bluuebunny/crossref_metadata_2025 huggingface dataset:
216 PRONUCLEAR FORMATION AND DEVELOPMENTAL COMPETENCE OF MATURE PORCINE OOCYTES DERIVED FROM SMALL- AND MEDIUM-SIZED FOLLICLES
Authors: Kohata C. , Funahashi H.
Published: August 2011

The maturation rate of oocytes derived from small follicles (SF) is known to be lower than that of oocytes from medium follicles (MF). The objective of this study was to assess the fertilizability and developmental competence of mature SF oocytes that were selected by the presence of the first polar body. Cumulus–oocyte complexes (COC) were aspirated from SF (1 to 2 mm in diameter) or MF (3 to 6 mm in diameter) of prepuberal ovaries. The COC were cultured in modified porcine oocyte medium supplemented with gonadotropins and dibutyryl cAMP for the first 20-h period and then in gonadotropin-free and dibutyryl cAMP-free porcine oocyte medium for another 24 h. Following IVM culture, mature oocytes with the first polar body were selected under a stereomicroscope, co-incubated with spermatozoa in a drop of modified TCM-199 containing 0.4% BSA and 5 mM caffeine for 6 h, and then incubated in porcine zygote medium-5 for 7 days. Sperm penetration, cleavage, and early development of the oocytes were examined before culture in porcine zygote medium-5 on Days 2 and 7 of culture. To analyse the fertilizability and developmental competence of oocytes from the SF and MF groups, sperm penetration, pronuclear formation, cleavage, blastocyst formation, and mean cell number in a blastocyst (as determined by fluorescence observation following Hoechst 33342 staining) were examined. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post-hoc test (P < 0.05). The percentages of oocytes in which the first polar body could be observed were 51.0 ± 4.5% and 78.5 ± 2.8% for SF- and MF-oocytes, respectively, whereas the maturation rates were 83.8 ± 4.0% and 62.8 ± 4.4% following fixation and staining. When only mature oocytes were co-cultured with sperm for 6 and 9 h, sperm penetration, monospermic penetration, and pronuclear formation were not different (P > 0.33) between mature SF- and MF-oocytes. Although there was no difference in cleavage rates between the mature SF- and MF-oocyte groups, blastocyst formation rate and mean cell number in the blastocyst were higher in mature MF-oocytes (31.0 ± 3.6% and 38.7 ± 1.9 cells, respectively) than in mature SF-oocytes (14.7 ± 3.2% and 31.2 ± 2.0 cells). From these results, we conclude that mature oocytes derived from SF have a similar fertilizability when compared with mature MF-oocytes, but the developmental competence to the blastocyst stage following IVF is significantly lower in mature SF-oocytes than in mature MF-oocytes.
217 EFFECTS OF PORCINE GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ON PORCINE IN VITRO-FERTILIZED EMBRYOS
Authors: Kwak S. S. , Biswas D. , Hyun S. H.
Published: August 2011

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in the female reproductive tract and is one of the regulatory molecules that mediate maternal effects on the growth and development of pre-implantation embryos in several species. The objective of the present study was to investigate the effects of porcine GM-CSF (pGM-CSF) on the developmental potential of porcine IVF embryos. All experiments were performed with zygotes that were produced in vitro and cultured in porcine zygote medium-3–polyvinyl alcohol-based medium. Data were analysed with PASW statistics-17 (SPSS Inc., Chicago, IL) using Duncan’s multiple range test. A total 865 zygotes in 4 replicates were used with different concentrations of pGM-CSF (0, 2, 10, 100 ng mL–1) in Experiment 1. It was demonstrated that 10 ng mL–1 of pGM-CSF could increase (15.1 ± 2.2) blastocyst development significantly (P < 0.05) compared with the control (6.1 ± 0.7). There was no effect on cleavage rate. In blastocyst formation, early and expanded blastocysts were significantly (P < 0.05) higher in the 10 ng mL–1 of pGM-CSF group compared with the control. In Experiment 2, a total 839 zygotes with at least 5 replicates in each group were used, and whether pGM-CSF would act to increase blastocyst yield before or after Day 4 development was tested. Zygotes were cultured with the following treatments: 1) zygotes cultured with fresh porcine zygote medium–polyvinyl alcohol medium from Days 0 to 7 post-insemination as a control; 2) medium supplemented with 10 ng mL–1 of pGM-CSF from Days 0 to 4 followed by no pGM-CSF from Days 4 to 7; 3) medium alone from Days 0 to 4 followed by supplementation with 10 ng mL–1 of pGM-CSF from Days 4 to 7; and 4) medium supplemented with 10 ng mL–1 of pGM-CSF from Days 0 to 7. As compared with the controls (7.8 ± 0.7), pGM-CSF influenced the percentage of blastocyst formation when pGM-CSF was added from Days 4 to 7 (14.6 ± 1.6) or Days 0 to 7 (15.2 ± 1.8), but not from Days 0 to 4 (8.7 ± 1.5). Similarly, the early blastocyst formation rates were significantly higher in the Day 4 to 7 culture period compared with the control, and expanded blastocyst formation was significantly higher in the Day 4 to 7 and Day 0 to 7 culture periods. There was no significant different in cleavage rate among these groups. In conclusion, these data suggest that supplementation of pGM-CSF in in vitro culture medium at Days 4 to 7 or Days 0 to 7 promotes the developmental potential of porcine IVF embryos.

