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The goal of this study was to determine whether traditional ergoespirometric incremental exercise testing carried out to the point of exhaustion could be useful in distinguishing the physiological profiles of elite runners that compete in races that lasted about 8 minutes versus those that lasted about 2 hours. Ten male marathon runners (performance time: 2:12:04, coefficient of variation (CV) = 2.33%) and 8 male 3000 m steeplechase runners (performance time: 8:37.83, CV = 2.12%) performed an incremental test on the treadmill (starting speed 10 km·h−1; increments, 2 km·h−1; increment duration, 3 min to exhaustion). Heart rate (HR), VO2, and lactate concentrations were measured at the end of each exercise level. At maximal effort, there were no differences between the groups regarding VO2maxand maximal HR; however, the workload time, vVO2maxand peak treadmill velocity were significantly higher in the 3000 m steeplechase group (p<0.05). At submaximal effort, there were no significant differences between groups for VO2(ml·kg−1·min−1), HR, or lactate. Our results show that this type of testing was not sufficient for discriminating the physiological profiles of elite runners who competed in middle-distance versus long-distance events (e.g. in the marathon and the 3000 m steeplechase).

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Embryo development of IVM-IVF prepubertal goat oocytes is low. The ICSI techniques may allow fertilization of all IVM oocytes. The objective of this study was to assess the efficacy of ICSI compared with IVF, for embryonc development of prepubertal goat oocytes. Prepubertal (1- to 2-month old) goat oocyte were recovered from a local slaughterhouse and matured in a conventional IVM medium (TCM199 with serum, hormones and cysteamine) for 27 h. Spermatozoa from fresh ejaculates were selected by swim-up and capacitated with heparin. IVM oocytes were divided into three groups: (1) oocytes fertilized using conventional IVF methodology, (2) ICSI oocytes activated by sequential treatment with 5 mM ionomycin for 5 min and 2 mM 6-DMAP for 4 h; and (3) parthenogenetic oocyte group used as a control for chemical activation. Presumptive zygotes and activated oocytes were cultured in TALP fertilization medium for 24 h. After fertilization, all groups were divided into two groups and cultured for 192 h in: (a) sequential media G1.3/G2.3; or b) TCM199 with granulosa cells. The number of cell nuclei in embryos was assessed by Hoescht staining. Each experiment was repeated seven times, and results were analyzed using chi-square or Fisher's test. The percentage of total embryos obtained after IVF, ICSI, and chemical activation was higher (P < 0.05) for ICSI than for IVF in G1.3/G2.3 (81.4 and 67.9%, respectively) but not in co-culture (61.9 and 58.2%, respectively). The percentage of total embryos was higher (P < 0.05) in G1.3/G2.3 than in co-culture for both IVF oocytes (67.9 and 58.2%, respectively) and ICSI oocytes (81.4 and 61.9%, respectively). The highest percentage of 8- to 16-cell embryos (28.1%) was obtained in the ICSI group cultured in G1.3/G2.3 medium. We did not find significant differences among the experimental groups in the percentage of morulae. In conclusion, ICSI improved the percentage of embryos developing beyond the 8-cell stage. However, this difference could be due to the additional effect of the chemical activation. Embryo development was also improved by using G1.3/G2.3 embryo culture medium rather than TCM-199 with granulosa cells. The ICSI procedure could be a useful tool to study more accurately the oocyte competence for embryo development.  This study was supported by MCYT (Spain), Grant AGL 2000-0353.

