Morphological, biochemical and functional studies to evaluate bovine oocyte vitrification.

The aim of the present study was to evaluate the effect of vitrification on morphological, biochemical and functional parameters of matured bovine oocytes at different recovery times. To this end, matured bovine oocytes were vitrified using the Cryotech® kit (a minimum-volume system) and then incubated in maturation medium for different post-warming durations (0 h, 3 h or 21 h). Morphology, viability and biochemical parameters were assessed at each time point mentioned above and the recovery of the metaphase plate was analyzed at 2 h, 3 h and 4 h post-warming. The vitrification-warming process did not affect the viability or morphology of oocytes at any time point. However, the recovery of the metaphase plate occurred mostly between 3 and 4 h rather than at 2 h after warming (P < 0.05). Both control and vitrified-warmed oocytes showed changes in cytosolic oxidative activity, quantification of active mitochondria, reactive oxygen species (ROS) levels and redox status at the different time points studied (P < 0.05). However, differences between control and vitrified-warmed oocytes were found only in the quantification of active mitochondria and ROS production (P < 0.05). Finally, in vitro fertilization and embryo culture were carried out as functional studies to establish whether vitrification-warming affected oocyte competence, and a significant decrease was found both in the cleavage rate and embryo development (P < 0.05). We concluded that major improvements in oocyte vitrification, at list with Cryotech® kit, are still needed to avoid variations in oocyte metabolism which could contribute to the reduction in the developmental competence of bovine oocytes.

The control of reactive oxygen species influences porcine oocyte in vitro maturation.

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H2O2 or O2∙(-) production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O2∙(-) and H2O2 scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O2∙(-) and H2O2 during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.

Evaluation of the Cryotech Vitrification Kit for bovine embryos.

The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit(®) and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production.

Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation.

The knowledge concerning redox and reactive oxygen species (ROS)-mediated regulation of early embryo development is scarce and remains controversial. The aim of this work was to determine ROS production and redox state during early in vitro embryo development in sperm-mediated and parthenogenetic activation of bovine oocytes. Sperm-mediated oocyte activation was carried out in IVF-modified synthetic oviductal fluid (mSOF) with frozen-thawed semen. Parthenogenetic activation was performed in TALP plus ionomycin and then in IVF-mSOF with 6-dimethylaminopurine plus cytochalasin B. Embryos were cultured in IVF-mSOF. ROS and redox state were determined at each 2-h interval (7-24 h from activation) by 2',7'-dichlorodihydrofluorescein diacetate and RedoxSensor Red CC-1 fluorochromes respectively. ROS levels and redox state differed between activated and non-activated oocytes (P<0.05 by ANOVA). In sperm-activated oocytes, an increase was observed between 15 and 19 h (P<0.05). Conversely, in parthenogenetically activated oocytes, we observed a decrease at 9 h (P<0.05). In sperm-activated oocytes, ROS fluctuated throughout the 24 h, presenting peaks around 7, 19, and 24 h (P<0.05), while in parthenogenetic activation, peaks were detected at 7, 11, and 17 h (P<0.05). In the present work, we found clear distinctive metabolic patterns between normal and parthenogenetic zygotes. Oxidative activity and ROS production are an integral part of bovine zygote behavior, and defining a temporal pattern of change may be linked with developmental competence.

Modulation of glycolysis and the pentose phosphate pathway influences porcine oocyte in vitro maturation.

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

Influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus-oocyte complexes during in vitro maturation.

The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A(1) (with a dense cumulus), A(2) (with a translucent cumulus), B(1) (with the corona radiata), B(2) (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A(1) COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.

Glass wool filtration of bull cryopreserved semen: a rapid and effective method to obtain a high percentage of functional sperm.

