomly allocated in 4 groups. At the time of intravaginal device insertion (Day 0) all animals received 2 mg of estradiol benzoate (EB) and a intravaginal progesterone-releasing device containing 0.558 g of P4 (Triu®, Biogenesis Bago, Buenos Aires, Argentina; group 0.5G, n = 4) or 1g of P4 (Cronipres®, Biogenesis Bago; group 1G, n = 4), or 0.558 g of P4 and PGF2α (150 μg of D-cloprostenol, group 0.5G/PGF, n = 4), or 1 g of P4 and PGF2α (group 1G/PGF, n = 3). Eight days later (Day 8), PGF2α (150 μg D-cloprostenol) was administered to all heifers, and the P4 source was removed. Twenty-four hours after removing the intravaginal device (Day 9), the heifers were treated with EB (1 mg, i.m.). Blood samples were collected on Days 0, 1, 2, 4, 8, and 10 to evaluate plasma P4 concentration. In exp. 2, cycling Nellore heifers (n = 292) were treated with the same protocols as in the exp. 1. However, 30 to 36 h later all animals were FTAI on Day 10. In exp. 3, heifers (n = 459) received the treatments described for groups 0.5G/PGF and 1G/PGF associated with 300 IU of eCG (i.m. groups 0.5G/PGF/eCG and 1G/PGF/eCG) at the time of intravaginal device removal (Day 8). In a subset of heifers from each group, ovarian ultrasonography was performed to determine the diameter of the dominant follicle on Day 9 (exp.3) and the ovulation rate on Day 12 (exp. 2 and 3). ANOVA for repeated measurements was used to compare P4 levels, and the binary variables (ovulation and pregnancy rates) were analyzed by logistic regression. In exp. 1, P4 concentrations were higher in animals that received 1.0 compared with 0.558 g of P4, and lower in heifers that received PGF2α on Day 0. In both exp. 2 and 3 there were no significant differences in ovulation (65 to 77%) and pregnancy (exp.2: 26 to 33%; exp.3: 39 to 43%) rates among the several groups. In exp. 3, the animals that received 0.558 g of P4 had a larger diameter of the dominant follicle on Day 9 compared with heifers treated with 1.0 g (10.3 ± 0.2 v. 9.3 ± 0.2 mm; P < 0.01). It is concluded that reduction of P4 concentration in the intravaginal device or the administration of PGF2α at the beginning of the treatments decreases blood levels of P4 between Days 1 and 8 of the FTAI protocols. However, although the use of intravaginal devices with lower P4 contributed to increase the dominant follicle diameter on Day 9, there was no significant increase on ovulation and pregnancy rates. Additionally, the association of eCG with FTAI protocols did not improve follicular growth or ovulation and pregnancy rates.

Supported by FAPESP (Sao Paulo, Brazil).
301 THE EFFECT OF EQUINE FOLICULLAR FLUID ON IN VITRO INDUCTION OF THE ACROSOME REACTION IN FROZEN - THAWED STALLION SPERMATOZOA
Authors: Silva D. S. , Rodriguez P. , Arruda N. S. , Rodrigues R. , Rodrigues J. L.
Published: August 2010

The capacitation process occurs in vivo upon exposure of the spermatozoa through the female reproductive tract, but can be induced in vitro in the presence of several compounds. This study was conducted to assess the effect of heparin or equine follicular fluid on hyperactivated motility and in vitro induction acrosome reaction swim-up method with frozen-thawed stallion semen. Two hundred microliters of frozen-thawed equine semen was placed in a tube (45°C) to increase contact area and incubated at 37°C for 1 h. After incubation 800 μL of the supernatant was collected by centrifugation (500 × g, 10 min) to collect spermatozoa. The resulting pellet was resuspended in capacitation medium Fert-TALP supplemented with 5.0 μg mL-1 heparin or 100% follicular fluid and incubated for different times (1, 2, 3, 4, and 5 h) at 37°C. After incubation the hyperactivated motility and acrosome-reacted spermatozoa were evaluated. Hoechst stain was used to differentiate live and dead spermatozoa, and chlortetracycline (CTC) fluorescent stain was used to assess the capacitation response of sperm; data were analyzed by ANOVA. The effect of equine follicular fluid resulted in improved percentage of spermatozoa with acrosome reaction at all times of incubation (60, 63, 57, 52, and 58%) but immediately after 3 h of incubation, the hyperactivated motility decreased in heparin group and follicular fluid (42 and 30%, respectively).
303 EFFECT OF SEMEN BATCHES AFTER SEXING ON PRODUCTION EFFICIENCY OF BOVINE EMBRYOS IN VITRO
Authors: Torregrossa T. L. G. , Fernandes M. B. , Prado R. B. , Vila R. A. , Elias F. P. , Netto H. A.
Published: August 2010