Developmental competence of prepubertal (1- to 2-month old) goat oocytes is compromised, probably because of an incomplete cytoplasmic maturation. Oocyte selection for IVM-IVF is based on morphological criteria. The main regulator of oocyte nuclear maturation is maturation promoting factor (MPF), and it could also be involved in cytoplasmic maturation. The present study tried to determined p34cdc2 (the catalytic subunit of MPF) expression in oocytes of different sizes before and after IVM. Prepubertal goat oocytes were matured in a conventional IVM medium (TCM199 with serum, hormones, and cysteamine) for 27 h. At collection time, a sample of oocytes was classified into 4 groups according to their diameter (<110 ?m; 110?125 ?m; 125?135 ?m; and >135 ?m), and nuclear stage was evaluated. After IVM, oocytes were classified by diameter (as described before) and stained to analyze nuclear stage. Before and after IVM, a sample of 10 oocytes of each diameter group was frozen at ?80C. These oocytes were used to perform the detection of p34cdc2-RNA by RT-PCR. Briefly, RNA of 10 oocytes was extracted with TriReagent (Sigma-Aldrich, Madrid, Spain), and used to perform RT using the ThermoScript kit (Invitrogen, Barcelona, Spain). The cDNA corresponding to two oocytes was amplified by PCR, and each amplification band was measured by densitometry using the Quantity One PC program. Rabbit globin mRNA was used as an extrinsic control of the whole process. Nuclear stage data were analyzed by Fisher's exact test, and RT-PCR data were analyzed by one-way ANOVA test. At collection time, a high percentage of prepubertal goat oocytes had resumed meiosis. After IVM, the percentage of MII-oocytes was higher in larger-sized oocytes. At collection time, significantly higher p34cdc2-RNA expression was found in 125?135 ?m oocytes. After IVM, no differences were found among oocyte groups. During maturation, a decrease of p34cdc2-RNA was found in 125?135 ?m oocytes. In contrast, an increase of p34cdc2-RNA was found in 110-125 ?m oocytes. The nuclear stage in the smallest oocytes show their reduced ability to resume meiosis. IVM-oocytes of different diameters showed no difference in p34cdc2-RNA expression.  This study was supported by MCYT (Spain), with the grant number AGL2000-0353.

Developmental competence of prepubertal goat oocytes recovered from a slaughterhouse is low, probably due to an incomplete cytoplasmic maturation. Regulation of cytoplasmic maturation is still unknown, although maturation-promoting factor (MPF) is suggested to play an important role in this process. To better understand the role of MPF in cytoplasmic maturation, we have studied MPF kinase activity in oocytes with different developmental competence. Ovaries were obtained from a slaughterhouse, and oocytes were recovered by slicing and were selected according to morphological criteria. Some oocytes were denuded and classified in diameter groups (<110 ?m, 110?125 ?m, 125?135 ?m, and >135 ?m), placed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM EDTA, 0.01? Brij35, 1 mM phenyl methyl sultonyl fluoride (PMSF), 0.05 mg/mL leupeptin, 50 mM 2-mercaptoethanol, 25 mM ?-glycerophosphate, 1 mM Na-orthovanadate) and frozen in liquid N2. Cell extracts were stored at -80C until use. The rest of oocytes were matured in vitro in medium TCM199 supplemented with hormones, 10? (DBS), and 400 ?M cysteamine, for 27 h in 5? CO2 in air and 38.5C. After IVM, a sample of oocytes were also denuded, classified by diameters, and frozen as described above. The rest of oocytes were used for IVF in mDM with spermatozoa capacitated with heparin and ionomicin. After 24 h, presumptive zygotes were cultured for 7 days in medium SOF in 5? CO2, 5? O2, and 90? N2 at 38.5C. At 48 h post-insemination, we added 0.1 ?L FBS per embryo. Embryonic development was evaluated with Hoechst staining after IVC. MPF kinase activity was detected using the MESACUP cdc2 kinase assay kit (MBL Woburn, MA, USA). Briefly, the oocyte extract corresponding to 10 oocytes was mixed with 10 reaction buffer (25 mM HEPES buffer (pH 7.5), 10 mM MgCl2) and 10? biotinylated MV peptide (SLYSSPGGAYC). We added 0.1 mM ATP to start the reaction. The mixture was incubated at 30C for 30 min. The reaction was finished by adding 200 ?L of PBS containing 50 mM EGTA. The phosphorylated MV peptide was detected by specific antibody using an ELISA procedure, and the OD was measured at 492 nm. Fisher's exact test was used to analyze IVC results, and ANOVA to analyze cdc2 kinase activity results. We considered differences statistically significant when P < 0.05. Results are shown in Table 1. We observed that embryonic cleavage and blastocyst rate increased with oocyte diameter. The MPF activity detected was higher in the largest oocytes after IVM. As a consequence, we could establish that oocytes with a higher MPF activity are more capable of maintaining embryonic development until the blastocyst stage, which may indicate the important role that MPF plays in cytoplasmic maturation.