Frozen-thawed bull sperm are widely used in assisted reproductive technologies, but cryopreservation negatively affects semen quality. Several sperm selection techniques have been developed to separate motile sperm from non-motile cells. The aim of the present study was to evaluate the effectiveness of the glass wool column filtration to select functional sperm from frozen-thawed bull semen samples. Frozen semen from six Holstein bulls was thawed and filtered through a glass wool column, followed by assessment of routine and functional sperm parameters. In a set of experiments, sperm aliquots were also processed by swim up to compare both selection methods. Samples recovered in the glass wool filtrate had high percentages of viable (94 ± 3%, mean ± SD), progressively motile (89 ± 4%), acrosome-intact (98 ± 1%), and non-capacitated (80 ± 10%) sperm; these values were higher (P < 0.05) than those obtained after performing the swim up procedure. Moreover, the glass wool filtration yielded 67 ± 19% motile cells, in comparison with 18 ± 8% obtained with swim up (P < 0.05), calculated as the concentration of progressively motile cells selected relative to their concentration in the sample before the selection procedure. Glass wool-filtered sperm were able to undergo capacitation-related events, based on the increase in the percentage of cells classified as capacitated by CTC staining (B-pattern) after incubation with heparin (50 ± 5%) in comparison with control conditions with no heparin (17 ± 4%) or heparin + glucose (16 ± 2%; P < 0.05). Moreover, they underwent acrosomal exocytosis in response to pharmacologic (calcium ionophore A23187 and lysophosphatidylcholine) and physiological (follicular fluid) stimuli, and they fertilized in vitro matured cumulus-oocyte complexes and denuded oocytes (two-cell embryos: 72 ± 4% and 52 ± 6%, respectively). We conclude that glass wool filtration is a low-cost, simple, and highly effective procedure to select functionally competent sperm for reproductive technologies in the bull, which may be useful for other domestic and farm animals, as well as for endangered species.

[Chronic musculo-skeletal pain in Switzerland: patient care from the view of physicians and patients]. / Chronische muskuloskelettale Schmerzen in der Schweiz: Die Betreuungsqualität aus Sicht der Arzte und der Patienten.

A considerable percentage of the population suffers from chronic musculoskeletal pain (CMP) and patient management does not appear to be optimal. The aim of the present investigations was to assess and evaluate epidemiologic data and discover eventual deficits in patient management. This investigation included several sequential steps: First a European study including Switzerland evaluated the prevalence and characteristics of patients with CMP as well as of the treating physicians. The results were discussed and elaborated in two workshops, where general practitioners and patients were included. In a further step the results of these workshops were evaluated again in a telephone survey addressing patients and physicians both in the French and German speaking parts of Switzerland. Considerable deficits were discovered in the management of patients with CMP: In 35% no firm diagnosis was established, the life quality was considerably reduced in about 13 of the patients, the patients' information on their disorders were found to be rather limited, furthermore, there were misconceptions about medical treatment. The two workshops confirmed the results of the first study. The causes of pain often remained unclear, there were considerable communication problems between patient and physician, medical treatment appeared to be inappropriate, and there were deficits in the time management during consultations. The telephone survey confirmed these deficits. In conclusion management of patients with CMP is characterized by considerable deficits such as missing or unclear diagnosis, misconceptions in medical contexts and treatment. Many of the deficits may be improved and call for measures for optimizing the management of patients with CMP.

Reactive oxygen species in bovine embryo in vitro production.

Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39 degrees C in 5% CO2: 95% humidified air. In vitro fertilization was carried out in IVF-mSOF with frozen-thawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90% N2: 5% CO2: 5% O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2',7'-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P < 0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time.

Reactive oxygen species in bovine embryo in vitro production

Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39°C in 5 CO2: 95 humidified air. In vitro fertilization was carried out in IVF-mSOF with frozenthawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90 N2: 5 CO2: 5 O2, ROS was determined in denuded oocytes and embryos at successive stages of development by the 2',7' -dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P<0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time

Reactive oxygen species in bovine embryo in vitro production

Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39ºC in 5 CO2: 95 humidified air. In vitro fertilization was carried out in IVF-mSOF with frozenthawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90 N2: 5 CO2: 5 O2, ROS was determined in denuded oocytes and embryos at su

Effect of alpha-tocopherol and ascorbic acid on bovine oocyte in vitro maturation.

In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.

Reactive oxygen species in bovine embryo in vitro production.

Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39 degrees C in 5

CO2: 95

humidified air. In vitro fertilization was carried out in IVF-mSOF with frozen-thawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90

N2: 5

CO2: 5

O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2,7-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P < 0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time.

Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro.

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.

Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro.

Little is known about the metabolic profile of cumulus-oocyte complexes (COCs) during maturation. The aim of this study was to determine the differential participation of enzymatic activity in cumulus cells and the oocyte during in vitro maturation of bovine oocytes, by measuring the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) and lipolysis (lipase). COCs were matured in medium 199 plus 10% (v/v) steer serum for 22-24 h at 39 degrees C in 5% CO(2):95% humidified air. Phosphofructokinase, glucose-6-phosphate dehydrogenase and lipase activities were measured in immature and in vitro matured COCs, denuded oocytes and cumulus cells, respectively. Phosphofructokinase and glucose-6-phosphate dehydrogenase activities (enzymatic units) remained constant during in vitro maturation of COCs, but there was a significant decrease in lipase activity (units) (P < 0.05), as activity in cumulus cells decreased significantly (P < 0.05). For the three enzymes studied, enzyme activity (units) remained unchanged in the oocyte during in vitro maturation. Specific activity increased in the oocyte (P < 0.05) and decreased in cumulus cells as a result of maturation (P < 0.05). In cumulus cells, phosphofructokinase was the most abundant of the three enzymes followed by glucose-6-phosphate dehydrogenase and then lipase (P < 0.05), whereas in the denuded oocyte this order was reversed (P < 0.05). Thus, the metabolism of cumulus cells is adapted to control the flow of metabolites toward the oocyte, which maintains its enzymatic activity even when dissociated from cumulus cells during maturation. The high activity of phosphofructokinase in cumulus cells indicates that glucose is metabolized mainly via the glycolytic pathway in these cells. The greater relative activity of glucose-6-phosphate dehydrogenase recorded in the oocyte indicates that glucose uptake could be directed mainly toward the pentose phosphate pathway. The marked lipolytic activity concentrated in the oocyte indicates an active participation in lipid catabolism during maturation.

Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation.

Reactive oxygen species (ROS) production is a normal process of cell metabolism. In vitro environments usually increase cell production of ROS, which has been implicated as a main cause of cell damage. Nevertheless, the role of ROS in oocyte in vitro maturation (IVM) is controversial. In most cells, enzymatic antioxidant systems can attenuate the effect of oxidative stress by scavenging ROS. The aim of this work was to determine whether: (1) standard conditions of bovine oocyte IVM are responsible for oxidative stress; (2) cumulus cells participate in protection against oxidative stress of the oocyte; and (3) enzymatic antioxidant activity is present in oocytes and cumulus cells. Cumulus-oocyte complexes (COCs) were matured in TCM-199 + 10% steer serum for 24 h at 39 degrees C in 5% CO2:95% humidified air. Oxidative stress was determined by the 2',7'-dichlorofluorescein diacetate assay. Superoxide dismutase (SOD), glutathione peroxidase, and catalase activities were measured spectrophotometrically. Under standard conditions of in vitro maturation, there was no increase in ROS production per COC (P > 0.05), but ROS level per cumulus cell diminished. There was no modification in ROS levels in oocytes matured in the presence versus the absence of their surrounding cumulus cells ( P > 0.05). To the best of our knowledge, the presence of SOD, glutathione peroxidase and catalase activities were detected in oocytes and cumulus cells for the first time. Enzymatic units were lower in denuded oocytes with respect to cumulus (P < 0.05), accounting for 37% for SOD, 25% for glutathione peroxidase, and 11% for catalase of the total COC units. Specific enzyme activity diminished in cumulus cells (P > 0.05) and increased in oocytes due to maturation (P > 0.05). The presence of activity of an enzymatic antioxidant system in the bovine oocyte would regulate in part ROS levels during IVM. Oocytes could be capable of controlling the increase in ROS because of the presence of their own enzymatic antioxidant system, SOD having the highest specific activity with respect to cumulus cells.