This work was supported by a grant (20070301034040) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.
22 INFLUENCE OF SEASONALITY ON OVULATORY FOLLICULAR WAVE DYNAMIC IN LONG PROTOCOLS IN SANTA INÊS SHEEP IN THE TROPICS
Authors: Oliveira M. E. F. , Ayres H. , Oliveira L. G. , Teixeira P. P. M. , Bicudo S. D. , Fonseca J. F. , Vicente W. R. R.
Published: August 2011

This study was designed to investigate the influence of seasonality on ovulatory follicular wave dynamics in long protocols, with or without P4 device replacement, in Santa Inês sheep. Seventy adult ewes were submitted to 2 synchronization protocols in 3 seasons (factorial 2 × 3; non-breeding: G-1CIDR, n = 12 and G-2CIDR, n = 11; transition: G-1CIDR, n = 12 and G-2CIDR, n = 12; breeding: G-1CIDR, n = 11 and G-2CIDR, n = 12). On D0 (randomised day of oestrus cycle), the oestrus was synchronized with a P4 device (CIDR™; Pfizer, New Zealand) for 14 days. However, in G-2CIDR, the CIDR was replaced by a new one on D7. At D0 and 14, 2.5 mg of dinoprost (Lutalyse™, Pfizer, New Zealand), IM, were administered, and on D14, all ewes received 300 IU of eCG (Novormon™, Syntex, Argentina). Ultrasonographic exam was performed daily between D0 and D14 and, every 8 h until D19. Data were analysed by GLIMMIX using SAS (SAS Institute Inc., Cary, NC, USA). There was no interaction between groups and seasons, being presented the main effects for the variables. There were effects of treatment on the day of emergence of an ovulatory wave (G-1CIDR: 8.28 ± 0.54 and G-2CIDR: 9.23 ± 0.44; P = 0.04), maximum diameter of first ovulatory follicle (G-1CIDR: 8.09 ± 0.22 v. G-2CIDR: 7.62 ± 0.19 mm; P = 0.02) and duration of follicular growth (G-1CIDR: 192.00 ± 11.27 v. G-2CIDR: 175.70 ± 9.92 h; P = 0.07). However, there were no effect of treatment on day of ovulation (G-1CIDR: 17.11 ± 0.11 v. G-2CIDR: 17.20 ± 0.10; P = 0.43), ovulatory diameter (G-1CIDR: 7.45 ± 0.21 v. G-2CIDR: 7.34 ± 0.19 mm; P = 0.59), and follicular growth rate (G-1CIDR: 0.69 ± 0.04 v. G-2CIDR: 0.71 ± 0.03 mm day–1; P = 0.65). Effects of season were observed on day of emergence (non-breeding: 7.07 ± 0.641a v. transition: 9.09 ± 0.61b v. breeding: 9.89 ± 0.48b; P < 0.0001), maximum diameter of first ovulatory follicle (non-breeding: 8.28 ± 0.24a v. transition: 7.71 ± 0.26b v. breeding: 7.54 ± 0.21b mm; P = 0.01), ovulatory diameter (non-breeding: 7.70 ± 0.23a v. transition: 7.59 ± 0.26a v. breeding: 6.88 ± 0.21b mm; P = 0.006) and duration of follicular growth (non-breeding: 216.11 ± 12.38a v. transition: 177.38 ± 13.67b v. breeding: 162.57 ± 11.28b h; P = 0.0004). Season had no effect on day of ovulation (non-breeding: 17.15 ± 0.12 v. transition: 17.05 ± 0.14 v. breeding: 17.28 ± 0.11; P = 0.40), and follicular growth rate (non-breeding: 0.66 ± 0.04 v. transition: 0.67 ± 0.05 v. breeding: 0.77 ± 0.04 mm day–1; P = 0.11). Therefore, both protocols showed efficiency in synchronization regardless of the season. Furthermore, protocols and seasons promoted effect on the day of emergence of ovulatory wave, thus influencing the other variables.

Supported by FAPESP.
220 RELATIONSHIP BETWEEN FOLLICULAR FLUID METABOLOME AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE
Authors: Matoba S. , Bender K. , Brennan L. , Lonergan P. , Fair T.
Published: August 2011

The follicle is a unique microenvironment within which the oocyte can develop and mature to a fertilizable gamete. The aim of this study was to examine the predictive value of the intrafollicular metabolomic profile on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from the ovaries of slaughtered animals. Following measurement of diameter (5 to 8 mm), follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba et al. 2010 Reprod. Fertil. Dev. 22, 839–851). Cleavage and blastocyst rates were assessed on Day 2 and Day 9, respectively. Follicular fluid was recovered and stored at –80°C until metabolomic analysis. Only samples that cleaved and developed to the blastocyst stage (n = 10) or that cleaved and subsequently degenerated (n = 10) were analysed. Aqueous and organic compounds were isolated and profiled separately using an Agilent 7890A gas chromatograph coupled with a 5975C mass spectrometer (Agilent, Palo Alto, CA). Both aqueous and organic data sets were divided into compounds identified and quantified using external standards and compounds semiquantified relative to the internal standard only. The percentage of oocytes that cleaved after IVF was 79.1% and the percentage that developed to the blastocyst stage was 28.3%. A total of 55 aqueous compounds were analysed, of which 19 were quantified and 36 were semiquantified. Principal component analysis of the quantified data showed separation between oocytes that formed blastocysts and oocytes that degenerated (R2 = 0.561). Further analysis using partial least squares–discriminant analysis generated a robust model that could predict which oocytes would form blastocysts (R2X = 0.552; R2Y = 0.874; Q2 = 0.722). Analysis of variance of the data confirmed a positive correlation of L-alanine, glycine, and L-glutamate with blastocyst formation. A total of 16 fatty acids were identified and quantified, and the total saturated fatty acids, total monounsaturated fatty acids, total polyunsaturated fatty acids (PUFA), n-3 PUFA, and n-6 PUFA contents were determined. Analysis of variance of the fatty acid data showed that follicular fluid from competent oocytes was significantly lower in palmitic acid (P = 0.023) and total saturated fatty acids (P = 0.031) than in follicular fluid from degenerate oocytes. No differences in total monounsaturated fatty acids, total PUFA, n-3 PUFA, and n-6 PUFA were found between blastocysts and oocytes that degenerated. In conclusion, our data suggest that aqueous and organic compounds in follicular fluid may be predictive of oocyte developmental outcome in an in vitro embryo production system.