Within an in vitro production system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effects are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation among frozen sexed semen batches (Sexing Technologies, Navasota, TX, USA; personal communication) when used with IVF. In an embryo commercial production system, 5058 viable oocytes obtained by ovum pick-up with ultrasound from 193 Nelore cows (Bos indicus) over a 12-month period were matured in vitro for 24 h in TCM-199 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 0.5 μg mL-1 FSH (Bioniche Animal Health, Belleville, Ontario, Canada), 50 μg mL-1 LH (Intervet, Boxmeer, the Netherlands) and 10% fetal bovine serum (Gibco). Mature oocytes were inseminated in vitro for 18 h in IVF-Talp medium (BSA-FAF 6 mg mL-1; 10 ng of heparin, Sigma, France), using 3 different batches (I, II, III) of frozen-thawed sexed semen from 4 bulls (A, B, C, D), separated with a Percoll gradient (45:90; Sigma, St-Quentin Fallavier, France). Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium droplets (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) with 3 mg mL-1 BSA-FV (Sigma) for 163 h in a humidified incubator at 39°C, with an atmosphere of 5% CO2 in air. Total number of oocytes, total number of blastocysts, and embryonic developmental rates for each bull and respective batch are reported in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is difference between the batches used with respect to developmental rates of blastocysts. Therefore, there is intra-bull variation in the ability to produce in vitro embryos according to the batch of frozen sexed semen.

Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF
309 MORPHOMETRICAL ANALYSIS OF THE LAMA GLAMA SPERM HEAD
Authors: Casaretto C. I. , Lombardo D. , Giuliano S. , Gambarotta M. , Carretero M. I. , Trasorras V. L. , Miragaya M. H.
Published: August 2010

During the last decades, the interest in breeding South American Camelids has increased, not only as companion animals but also for their high- quality fiber. Although several studies have been carried out on artificial insemination in Lama glama, this technique has not been widely applied in reproductive programs, principally due to the difficulty in collecting raw semen from males and the lack of knowledge about freezing/thawing techniques and semen characteristics. The aim of the present study was to objectively characterize llama sperm morphometry by a computer-assisted system, thereby increasing the knowledge on male llama physiology, leading to further developement of reproductive biotechnologies such as artificial insemination. Five semen samples were obtained by electroejaculation from each of 8 males, 6- to 10-year-old llamas of proven fertility. Smears were prepared from each sample and stained with Tinción 15® (Biopur S.R.L., Rosario, Argentina) and observed at x 1000 magnification. Images of sperm heads were captured by a Leica DC180 camera (Leica Microsystems Co., Wetzlar, Germany), obtaining 200 images from each sample. Binary images were obtained and area, length, width, equivalent circle diameter, curve length, curve width, perimeter, convex perimeter, roundness, and elongation were measured using QWin Plus (Leica Microsystems Co.). A total of 8005 sperm heads were measured. Descriptive statistics of the complete population was performed, with the following results obtained (mean ± SD): area (μm2) 20.09 ± 0.6, length (μm) 6.6 ± 0.3, width (μm) 4.14 ± 0.1, equivalent diameter ((μm) 5.06 ± 0.1, curve length (μm) 5.8 ± 0.3, curve width (μm) 3.48 ± 0.3, perimeter (μm) 18.54 ± 0.1, convex perimeter (μm) 17.34 ± 0.3, roundness 1.28 ± 0.04, and elongation 1.6 ± 0.01. Coefficients of variation were between 0.47 and 8.72%. A design considering the male as a fixed factor and the ejaculate as a nested factor was used for the purpose of identifying differences in morphometry between ejaculates of the same male and/or between males. Normality was tested using the Kolmogorov test. Significant differences between ejaculates of some males were found for curve length, curve width, perimeter, roundness, and elongation (P < 0.05). There were no intra-male differences for sperm head area, length, width, equivalent circle diameter, and convex perimeter. Of the parameters, there were significant differences between males for sperm area, length, equivalent circle diameter, and convex perimeter (P < 0.05). The differences found in sperm morphometry confirm the great polymorphism observed when subjectively evaluating llama semen morphology and make the establishment of a single pattern of normal llama sperm morphometry impossible.
310 SEASON INFLUENCE UPON SEMINAL PLASMA PROTEINS IN BROWN SWISS BULLS
Authors: Chacur M. G. M. , Sirchia F. P. , Ruiz A. C. L. , Guaberto M. L.
Published: August 2010