Embryo development of IVM-IVF prepubertal goat oocytes is low. The ICSI techniques may allow fertilization of all IVM oocytes. The objective of this study was to assess the efficacy of ICSI compared with IVF, for embryonc development of prepubertal goat oocytes. Prepubertal (1- to 2-month old) goat oocyte were recovered from a local slaughterhouse and matured in a conventional IVM medium (TCM199 with serum, hormones and cysteamine) for 27 h. Spermatozoa from fresh ejaculates were selected by swim-up and capacitated with heparin. IVM oocytes were divided into three groups: (1) oocytes fertilized using conventional IVF methodology, (2) ICSI oocytes activated by sequential treatment with 5 mM ionomycin for 5 min and 2 mM 6-DMAP for 4 h; and (3) parthenogenetic oocyte group used as a control for chemical activation. Presumptive zygotes and activated oocytes were cultured in TALP fertilization medium for 24 h. After fertilization, all groups were divided into two groups and cultured for 192 h in: (a) sequential media G1.3/G2.3; or b) TCM199 with granulosa cells. The number of cell nuclei in embryos was assessed by Hoescht staining. Each experiment was repeated seven times, and results were analyzed using chi-square or Fisher's test. The percentage of total embryos obtained after IVF, ICSI, and chemical activation was higher (P < 0.05) for ICSI than for IVF in G1.3/G2.3 (81.4 and 67.9%, respectively) but not in co-culture (61.9 and 58.2%, respectively). The percentage of total embryos was higher (P < 0.05) in G1.3/G2.3 than in co-culture for both IVF oocytes (67.9 and 58.2%, respectively) and ICSI oocytes (81.4 and 61.9%, respectively). The highest percentage of 8- to 16-cell embryos (28.1%) was obtained in the ICSI group cultured in G1.3/G2.3 medium. We did not find significant differences among the experimental groups in the percentage of morulae. In conclusion, ICSI improved the percentage of embryos developing beyond the 8-cell stage. However, this difference could be due to the additional effect of the chemical activation. Embryo development was also improved by using G1.3/G2.3 embryo culture medium rather than TCM-199 with granulosa cells. The ICSI procedure could be a useful tool to study more accurately the oocyte competence for embryo development.  This study was supported by MCYT (Spain), Grant AGL 2000-0353.

Developmental competence of prepubertal (1- to 2-month old) goat oocytes is compromised, probably because of an incomplete cytoplasmic maturation. Oocyte selection for IVM-IVF is based on morphological criteria. The main regulator of oocyte nuclear maturation is maturation promoting factor (MPF), and it could also be involved in cytoplasmic maturation. The present study tried to determined p34cdc2 (the catalytic subunit of MPF) expression in oocytes of different sizes before and after IVM. Prepubertal goat oocytes were matured in a conventional IVM medium (TCM199 with serum, hormones, and cysteamine) for 27 h. At collection time, a sample of oocytes was classified into 4 groups according to their diameter (<110 ?m; 110?125 ?m; 125?135 ?m; and >135 ?m), and nuclear stage was evaluated. After IVM, oocytes were classified by diameter (as described before) and stained to analyze nuclear stage. Before and after IVM, a sample of 10 oocytes of each diameter group was frozen at ?80C. These oocytes were used to perform the detection of p34cdc2-RNA by RT-PCR. Briefly, RNA of 10 oocytes was extracted with TriReagent (Sigma-Aldrich, Madrid, Spain), and used to perform RT using the ThermoScript kit (Invitrogen, Barcelona, Spain). The cDNA corresponding to two oocytes was amplified by PCR, and each amplification band was measured by densitometry using the Quantity One PC program. Rabbit globin mRNA was used as an extrinsic control of the whole process. Nuclear stage data were analyzed by Fisher's exact test, and RT-PCR data were analyzed by one-way ANOVA test. At collection time, a high percentage of prepubertal goat oocytes had resumed meiosis. After IVM, the percentage of MII-oocytes was higher in larger-sized oocytes. At collection time, significantly higher p34cdc2-RNA expression was found in 125?135 ?m oocytes. After IVM, no differences were found among oocyte groups. During maturation, a decrease of p34cdc2-RNA was found in 125?135 ?m oocytes. In contrast, an increase of p34cdc2-RNA was found in 110-125 ?m oocytes. The nuclear stage in the smallest oocytes show their reduced ability to resume meiosis. IVM-oocytes of different diameters showed no difference in p34cdc2-RNA expression.  This study was supported by MCYT (Spain), with the grant number AGL2000-0353.