Supported by Science Foundation Ireland (07/SRC/B1156).
221 EFFECT OF STAGE OF CORPUS LUTEUM DEVELOPMENT ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Miyashita S. , Miyata K. , Tachibana C. , Inaba Y. , Koyama H. , Dochi O.
Published: August 2011

The objective of this study was to investigate the effect of stage of corpus luteum (CL) development on the in vitro production of bovine embryos. Ovaries were classified according to the expected day of the oestrous cycle based on the morphology of the ovaries. Ovaries with a corpus hemorrhagicum and the remnant of the follicular lumen filled with blood were considered the early luteal stage (Days 2 to 4; Day 0 = day of ovulation, n = 46). Ovaries with a large mass of orange tissue in the CL were classified as the midluteal stage (Days 7 to 10, n = 42). Cumulus–oocyte complexes (COC) were collected by aspiration of 2- to 6-mm follicles. The COC were classified into the following grades: COC with >3 compact layers of cumulus cells and evenly granulated cytoplasm were classified into Grade 1; COC with >3 layers cumulus cells and evenly granulated cytoplasm were classified into Grade 2; COC with partially remaining cumulus cells and abnormal cytoplasm were classified into Grade 3; COC without cumulus cells or those with expanded cumulus cells were classified into Grades 4 and 5, respectively. Grades 1 and 2 COC were in vitro matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 mg mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air. Matured COC were inseminated with 5 × 106 sperm for 18 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). The mean number of COC and the proportion of COC classified as Grades 1 and 2 were analysed by ANOVA. Cleavage rates on Day 3 and blastocyst rates on Days 7 to 9 were analysed by a chi-square test. The mean number of recovered oocytes in the early luteal stage (18.7 ± 9.5) was significantly higher (P < 0.05) than the number in the midluteal stage (12.2 ± 5.7). The proportion of Grades 1 and 2 oocytes in the early luteal stage [66.7% (531/789)] was significantly higher (P < 0.01) than that in the midluteal stage [51.6% (252/484)]. The cleavage and blastocyst rates in the early luteal stage [60.9% (181/297) and 32.7% (97/297), respectively] were significantly higher (P < 0.05) than those in the midluteal stage [50.7% (76/150) and 20.7% (31/150) respectively].The present study suggests that the stage of development of the CL in bovine ovaries influences the number of recovered oocytes per ovary and the development of in vitro production of bovine embryos.
226 EVALUATION OF IN VITRO EMBRYO DEVELOPMENT OF BOVINE OOCYTES EXPERIMENTALLY EXPOSED TO MYCOPLASMA BOVIS
Authors: Pavao D. , Alves M. , Qureiroz R. , Souza F. , Angelo M. D.
Published: August 2011

Reproductive biotechnologies are known to improve the quality and productivity of herds. Research on cattle health and on the conditions of oocytes and embryos produced in vitro and in vivo are carried out worldwide to prevent the transmission of infectious agents, particularly those associated with ovarian tissues, the uterine tubes, and the uterus. Thus, there is concern regarding the possibility of transmitting Mycoplasma bovis through animal reproduction procedures. The aim of this study was to evaluate morphological changes by means of optical microscopy, cleavage rate, and blastocyst formation during the in vitro development of bovine embryos experimentally exposed to M. bovis. Oocytes were aspirated from ovaries of slaughtered cows, and oocytes with an intact zona pellucida were selected and matured. After 20 h, the oocytes were divided in 2 groups: a control group (n = 260), and oocytes exposed to 2.5 × 104 cfu mL–1 of M. bovis (n = 261). The semen was treated by a discontinuous Percoll gradient technique, and the sperm concentration was adjusted to approximately 100 000 sperm for each oocyte. Statistical analysis of the experiments was conducted using Student’s t-test (P < 0.05). Embryos exposed to M. bovis showed a cleavage rate of 21.07% (55/261), whereas the control group showed a cleavage rate of 64.6% (168/260). In relation to blastocyst formation, control embryos had a rate of 39.60% (103/260) v. 12.20% (32/261) for the exposed group. Statistical analysis of the experiments revealed a significant difference in the final results. Regarding morphological aspects, we observed failures in the division and asymmetry of blastomeres, cytoplasmic shrinkage, and degeneration and disruption of the zona pellucida in embryos exposed to the pathogen. These results are important to support the development of parameters for evaluating oocytes and in vitro-produced bovine embryos and to establish more efficient methods to control the spread of disease by animal reproduction biotechnologies. Further studies will be done with oocytes exposed to the pathogen and subsequently evaluated according to the Manual of the International Embryo Transfer Society.
227 THE EFFECTS OF SEXED SEMEN ON EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE
Authors: Plummer W. , Beckett D. , Schaffner A. , Layport B. , Norman J. , Grimbleby C. , Wessinger P. , Pace E. , Kirscher L. , Matthews K. , Dorshorst T.
Published: August 2011