Seminal plasma is a complex of secretions of the male accessory reproductive organs and appears to exert important effects on sperm function (Shivaji et al. 1990 Proteins of Seminal Plasma, Wiley, New York, NY, USA). The protein quality of the seminal plasma may affect positively the bulls’fertility (Killiam et al. 1993 Biol. Reprod. 49, 1202-1207). Peptides of 55 and 66 kDa were present in bulls with excellent spermatic conditions for example motility and vigor. On the other hand, 16- and 36-kDa peptides were observed with unfavorable spermatic conditions (Chacur et al. 2009 Anim. Reprod. 6, 339). The objective was to determine the influence of season on seminal plasma proteins in Brown Swiss bulls. Semen from 33 Brown Swiss bulls 24 months of age was collected by electroejaculation during winter (from June to August) and summer (from December to February) in the southern hemisphere in 2008. Semen samples were collected with 14-day intervals totalizing 196 ejaculates. Samples of semen were centrifuged (1500g/15 min) and the seminal plasma was conditioned in cryotubes and stored at -20°C until further processing. Proteins were extracted from 200 μL of each sample in 2 mL of extraction buffer composed of 0.625 M Tris-HCl, at pH 6.8, in 2% SDS, 5% fi-mercaptoethanol, and 20% of glycerol. Percentages of different plasma proteins by season were statistically compared by the chi-square test with significance level at P < 0.05). Proteins were quantified according to Bradford (1976 Anal. Biochem. 72, 248-254) and electrophoresis was performed according to Laemmili (1970 Nature 227, 680-685). Gels were fixed with isopropanol: acetic acid: water (4:1:5 v/v) for 30 minutes and stained in the same solution with 2% of Coomassie Blue R250. In 26 bulls, the absence of high molecular weight (HMW; 55 kDa, 66 kDa, and 80 kDa) proteins was found in the summer. There was a significant increase (P < 0.05) in total spermatic defects, acrosome defects, and distal cytoplasmatic droplets in these bulls. The 40-kDa protein that reflected low fertility was observed in 10 bulls in the summer with semen quality decreases. The 11 bulls showed presence of HMW (55 kDa) in the winter. In 11 bulls, HMW (55 kDa, 66 kDa, or 80 kDa) proteins were present with a satisfactory semen condition according to Killiam et al. (1993 Biol. Reprod. 49, 1202-1207). In conclusion, the seasons of the year may influence the presence of proteins in seminal plasma. There was a direct relationship of the season with seminal plasma proteins. The presence of the proteins of 20 kDa, 55 kDa, 66 kDa, and 80 kDa suggested an increase of the semen quality during the winter.

UNOESTE and Bartira Farm.
317 RESISTANCE AGAINST DIFFERENT REACTIVE OXYGEN SPECIES IN BOVINE EPIDIDYMAL SPERM UNDER DISTINCT MATURATION STATUS
Authors: Nichi M. , Perez E. G. A. , Viana C. H. C. , Teodoro A. C. , Goes P. A. A. , Bols P. E. J. , Dalmazzo A. , Ferreira G. H. , Silva R. O. C. , Barnabe R. C. , Barnabe V. H.
Published: August 2010