Study of evaluation criteria used for in vitro bovine oocyte selection and maturation

Production of bovine preimplantation embryos in vitro requires beneficial maturation conditions and high quality oocytes at the germinal vesicle stage. The current classification of oocytes is based on character of the cumulus cell investment around the oocyte. We wished to study the nuclear stage of immature oocytes selected for in vitro maturation according to cumulus cell character and, in the other hand, to compare the relationship among 3 parameters utilized to evaluate in vitro maturation of bovine oocytes (degree of cumulus expansion, meiotic maturation rate and in vitro fertilization rate) when fetal calf serum, steer serum and bovine follicular fluid supplementation were used. Ovaries were collected at an abattoir and the oocytes harvested. As regards selection criteria, immature oocytes were classified as Class A, B, C and D according to the character of the cumulus cells. A high percentage of Class A oocytes (87.7) were in the germinal vesicle stage with respect to the other classes (p < 0.05). Significant differences were found in the meiotic maturation rate in Class A oocytes (76.5) versus those of the other classes (p < 0.05). The meiotic maturation rate diminished to 47.5 when Class A oocytes were denuded and then matured in vitro (p < 0.05). As regards maturation criteria, there was no cumulus expansion when oocytes were matured in TCM-199 without supplementation, partial expansion with the addition of fetal calf serum and full expansion when supplemented with steer serum or bovine follicular fluid. No significant differences were found in the meiotic maturation rate for the various treatments. In vitro fertilization rate was significantly lower in media without supplementation versus supplemented media (p < 0.05), but no significant differences were found between the supplemented media inter se. There is no direct relationship between the three studied parameters to evaluate in vitro maturation. Class A oocytes are the most likely to mature in vitro as they not only have a close association with their surrounding cumulus cells, but are also very numerous in the germinal vesicle stage. The degree of cumulus expansion and the meiotic maturation rate have a relative importance in evaluating in vitro maturation, as oocyte maturation implies not only nuclear events but also at other cellular levels, as evaluated by in vitro fertilization

Study of evaluation criteria used for in vitro bovine oocyte selection and maturation

Production of bovine preimplantation embryos in vitro requires beneficial maturation conditions and high quality oocytes at the germinal vesicle stage. The current classification of oocytes is based on character of the cumulus cell investment around the oocyte. We wished to study the nuclear stage of immature oocytes selected for in vitro maturation according to cumulus cell character and, in the other hand, to compare the relationship among 3 parameters utilized to evaluate in vitro maturation of bovine oocytes (degree of cumulus expansion, meiotic maturation rate and in vitro fertilization rate) when fetal calf serum, steer serum and bovine follicular fluid supplementation were used. Ovaries were collected at an abattoir and the oocytes harvested. As regards selection criteria, immature oocytes were classified as Class A, B, C and D according to the character of the cumulus cells. A high percentage of Class A oocytes (87.7) were in the germinal vesicle stage with respect to the other classes (p < 0.05). Significant differences were found in the meiotic maturation rate in Class A oocytes (76.5) versus those of the other classes (p < 0.05). The meiotic maturation rate diminished to 47.5 when Class A oocytes were denuded and then matured in vitro (p < 0.05). As regards maturation criteria, there was no cumulus expansion when oocytes were matured in TCM-199 without supplementation, partial expansion with the addition of fetal calf serum and full expansion when supplemented with steer serum or bovine follicular fluid. No significant differences were found in the meiotic maturation rate for the various treatments. In vitro fertilization rate was significantly lower in media without supplementation versus supplemented media (p < 0.05), but no significant differences were found between the supplemented media inter se. There is no direct relationship between the three studied parameters to evaluate in vitro maturation. Class A oocytes are the most likely to mature in vitro as they not only have a close association with their surrounding cumulus cells, but are also very numerous in the germinal vesicle stage. The degree of cumulus expansion and the meiotic maturation rate have a relative importance in evaluating in vitro maturation, as oocyte maturation implies not only nuclear events but also at other cellular levels, as evaluated by in vitro fertilization

Study of evaluation criteria used for in vitro bovine oocyte selection and maturation.