Sexed semen (SS) exhibits approximately 80% of the fertilizing ability of conventional semen (CS), and studies have shown that this continues through the 8-cell stage of bovine embryo development. At the time of this study, no information could be found that, when used for IVF and intracytoplasmic sperm injection (ICSI) development, had been carried to the blastocyst stage. In addition, questions have arisen regarding which of the measured sperm parameters are responsible for the difference between the SS and CS and contribute to this decline in fertility. The goals of this project were to evaluate the effects of using sexed sperm as it relates to embryonic development and to determine if any of the differences in sperm parameters affect embryonic development. A preliminary project evaluated SS and CS from 5 bulls for IVF and ICSI. One bull was selected to provide the sperm (both SS and CS) for the trial, and 1752 oocytes were assigned to either IVF or ICSI. The SS and CS were divided among the available oocytes used for IVF and ICSI. Straws were thawed for 30 s at 37°C, and sperm were then evaluated for motility (provided by CASA, SpermVision, MiniTube of America, Verona, WI), morphology, acrosomal integrity (Coomassie and Pope stains), viability, and nuclear decondensation (SYBR Green and HALO). Results for SS v. CS were as follows: motility, 8 v. 26%; viability, 40.6 v. 30%; nuclear decondensation, 40 v. 30%; normal morphology and acrosomal integrity, no differences. Oocytes were obtained from Applied Reproductive Technologies, LLC (Madison, WI). The fertilization rate was consistently lower (Table 1) for both IVF and ICSI when SS were used (Z = 3.65; P = 0.0003), and there was no evidence that this decline in fertilization rate differed for the 2 methods (Z = 0.18; P = 0.86). Nor was there any evidence that the method affected the fertilization rate in general (Z = 0.75; P = 0.45). Thus, the difference was specific for fertilization rate and had no effect on Day 3 cells or Day 7 blastocysts. A higher fertility rate using ICSI would have indicated that a surface membrane factor may have been decreasing the fertility rate with SS because of the elimination of binding factors associated with ICSI. Thus, it may not be the sperm surface membrane that is distorted in the sexing procedure, but likely the integrity of the spermal DNA, as indicated by the increased nuclear decondensation of SS.

Table 1.Comparison of sexed sperm with conventional sperm when used for IVF and intracytoplasmic sperm injection (ICSI)

Partial funding for this project was made available by the California State University Agricultural Research Institute (ARI). Appreciation is extended to Sexing Technologies, Inc. (Navasota, TX) for donating the semen.
228 EVALUATION OF EMBRYO VIABILITY AFTER PROLONGED TRANSPORT IN DIFFERENT MEDIA AND AT DIFFERENT TEMPERATURES
Authors: Puelker R. Z. , Pupulim A. G. R. , Constant C. H. O. , Andreazzi M. A. , Emanuelli I. P.
Published: August 2011

In vitro-produced embryos (IVP) are known to have poor quality and be susceptible to heat and oxidative stress. In addition, they constantly undergo long-distance transport, which causes low conception rates. The aim of this study was to search for alternatives to long-distances transport of embryos produced in vitro by evaluating the use of different media and different temperatures. Bos indicus cumulus–oocyte complexes (COC; 823, quality I and II) were matured in TCM-199 bicarbonate–10% FBS (38.5°C, 5% CO2, in air) for 24 h. The fertilization was performed in TALP-IVF medium for 18 h of incubation. Presumptive zygotes were transferred to SOF medium and in vitro culture in a controlled atmosphere (5% O2, 5% CO2, 90% N2) for 7 days at 38.5°C. At Day 7, 366 embryos (quality I and II, IETS), were randomly allocated to the designated experimental groups. For Experiment 1, blastocysts were filled into straws and kept for 12 h at 36°C, using the medium as an independent variable according to the following groups: GSup, embryo support medium without FCS and amino acids (n = 115); and GHSOF, embryo culture medium SOF (HSOF) with FCS and amino acids (n = 105). Both media were buffered with HEPES. For Experiment 2, blastocysts were filled into straws with HSOF medium and sustained in transportation for 12 h, using temperature as the independent variable according to the following groups: G36, 36°C (n = 65); and G38, 38°C (n = 81). After 12 h of transport in both experiments, embryos were evaluated and classified as viable or nonviable blastocysts and were recultured in the same conditions mentioned. On Day 10, hatching rates and degeneration were evaluated. Logistic regression was used to compare the groups in each experiment. In Experiment 1, blastocyst viability after 12 h of transport was higher in the culture medium (GHSOF: 91.4%) than the support medium (GSup: 75.6%; P < 0.001). The hatching capacity in the culture medium (GHSOF: 72.4%) was higher than that in the support medium (GSup: 34.5%; P < 0.001). In Experiment 2, blastocyst viability post-transport was the same in both temperatures (G36: 92.3%, G38: 82.7%; P > 0.09). Blastocyst hatching capacity at 36°C (G36: 75.4%) was superior to that at 38°C (G38: 49%; P < 0.001). The hatching capacity immediately after 12 h of transport was minimal, so it was not possible to apply the logistic regression method in any experiment. In conclusion, the culture medium was more efficient for long-distance transport than the support medium; moreover, a temperature of 36°C for the culture medium increased embryo development compared with a temperature of 38°C during transport.

Financial support was provided by CESUMAR.
229 EVALUATION OF OOCYTE QUALITY RECOVERED FROM Gir BREED COWS WITH SYNCHRONIZED FOLLICULAR WAVE ON IN VITRO EMBRYO PRODUCTION
Authors: Iguma L. T. , Sales J. N. S. , Batista R. I. T. P. , Pereira M. M. , Quintão C. C. R. , Verneque R. S. , Camargo L. S. A. , Viana J. H. M. , Baruselli P. S.
Published: August 2011