Oxidative stress is caused by reactive oxygen species (ROS) that may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components. Evidence indicates that oxidation products are also deleterious to biological systems. Spermatozoa are particularly susceptible the oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in its membrane. The mechanisms by which sperm acquire antioxidant capacity are still not completely elucidated. The aim was to study the resistance of sperm derived from different epididymal compartments (caudae and head) to the different ROS and to the lipid peroxidation product malondialdehyde (MDA). Epididymal sperm samples from 4 testicles were collected from the head and caudae epididymides. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical), and MDA. Samples were analyzed for 3-3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin nigrosin stain, as an index of membrane integrity; the simple stain (fast green/Bengal rose), as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Pearson correlation). Results showed that immature sperm (head epididymides) were significantly more susceptible to the MDA and to the hydroxyl radical in all studied variables, especially acrosomes, membranes, and mitochondrial potential. Semen derived from the caudae epididymides was more susceptible to the hydrogen peroxide and to the MDA, especially regarding mitochondrial potential. In semen from the epididymal head, a positive correlation was found between TBARS and sperm showing no mitochondrial potential (r = 0.66, P = 0.01). On the other hand, negative correlations were found between TBARS and sperm with damaged acrosome and membrane (r = -0.63, P = 0.01 and r = -0.58, P = 0.02, respectively) in samples collected from the caudae epididymides. The present results suggest that sperm susceptibility to the attack of ROS is different throughout maturation. Although immature sperm are more susceptible to the hydroxyl radical, mature sperm are more susceptible to the hydrogen peroxide. Furthermore, MDA, a product of lipid peroxidation, is also deleterious to the sperm, indicating that once oxidative stress starts, further damage may be caused by their products.
The authors thankNutricell for the media used in the experiment andFAPESP for financial support (process #06/05736-1).
322 EFFECT OF PLASMIN ON ACROSOME REACTION OF BUFFALO (BUBALUS BUBALIS) SPERMATOZOA IN VITRO
Authors: Venditto I. , Mariotti E. , Boccia L. , Rubessa M. , De Blasi M. , Sattar A. , Gasparrini B.
Published: August 2010

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolytic enzymes are involved in different steps of the fertilization process; among these, a critical role may be played by the plasminogen activator-plasmin system. It has been demonstrated that plasmin, the active enzyme of this system, induces acrosome reaction (AR) in bull spermatozoa (Taitzoglou IA et al. 2003 Andrologia 35, 112-116). The aim of this study was to investigate the effect of plasmin on the ability of buffalo sperm to undergo the AR. Frozen- thawed sperm from 4 buffalo bulls were treated by swim-up and incubated with 0.01 mM heparin for 4 h. At 0, 2, and 4 h, aliquots of spermatozoa were exposed for 10 min to 60 μg mL-1 of lysophosphatidylcholine (LPC), as positive control, and to 0.01 μg mL-1 of plasmin. This concentration was chosen after a preliminary dose-response trial. Another sample from each treatment was incubated with IVF medium (negative control). After 10 min, sperm motility was evaluated and sperm were fixed in 37% formaldehyde and stained with trypan blue-Giemsa for subsequent microscopic examination. The total number of sperm counted, over 3 replicates, was 1269 for the negative control, 1293 for LPC, and 1238 for plasmin, equally distributed among incubation times. Differences among groups were analyzed by chi-square test. After swim-up, acrosomal loss was observed only in 4% of the sperm. The addition of 0.01 μg mL-1 of plasmin for 10 min to buffalo spermatozoa at time 0 significantly (P < 0.01) enhanced (23%) AR compared with the control (7.8%), with the same efficiency of LPC (17.1%). After 2 h of incubation with heparin, both plasmin and LPC increased the AR compared to the control (24.4, 20.1, and 14.0%, respectively; P < 0.01). After 4 h, plasmin gave higher percentages of AR (27.2%) compared to both the control (21.0%; P < 0.05) and LPC (19.2%; P < 0.01). Another interesting result is the improved motility recorded with plasmin compared to both the control and LPC groups at 2 h of incubation (90, 75, and 75%, respectively; P < 0.05) and at 4 h of incubation (75, 60, and 60%, respectively; P < 0.05). Finally, no differences in sperm viability were observed between plasmin and the control, whereas a decreased viability was found when LPC was used at 0 h (96.2, 95.0, and 89.0%, respectively, for plasmin, control, and LPC; P < 0.05), at 2 h (85.0, 87.5, and 77.0%, respectively, for plasmin, control, and LPC; P < 0.01), and at 4 h (85.0, 93.3, and 81.1%, respectively, for plasmin, control, and LPC; P < 0.01). In conclusion, we found that addition of plasmin to capacitated sperm increases the percentage of acrosome-reacted spermatozoa and improves motility. Our results suggest that plasmin may play a role in events surrounding fertilization and suggest to evaluate in further studies whether the addition of plasmin during IVF improves the IVEP efficiency in buffalo.
323 POSSIBLE BENEFICIAL EFFECT OF REACTIVE OXYGEN SPECIES ON BOAR SPERM FUNCTION
Authors: Viana C. H. C. , Nichi M. , Silva R. O. C. , Perez E. G. A. , Garcia R. N. , Lima P. F. , Cabral R. P. , Silva D. G. , Teodoro Â. C. , Barnabe V. H. , Barnabe R. C.
Published: August 2010