Production of bovine preimplantation embryos in vitro requires beneficial maturation conditions and high quality oocytes at the germinal vesicle stage. The current classification of oocytes is based on character of the cumulus cell investment around the oocyte. We wished to study the nuclear stage of immature oocytes selected for in vitro maturation according to cumulus cell character and, in the other hand, to compare the relationship among 3 parameters utilized to evaluate in vitro maturation of bovine oocytes (degree of cumulus expansion, meiotic maturation rate and in vitro fertilization rate) when fetal calf serum, steer serum and bovine follicular fluid supplementation were used. Ovaries were collected at an abattoir and the oocytes harvested. As regards selection criteria, immature oocytes were classified as Class A, B, C and D according to the character of the cumulus cells. A high percentage of Class A oocytes (87.7%) were in the germinal vesicle stage with respect to the other classes (p < 0.05). Significant differences were found in the meiotic maturation rate in Class A oocytes (76.5%) versus those of the other classes (p < 0.05). The meiotic maturation rate diminished to 47.5% when Class A oocytes were denuded and then matured in vitro (p < 0.05). As regards maturation criteria, there was no cumulus expansion when oocytes were matured in TCM-199 without supplementation, partial expansion with the addition of fetal calf serum and full expansion when supplemented with steer serum or bovine follicular fluid. No significant differences were found in the meiotic maturation rate for the various treatments. In vitro fertilization rate was significantly lower in media without supplementation versus supplemented media (p < 0.05), but no significant differences were found between the supplemented media inter se. There is no direct relationship between the three studied parameters to evaluate in vitro maturation. Class A oocytes are the most likely to mature in vitro as they not only have a close association with their surrounding cumulus cells, but are also very numerous in the germinal vesicle stage. The degree of cumulus expansion and the meiotic maturation rate have a relative importance in evaluating in vitro maturation, as oocyte maturation implies not only nuclear events but also at other cellular levels, as evaluated by in vitro fertilization.

Effect of lactate dehydrogenase activity and isoenzyme localization in bovine oocytes and utilization of oxidative substrates on in vitro maturation.

Oocyte nutritional metabolism changes during maturation in order to increase the energy available to support metabolic requirements. The aim of this work was to study pyruvate and lactate utilization as oxidative substrates on IVM and lactate dehydrogenase (LDH) activity and localization of their isoenzymes in bovine oocytes. Immature cumulus-oocyte complexes (COCs) were recovered by aspiration of antral follicles in ovaries obtained from slaughtered cows. The COCs and denuded oocytes were separately cultured in TCM-199 with steer serum (controls) and were supplemented with pyruvate, lactate or lactate plus NAD for 24 h at 39 degrees C in 5% CO2:95% humidified air. No significant differences were found in IVM rates of COCs matured according to the various treatments (P>0.05). The IVM rate in denuded oocytes without supplementation was 47.8%. The presence of pyruvate in the culture medium resulted in an increased number of matured denuded oocytes (59.4%; P<0.05), but the addition of lactate failed to improve the IVM rate of matured denuded oocytes (47.6%, P>0.05). When the medium was supplemented with lactate plus NAD, the IVM rate of denuded oocytes likewise failed to differ from that obtained with the addition of pyruvate (59.9%, P>0.05). The LDH activity in immature and matured COCs and denuded oocytes was (3.1+/-1.6) 10(-3), (3.3+/-1.6) 10(-3) U/COC, (5.2+/-2.0) 10(-5), (5.4+/-3.5) 10(-5) U/oocyte with pyruvate as substrate, and (1.2+/-0.5) 10(-3), (1.0+/-0.5) 10(-3) U/COC, (2.2+/-0.1) 10(-5), (2.5+/-1.4) 10(-5) U/oocyte respectively, with lactate; no significant differences due to maturation status were observed (P>0.05; n = 9 for each LDH activity). Electrophoresis disclosed that the principal band corresponded to the LDH-1 isoenzyme in oocytes, while there was no predominance of any isoenzyme in cumulus cells. Due to the fact that LDH-1 is the main oocyte isoenzyme, the pyruvate used during oocyte maturation could be partly produced from lactate when the NAD supply is adequate. Cumulus cells would be responsible for providing pyruvate and/or lactate as oxidative substrates to be used by the bovine oocyte and this supply would be regulated by the LDH activity in these cells.