Recent data reveal that approximately 80% of bovine embryos produced in vitro worldwide are from (Brazil J. H. M. Viana, personal communication). Adoption of this biotechnology by Brazilian producers might be attributed to particularities of the Bos indicus subspecies. Zebu breeds provide 2 to 3 times more viable oocytes than Bos taurus breeds per ovum pickup (OPU) session. This work aimed to evaluate the quality of cumulus–oocyte complexes (COC) retrieved from Gir breed cows (B. indicus) by OPU with a synchronized follicular wave and subjected to an in vitro embryo production (IVP) technique. All COC were obtained by OPU of 14 Gir cows performed every 14 days. There were 4 OPU sessions, preceded by synchronization of the follicular waves. Wave synchronization was achieved by an injection of 2 mg of oestradiol benzoate followed by the insertion of a norgestomet implant that stayed in place for 5 days. On Day 5, follicles of 2 to 8 mm were aspirated. The oocytes recovered were distributed according to classification: Group 1, with ≥3 cumulus cell layers and homogeneous or slightly heterogeneous cytoplasm (n = 314); and Group 2, ≤2 cumulus cells layers and slightly or coarsely granulated cytoplasm (n = 236). Oocytes from both groups were followed to in vitro maturation–IVF–fertilization–in vitro culture. Two (Day 2) and 8 (Day 8) days after the beginning of fertilization, the cleavage and blastocyst rates were measured, respectively. The statistical analyses were performed using a chi-square test (P < 0.05). The mean cleavage rates observed were 72.93 and 69.07% (not significant), whereas the mean blastocyst rates were 37.90 and 17.80%, and were significantly different for Groups 1 and 2, respectively. The results suggest that the morphology of oocytes (number of cumulus cell layers and cytoplasm homogeneity) is a strong indicator of the in vitro developmental capacity of oocytes recovered from Gir cows with synchronized follicular waves, although we did not observe compromised cleavage rates.

Financial support was provided by Fapesp/Fapemig/CNPq/Embrapa. Epamig/Faz. Calciolândia/Nutricell–Nutrientes Celulares is acknowledged.
23 EFFECTS OF THE NUMBER OF SERVICES FERTILITY IN DAIRY COWS
Authors: Yamaguchi M. , Tanisawa M. , Koyama H. , Takahashi S. , Dochi O.
Published: August 2011

In recent years, reproductive performance of dairy cows has been declining worldwide, especially among cows of high genetic merit for milk production. The cause of the low fertility may considerably vary across countries and is probably multifactorial. This problem remains unsolved. Further, the first-service conception rate of dairy cows has remarkably decreased worldwide. The number of services required for conception has increased due to the low fertility in dairy cows. However, there are few reports about the relationship between conception rates and the number of services in the current dairy cows. The objective of this study was to investigate whether the number of services affects the conception rates of dairy cows. Data concerning the conception rates was obtained for 8386 Holstein cows from 40 commercial dairy herds in eastern Hokkaido, Japan, from 2006 to 2009. The diagnosis of pregnancy was confirmed by rectal palpation between 30 and 45 days after insemination. The average interval between calving and the first-service was 87.3 days. The average milk yield was 8500 kg. Number of conducting services was from the first to the seventh service. The conception rate was analysed using chi-square test. The results are presented in Table1. The conception rate from the first to the seventh service was 40.2 to 54.0%. The conception rate at the first service was significantly low (40.2%); however, the conception rates did not significantly differ after the second-service. A similar tendency was observed during each year. Moreover, the total percentage of conceptions from the first to the third service was 83.8%. The average number of services per conception was 2.2. The results of this study indicate that the first service yielded the lowest conception rates. The conception rates after the second service did not significantly differ. Moreover, ∼84% of the dairy cows became pregnant between the first service and the third service. However, this result shows that ∼16% of the dairy cows in the herds were repeat breeding. In order to improve fertility, it is necessary to study the factors that affect the first-service conception rates of dairy cows. Moreover, to improve the conception rate of dairy cows, it is important to elucidate the cause of these problems.

Table 1.The number and percentage of dairy cows conceiving at each of the services for consecutive services
230 OVUM PICKUP - IN VITRO PRODUCTION EFFICIENCY FOR CONSERVATION OF THE FLAMENGA CATTLE BREED
Authors: Zago F. C. , Gaudêncio Neto S. , Aguiar L. H. , Schutz L. F. , Forell F. , Marinho L. R. S. , Machado J. , Mezzalira A. , Bertolini M.
Published: August 2011

The introduction of more specialised and productive dairy breeds into commercial herds in recent decades has caused a gradual loss of interest in dual-purpose cattle breeds, such as the Flamenga. Because of its importance to biodiversity, the high risk of loss of this small Brazilian herd, located in one geographic region in Southern Brazil, justifies the need for and efforts in genetic conservation of the Flamenga breed. The ovum pickup (OPU) procedure is a potential tool for such conservation purposes. Thus, the aim of this study was to evaluate the oocyte and embryo yield potential of the Flamenga breed, as compared with Holstein control counterparts, after in vivo oocyte retrieval followed by in vitro embryo production (IVP). Four Flamenga and 4 Holstein multiparous milking cows from the same herd were subjected to weekly ultrasound-guided OPU sections for 10 consecutive weeks. The number of visible follicles per ovary per female was recorded before the aspiration of ≥3-mm follicles. Retrieved cumulus–oocyte complexes (COC) were morphologically graded according to the method of (Leibfried and First 1979 J. Anim. Sci. 48, 76–86), and viable COC (Grades I, II, and III) from both breeds and from slaughterhouse ovaries (IVP controls) were used for the IVP of embryos, based on our established procedures (Vieira et al. 2002 Cryobiology 45, 91–94). Frozen semen from the same Flamenga breed bull was used in all groups. Quantitative (visible and aspirated follicles, collected COC), and qualitative (COC recovery efficiency, cleavage and blastocyst rates) data were compared between breeds and IVP controls (when pertinent) by Student’s t-test and chi-square test, respectively, for P < 0.05. Results are presented in Table 1. The pools of visible and aspirated follicles were similar between breeds. However, the COC recovery efficiency was higher for Flamenga than for Holstein females. In vitro embryo development was also different between breeds, with cleavage rate being significantly lower for Holsteins than for Flamenga and IVP controls. Blastocyst rates also differed between treatments. Nonetheless, development to the blastocyst stage, when based on cleavage, was similar between Flamenga and Holstein embryos, but with both breeds being lower than the IVP controls. Oocyte quality was likely the cause of such differences in embryo developmental potential between groups, with Flamenga females yielding more oocytes and IVP embryos than Holstein cows. In summary, the use of OPU-IVP procedures was demonstrated to be suitable for the preservation of the Flamenga breed in Southern Brazil.