Boar spermatozoa are sensitive to the attack of reactive oxygen species (ROS) due to the high content of unsaturated fatty acids in its plasma membrane and the relative low antioxidant capacity of boar seminal plasma. The benefical effect of antioxidant supplementation to the semen of boars is still a matter of debate, with different results in the literature, probably due to the empirical methods used to determine the more adequate antioxidant, and to the physiological role or the ROS. The aim of the present experiment was to study which ROS is the most deleterious to the boar sperm. Sperm samples from 4 boars were collected by digital manipulation. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produced hydroxyl radical), and malondialdehyde (lipid peroxidation product). Samples were analyzed for the 3-3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin/nigrosin staining, as an index of membrane integrity; the simple stain (fast green/Bengal rose), as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation. Results showed that the boar sperm is relatively resistant to all ROS studied in the present experiment. Only for the percentage of sperm showing intact membrane, a difference was found between incubation with superoxide anion and hydroxyl radical (Table 1). The TBARS production was higher in samples incubated with hydroxyl radical, indicating that these samples were more susceptible to the attack of this ROS. Surprisingly, a positive correlation was found between TBARS and membrane integrity (r = 0.51, P = 0.04), indicating that samples more susceptible to the oxidative stress showed higher membrane integrity. A hypothesis to explain such conflicting results would be the physiological role of the ROS on several spermatic events, especially in this particular species. It is well known that boar semen, in order to achieve ideal motility, requires aerobic conditions. Further studies are necessary to study the effect of each ROS on each physiological or pathological event on boar sperm function.

Table 1.Effect of different ROS in boar semen

The authors thank Nutricell for the media used in the present experiment.
329 EXPRESSION AND REGULATION OF GENOMIC AND NON-GENOMIC PROGESTERONE RECEPTORS IN BOVINE CUMULUS - OOCYTE COMPLEXES DURING MATURATION IN VITRO
Authors: Aparicio I. M. , Hensey C. , Lonergan P. , Fair T.
Published: August 2010