Table 1.Follicular pool and oocyte and embryo yield between Flamenga and Holstein cows following ovum pickup–in vitro production (IVP) procedures

This study was supported by CAPES and CNPq/Brazil.
237 PROTEIN-TYROSINE PHOSPHORYLATION AND CALCIUM UPTAKE IN BOAR SPERM SUBPOPULATIONS AFTER DIFFERENT DISCONTINUOUS PERCOLL GRADIENT CENTRIFUGATIONS
Authors: López-Úbeda R. , Luño V. , Vieira L. , Gadea J. , Matás C.
Published: August 2011

Semen is composed of an heterogeneous population of sperm with varying degrees of structural and functional differentiation. To optimize in vitro fertilization (IVF), different selection methods have been used to isolate sperm subpopulations with high fertilizing capacity. The aim of this study was to evaluate protein-tyrosine phosphorylation (P-Tyr) and calcium uptake in boar sperm subpopulations from the same ejaculate. Spermatozoa from fertile and normozoospermic boars were treated by 2-step discontinuous gradient centrifugation consisting of 3 different combinations of isotonic Percoll (45/60, 60/75, and 45/90%). An aliquot of semen was kept without centrifugation as a control group. Immunofluorescence was employed to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al. 2001 Biol. Reprod. 65, 784–792). In order to measure intracellular free [Ca2+], spermatozoa treated as described above were incubated with 2.5 μM Fura-2AM in a noncapacitating medium (Tardif et al. 2003 Biol. Reprod. 68, 207–213) for 45 min at 37°C. Then, spermatozoa were resuspended in TALP medium (Rath et al. 1999. J. Anim. Sci. 77, 3346–3352), incubated (5% CO2, 38.5°C) for a further 10 min and then analysed in a fluorescence spectrofluorometer during 30 min with excitation wavelength set at 340–380 nm and emission held at 510 nm. The calculation of intracellular free [Ca2+] was performed according to the equation of (Grynkiewicz et al. 1985 J. Biol. Chem. 260, 3440–3450). The results showed that Percoll treatment increased the percentage of cells with P-Tyr v. control group (P < 0.01). Among Percoll groups, 45/90% showed the highest pattern of cells with signal in equatorial region, acrosome, and tail. As was observed with P-Tyr results, the intracellular free Ca2+ concentration (nM) was different between control group and spermatozoa treated with Percoll gradient (P < 0.01). Among Percoll groups, 45/90% showed the highest levels in calcium uptake and 45/60 showed the lowest levels (299.26; P < 0.01). In conclusion, it could be stated after Percoll centrifugation that sperm subpopulations have different abilities to protein-tyrosine phosphorylation and calcium uptake that could be related to different levels of in vitro fertility.

Supported by Fundación Séneca 08752/PI/08.
239 POSSIBLE MECHANISMS OF SPERM DAMAGE CAUSED BY HEAT STRESS IN EUROPEAN BULLS RAISED IN TROPICAL ENVIRONMENTS
Authors: Nichi M. , Bertolla R. P. , Soler T. B. , Cortada C. N. M. , Zuge R. M. , Bols P. E. J. , Barnabe R. C. , Barnabe V. H.
Published: August 2011

Previous studies have indicated that semen of heat-stressed bulls shows impaired mitochondrial activity and high levels of oxidative stress, which may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components (Nichi et al. 2006 Theriogenology 66, 822–828). Disruption of the sperm mitochondria could have a potential damaging effect not only on an individual sperm cell but also on the surrounding cells, especially regarding the sperm membrane, possibly due to the release of a high amount of reactive oxygen species (ROS) produced in this environment (rich in electrons) that would then lead to oxidative stress. To test this hypothesis, semen samples of 11 Simmental bulls kept in tropical environments were collected during the summer months. Semen was evaluated as follows: the 3-3′ diaminobenzidine stain (DAB) as an index of mitochondrial activity, the hypo-osmotic swelling test (HOST) as an index of membrane integrity, measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation, and measurement of the enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase activities. For correlation analysis, the Pearson test was used (variables were transformed when necessary), and for nonparametric variables, the Spearman rank test was used. A high positive correlation was found between sperm cells with highly active mitochondria (DAB class I) and the percentage of cells with intact membrane by HOST (r = 0.93; P < 0.05), and a negative correlation between the latter and the percentage of inactive mitochondria (r = –0.91; P < 0.05), indicating that the higher the percentage of cells showing impaired mitochondrial activity, the higher the percentage of cells with damaged membrane. There was also a positive correlation between TBARS and the percentage of cells with disrupted mitochondria (r = 0.86; P < 0.05), indicating that the higher the percentage of sperm with impaired mitochondrial activity, the higher the oxidative stress. No correlation existed between the enzymatic antioxidants and any of the variables studied. The results indicate that heat stress may lead to an increase in testicular ROS levels, overcoming the seminal antioxidant protection. This, in turn, may cause damage of the mitochondria and a subsequent release of more pro-oxidative substances, and an exponential increase of oxidative stress. Understanding these mechanisms may lead to more tailored antioxidant therapies in the future.

The authors thank FAPESP for the scholarship and financial support.
24 EFFECT OF CULTURE OF SEMEN IN A LOW PRESSURE CONDITION AT ROOM TEMPERATURE ON VIABILITY AND CAPACITATION STATUS OF BOAR SPERM
Authors: Yamashita K. , Ishida S. , Funahashi H.
Published: August 2011

Sperm are affected by physical conditions, such as centrifugation and temperature. The objective of this study was to examine the effect of a low atmospheric pressure on viability and capacitation status of boar sperm during semen preservation at room temperature. Sperm-rich fraction from Berkshire boars was diluted at cells mL–1 with modified Modena containing 20% seminal fluid after washing with centrifugation (300 × g for 35 min at room temperature) in a Percoll gradient (45%/90%). The sperm suspension was stored at a pressure of 0.5 or 1.0 atmospheres in the dark at room temperature (25°C). Following storage for 4 h or 4 days, the semen samples were analysed for viability, intracellular calcium level, and acrosome status of the sperm. Viability and intracellular calcium level of sperm were assessed by flow cytometry following staining with SYBR-Green/PI and Furo-3/PL, respectively. Sperm status associated with capacitation and acrosome reaction was analysed by CTC-assay under fluorescence microscope. Statistical analyses of data from 4 or 5 replicated trials were carried out by ANOVA and with a Bonferroni-Dunn post hoc test (significance, P < 0.05). Viability of sperm was not different (P = 0.50) between 2 pressures (0.5 and 1.0 atm) 4 h and 4 days after the start of storage (94.6% v. 95.6% and 92.7% v. 94.3%, respectively). Although the percentage of live sperm with high intracellular calcium levels drastically increased (P < 0.01) 4 days after the start of storage (20.2% v. 23.4%) compared with 4 h of storage (5.5% v. 4.9%), there were no differences between sperm stored in 0.5 and 1.0 atm at 4 h (P = 0.80) and 4 days of storage (P = 0.40). After 4 h of storage, there were no differences in the percentage of intact (93.3% v. 94.7%), capacitated (5.5% v. 4.3%), and acrosome-reacted sperm (1.5% v. 1.5%) between sperm stored in 0.5 and 1.0 atm. After 4 days of storage, however, the percentage of intact sperm decreased when the sperm suspension was cultured in 0.5 atm (71.8%) compared with 1.0 atm (88.5%), and the incidence of capacitated sperm increased (14.3% v. 7.8%, respectively), whereas there was no difference in the acrosome-reacted cells. These results demonstrate that the status of sperm associated capacitation is stimulated in a low atmospheric pressure without any effects of the viability of sperm, during storage for 4 days.
246 EVIDENCE OF FOCAL ADHESION ASSEMBLY IN BOVINE OOCYTES
Authors: Sessions B. R. , Rutigliano H. , Davies C. J. , White K. L.
Published: August 2011

Integrins are located on the plasma membrane of bovine oocytes and have been implicated in bovine fertilization and activation (Campbell et al. 2000; Sessions et al. 2006). In response to signals from the extracellular matrix, activated integrins cluster at focal adhesions and interact with the cytoskeleton via signalling molecules, including focal adhesion kinase (FAK), α-actinin, phosphotyrosine, and talin. Immunohistochemistry was used to illustrate the formation of focal adhesions at the site of sperm binding in bovine oocytes during fertilization. To confirm antibody specificity, proteins were recovered from bovine oocyte lysate, separated by electrophoreses, and exposed to Western blotting. Specificity was confirmed based on antibody binding to the correctly sized bovine protein in the blot. Bovine oocytes were recovered, matured in vitro, and exposed to spermatozoa as described previously. Antibodies were then used to directly identify co-localization of FAK, α-actinin, phophotyrosine, and talin with each other in addition to localization with β-integrin isotypes within the bovine oocyte plasma membrane. Appropriate controls were performed in which all steps were duplicated with the elimination of the primary antibodies, to confirm that immunohistochemistry was specific for the proteins of interest. The results of immunohistochemistry indicate a co-localization of FAK protein clusters that involves specific oocyte membrane integrin proteins during fertilization. These findings illustrate the formation of focal adhesions via colocalization of signalling molecules with integrin β subunits in bovine oocytes and further support the hypothesis that induction of signalling pathways is initiated with sperm–oocyte binding in the bovine.
249 EXPRESSION OF mRNA ENCODING EPIDERMAL GROWTH FACTOR-LIKE FACTORS IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FOLLICLE-STIMULATING HORMONE
Authors: Caixeta E. S. , Machado M. F. , Ripamonte P. , Lima P. F. , Castilho A. C. S. , Bueno da Silva R. , Barros C. M. , Price C. A. , Buratini J.
Published: August 2011

Epidermal growth factor (EGF)-like family members [amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC)] have been shown to be important regulators of cumulus–oocyte complex (COC) maturation, particularly cumulus expansion. The aim of this study was to determine the temporal expression patterns of mRNA encoding EGF-like growth factors in bovine cumulus cells (CC) during COC in vitro maturation and to assess the effects of grading doses of FSH on EGF-like mRNA expression in CC. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries. In the first experiment, CC were separated from 20 COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 h with (10 ng mL–1) or without FSH. In the second experiment, pools containing 20 COC were matured for 12 h with grading doses of FSH (0, 0.1, 1, 10, and 100 ng mL–1). After culture, CC were mechanically separated and stored at –80°C. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed. Expression of target genes was assessed by real-time PCR and normalized by Cyclophilin (CYC-A). Relative quantification of mRNA abundance was determined by the Pfaffl equation. Effects of time of culture and FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer honestly significant difference test. Nonparametric analysis was used when data were not normally distributed. Differences were considered significant when P < 0.05. In the presence of FSH, AREG and EREG mRNA abundance was increased at 4 h of culture, whereas in the absence of FSH, AREG but not EREG mRNA levels were increased by 4 h of culture. The addition of FSH stimulated AREG mRNA expression from 4 to 16 h of culture. In contrast, BTC mRNA was more expressed in immature CC, decreased after 4 h of culture with FSH, and did not vary during maturation in the absence of FSH. In the dose–response experiment, AREG and EREG mRNA expression was stimulated by FSH starting from 10 ng mL–1 and did not increase from 10 ng mL–1 to 100 ng mL–1. Again in contrast, BTC mRNA expression was inhibited by FSH at 100 ng mL–1. In conclusion, the present data suggest that FSH differently regulates the expression of EGF-like factors during bovine COC maturation, although AREG and EREG are stimulated, BTC is inhibited by FSH.