The steroid hormone, progesterone (P4) plays an essential role in the regulation of female reproductive events. It acts by binding to specific receptors classified as genomic (nPR-A, nPR-B) and non-genomic (mPRα, mPRβ, PGRMC1, PGRMC2) progesterone receptors (PRs). The aim of the present work was to identify and characterize the PRs that are expressed in bovine COCs during IVM. Immature COCs collected from the ovaries of slaughtered animals and either allocated to analysis at the immature, germinal vesicle stage (GV) or processed for IVM and collected at the metaphase II stage (MII) of meiosis. Groups of up to 50 COCs were placed in 500 mL of maturation medium (TCM-199) supplemented with 10% (v/v) FCS and 10 ng mL-1 epidermal growth factor without or with FSH (100 ng mL-1), LH (100 ng mL-1), or P4 (10, 100, 500 ng m-1), in 4-well dishes and cultured at 39°C for 24 h in a humidified atmosphere containing 5% CO2. GV and MII COCs were stripped of their cumulus cells and 100 oocytes and 10 μg of protein from cumulus cells were resolved by SDS-PAGE using specific antibodies against the different PRs. In addition, the pattern of PR expression was characterized in wholemount preparations of GV and MII COCs by immunocytochemistry using the same antibodies. nPR-A, mPRα, and mPRβ were expressed in both bovine oocytes and their surrounding cumulus cells, while expression of nPR-B, PGRMC1, and PGRMC2 was detected solely in the cumulus cells. Cumulus cell expression of nPR-A and nPR-B, mPRα, and mPRβ increased following IVM, while PGRMC1 and PGRMC2 expression decreased. In contrast to the expression profile of the cumulus cells, nPR-A, mPRα, and mPRβ expression was downregulated following IVM in the corresponding oocytes. Supplementation of the IVM media with FSH or LH resulted in an increase in the expression of nPR-A and nPR-B in cumulus cells and a decrease of nPR-A in oocytes. In contrast, P4 supplementation (500 ng mL-1) during IVM resulted in increased cumulus cell nPR-B expression and decreased nPR-A, mPRα, and mPRβ expression in oocytes. Oocyte mPRβ expression was also downregulated by LH and FSH. PGRMC1 and PGRMC2 were upregulated by 500 and 10 ng mL-1 of P4 respectively. In conclusion, the dynamic changes observed in the protein expression of genomic and non-genomic P4 receptors in bovine COCs after IVM and after treatment with LH, FSH, and P4 suggest that some or all of these receptors may be involved in oocyte maturation in cattle.
Supported by Science Foundation Ireland (07/SRC/B1156). IMAparicio was supported by a postdoctoral contract (POS07011) from Consejeria de Economia, Comercio e Innovacion, Junta de Extremadura, Spain.
337 MITOCHONDRIA AND REACTIVE OXYGEN SPECIES IN PREBUPERTAL LAMB OOCYTES BEFORE AND AFTER IN VITRO MATURATION
Authors: Dell'Aquila M. E. , Ambruosi B. , Guastamacchia R. , Binetti F. , Ciani E. , Cianci D. , Lacalandra G. M.
Published: August 2010

Juvenile in vitro embryo transfer (JIVET) reduces the generation interval and increases the rate of genetic gain. The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Oxidative stress in the oocyte is an emerging problem in reproductive in vitro technologies, due to the gas atmosphere used to incubate oocytes and the lack of physiological defense mechanisms available in the female reproductive tract. The major source of reactive oxygen species (ROS) is represented by mitochondria where ROS are produced during oxidative phosphorylation. The aim of the present study was to analyze mitochondria and ROS in ovine prepubertal oocytes before and after IVM in order to clarify their suitability in JIVET programs. Cumulus-oocyte complexes from the ovaries of 38 slaughtered prepubertal (less than 8 months of age) lambs of the Comisana breed were analyzed at retrieval (group A) or after IVM (group B; Ambruosi et al. 2009 Theriogenology 71, 1093-1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mitochondria and ROS evaluation by confocal analysis of fluorescence distribution and intensity. Hoechst 33258 and Mitotracker Orange CMTM Ros (Molecular Probes Inc., Eugene, OR) were used to label nuclear chromatin and mitochondria (Ambruosi et al. 2009) and 2′,7′-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353-360). Out of 65 oocytes from group A, 38 oocytes with regular size (>130 μm in diameter), morphology and nuclear chromatin at the GV stage were selected for analysis. One-hundred-thirty-eight oocytes underwent IVM (group B). Nuclear maturation rate (metaphase II with 1st polar body extruded) was 54%, 75/138. All MII oocytes were used for analysis. Significantly higher rate of oocytes from group B showed heterogeneous (large aggregates, clusters, pericortical, perinuclear) mitochondrial (mt) distribution pattern than oocytes from group A (55%, 41/75 v. 29%, 11/38, respectively; P < 0.05) which showed uniform distribution of small mt aggregates. Fluorescent intensity of mt labeling did not differ between groups (43.05 ± 16.15 v. 45.89 ± 10.36, for group A and B respectively; NS). In most of the oocytes from both groups, intracellular ROS were distributed in small or large aggregates (35/38, 92% and 62/75, 83%). No statistical difference was observed for intracellular ROS levels between oocytes from group A (66.36 ± 13.2) and group B (72.84 ± 20.63; NS). The culture conditions used in this study provided normal mt distribution and intracellular ROS levels. Qualitative and quantitative evaluation of mitochondria and intracellular ROS could be useful to improve in vitro culture methods in ovine prepubertal oocytes.