This work was supported by FAPESP.
255 SPERM NUCLEAR DECONDENSATION ABILITY OF IN VITRO MATURED CANINE OOCYTES
Authors: De los Reyes M. , Vergara J. , Palomino J.
Published: August 2011

The sperm chromatin decondensation occurs when a spermatozoon enters into an oocyte during fertilization, and the effectiveness of this process is connected to the grade of oocyte cytoplasmic maturation. In this study chromatin sperm decondensation was evaluated after IVF of canine oocytes matured in vitro (IVM), comparing different durations of maturation. Cumulus–oocytes complexes (COC) for IVM were obtained from bitch ovaries after ovariohysterectomy, selecting those COC with compact cumulus cells and a homogeneous dark cytoplasm. The COC were matured in vitro for 0, 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM HEPES, 10% FCS, 0.25 mM pyruvate, 10 IU mL–1 of hCG, 300 IU mL–1 of penicillin, and 20 mg mL–1 of streptomycin at 38.5°C and 5% CO2. Fresh ejaculates from 3 adult dogs were centrifuged, and the sperm pellet was resuspended in fert-TALP medium. In each replicate, 100-μL fert-TALP drops containing 10 to 12 IVM oocytes after each culture time were co-culture with 2.5 × 106 spermatozoa mL–1 for 24 h under culture conditions. Soon after co-culture, all oocytes were denuded from cumulus cells and fixed in 3% paraformaldehyde. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined by 4′,6-diamidino-2-phenylindole staining under a fluorescence inverted microscope. For each treatment, at least 4 replicates were performed, and the data were compared statistically by chi-square test, using InfoStat Professional Program. A total of 800 oocytes were evaluated, and the percentages of oocytes with sperm penetration were 58% (138/238), 61% (108/177), 72% (118/165), and 70% (153/220) at 0, 48, 72, and 96 h of IVM, respectively. The percentage of sperm nuclear decondensation at each time point significantly increased up to 72 h of culture, showing 12, 34, 81, and 85% of sperm nuclei deconsated, respectively. The percentages of nuclear maturation also increased (P ≤ 0.05) with time, showing 0, 8, 20, and 27% of oocytes at second metaphase (MII) stage at 0, 48, 72, and 96 h of culture. The percentage of MII stage was much lower than that of chromatin decondensation in all maturing groups. These results suggest that canine oocytes matured in vitro are able to decondense the sperm chromatin during IVF, and this ability increases up to 72 h of culture. Nevertheless, cytoplasmic maturation, as evaluated by sperm chromatin decondensation, in canine oocytes matured in vitro may not be completely connected with nuclear development.

This work was supported by Grant FONDECYT 1080618.
256 SYNERGISTIC EFFECT ON EMBRYO DEVELOPMENT AND EMBRYO QUALITY WHEN BOVINE DENUDED OOCYTES ARE CO-CULTURED WITH CUMULUS - OOCYTE COMPLEXES DURING IN VITRO MATURATION
Authors: Dey S. R. , Deb G. K. , Bang J. I. , Cho S. J. , Choi B. H. , Kong I. K.
Published: August 2011

The oocyte and its surrounding somatic cells are metabolically coupled to each other through gap junctions. This phenomenon allows intercellular communication and transfer of different low-molecular-weight substrates between the cells necessary for oocyte growth. The oocyte itself regulates the cumulus cell microenvironment through oocyte-secreted factors. The development competence of the bovine oocytes is increased when denuded oocytes (DO) are co-cultured with cumulus–oocyte complexes (COC) during in vitro maturation (IVM). However, the fate of the DO, which are usually discarded after IVM, has not been determined. The present study aimed to investigate whether there is a synergistic effect of co-culturing COC and DO during IVM. We performed 3 IVM schemes: 1) COC and DO co-culture, with 12 COC and 60 DO; 2) COC control, with 12 COC; and 3) DO control, with 60 DO in 120-μL drop of TCM-199 for 22 to 24 h. Following IVM, IVF and in vitro culture were separately performed for the COC (COC co-culture) and DO (DO co-culture) from the IVM co-culture group. In vitro fertilization and in vitro culture (modified CR1aa) were done in 60-μL drops. Embryos were cultured at 38.5°C and 5% CO2 in air. Cleavage and blastocyst rates were checked at Day 3 and 8 from IVF on total COC/DO placed in IVM drop. Day 8 blastocysts were used for TUNEL staining using In Situ Cell Death Detection Kit (Roche, Budapest, Hungary). Data were analysed by one-way ANOVA, and significant differences among groups were tested by DMRT. Compared with the respective control treatments, co-culture has no effect on cleavage rates of COC and DO (see Table 1). However, blastocyst rates and total cell numbers of blastocysts were increased in COC co-culture and DO co-culture group compared with their respective control groups (see Table 1). Co-culture had no effect on apoptosis of blastocysts. These data show that co-culture of COC and DO improved developmental competence and quality of embryos from the COC co-culture and DO co-culture group.

Table 1.Development competence and blastocyst quality of intact and denuded bovine oocytes

This work was partly supported by the BK21 program, the KRF (KRF-2008-211-F00011), the IPET (108068-03-1-SB010), and the KOSEF (10525010001-05N2501-00110).