Fatty Acids and Metabolomic Composition of Follicular Fluid Collected from Environments Associated with Good and Poor Oocyte Competence in Goats

In goats, embryo oocyte competence is affected by follicle size, regardless of the age of the females. In previous studies, we found differences in blastocyst development between oocytes coming of small ( 3 mm) in prepubertal (1-2 month-old) goats. Oocyte competence and follicular fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze the fatty acids (FAs) composition and metabolomic profiles of FF recovered from small and large follicles of prepubertal goats and follicles of adult goats. FAs were analyzed by chromatography and metabolites by 1H-nuclear magnetic resonance (1H-NMR) spectrometry. The results showed important differences between adult and prepubertal follicles: a) the presence of α,β-glucose in adult and no detection in prepubertal; b) lactate, -N-(CH3)3 groups and inositol were higher in prepubertal; c) the percentage of linolenic acid, total saturated fatty acids and n-3 PUFAs were higher in adults; and d) the percentage of linoleic acid, total MUFAs, PUFAs, n-6 PUFAs and n-6 PUFAs:n-3 PUFAs ratio were higher in prepubertal goats. No significant differences were found in the follicle size of prepubertal goats, despite the differences in oocyte competence for in vitro embryo production

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulusoocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats

Resveratrol supplementation during in vitro maturation improves embryo development of prepubertal goat oocytes selected by brilliant cresyl blue staining

This study aimed to investigate the effect of resveratrol supplementation in maturation medium on the developmental ability and bioenergetic\oxidative status of prepubertal goat oocytes selected by brilliant cresyl blue (BCB). Oocytes collected from slaughterhouse-derived ovaries were selected by 13 µM BCB staining and classified as grown BCB+ and growing BCB- oocytes. All oocytes were matured in vitro in our conventional maturation medium and supplemented with 1 µM (BCB+R and BCB-R) and without (Control groups: BCB+C and BCB-C) resveratrol. After 24 h, IVM-oocytes were fertilized with fresh semen and presumptive zygotes were in vitro cultured for 8 days. Oocytes were assessed for blastocyst development and quality, mitochondrial activity and distribution, and levels of GSH, ROS, and ATP. BCB+R (28.3%) oocytes matured with resveratrol presented significantly higher blastocyst development than BCB+C (13.0%) and BCB- groups (BCB-R: 8.3% and BCB-C: 4.7%). Resveratrol improved blastocyst development of BCB-R oocytes at the same rate as BCB+C oocytes. No differences were observed in blastocyst quality among groups. GSH levels were significantly higher in resveratrol groups (BCB+R: 36554.6; BCB-R: 34946.7 pixels/oocyte) than in control groups (BCB+C: 27624.0; BCB-C: 27655.4 pixels/oocyte). No differences were found in mitochondrial activity, ROS level, and ATP content among the groups. Resveratrol-treated oocytes had a higher proportion of clustered active mitochondria in both BCB groups (BCB+R: 73.07%; BCB-R: 79.16%) than control groups (BCB+C: 19.35%; BCB-C: 40%). In conclusion, resveratrol increased blastocyst production from oocytes of prepubertal goats, particularly in better quality oocytes (BCB+)

Gestión del conocimiento: perspectiva multidisciplinaria. Volumen 11

El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 11, de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro cuenta con el apoyo de los grupos de investigación: Universidad Sur del Lago “Jesús María Semprúm” (UNESUR), Zulia – Venezuela; Universidad Politécnica Territorial de Falcón Alonso Gamero (UPTAG), Falcón – Venezuela; Universidad Politécnica Territorial de Mérida Kleber Ramírez (UPTM), Mérida – Venezuela; Universidad Guanajuato (UG) - Campus Celaya - Salvatierra - Cuerpo Académico de Biodesarrollo y Bioeconomía en las Organizaciones y Políticas Públicas (C.A.B.B.O.P.P), Guanajuato – México; Centro de Altos Estudios de Venezuela (CEALEVE), Zulia – Venezuela, Centro Integral de Formación Educativa Especializada del Sur (CIFE - SUR) - Zulia - Venezuela, Centro de Investigaciones Internacionales SAS (CIN), Antioquia - Colombia.y diferentes grupos de investigación del ámbito nacional e internacional que hoy se unen para estrechar vínculos investigativos, para que sus aportes científicos formen parte de los libros que se publiquen en formatos digital e impreso

Oocyte competence: Study of melatonin and meiotic inhibitors to improve in vitro embryo production in juvenile goats

La producción in vitro de embriones (PIVE) tiene su mayor limitación en la eficacia de la maduración in vitro (MIV) de los oocitos. La MIV altera la competencia de los oocitos debido al efecto perjudicial de los radicales de oxígeno (ROS) y a la reanudación rápida y espontanea de la meiosis. En oocitos de cabras de 1 a 2 meses de edad, estos dos factores son más intensos y perjudiciales. En esta tesis, se plantearon dos estrategias para mejorar los protocolos de MIV en estos oocitos: A) Suplementar el medio de MIV con melatonina como potente antioxidante para disminuir los ROS; B) Diseñar un sistema bifásico de MIV con una fase de pre-maduración (pre-MIV) con inhibidores de la meiosis, seguida por una segunda fase de MIV convencional. El efecto de la melatonina fue evaluado en dos estudios. En el primero, se testaron diferentes concentraciones de melatonina (10-11, 10-9, 10-7, 10-3 M) en el medio de MIV. La suplementación con 10-7 M de melatonina aumentó el porcentaje de blastocistos (28.9% vs. 11.7% en el control) y redujo el nivel intra-oocitario de ROS. En el segundo estudio, evaluamos los mecanismos de acción de la melatonina (10-7 M) en los oocitos y detectamos la presencia del receptor de melatonina 1 (MT1) en oocitos y células del cúmulo mediante inmunocitoquímica. La melatonina incrementó la actividad mitocondrial y el ATP de los oocitos, redujo los ROS y mejoró la calidad de los blastocistos producidos in vitro (55.8 vs. 30.4 células de la masa celular interna), comparado con el grupo control sin antioxidantes. No pudimos determinar si estos efectos son mediados por el MT1 porqué el grupo melatonina más lucindol (antagónico del MT1) no mostró diferencias significativas respeto los grupos melatonina y control. La estrategia para mejorar la competencia de los oocitos mediante una pre-MIV con inhibidores meióticos se desarrolló en dos estudios. El primer estudio se llevó a cabo en la Universidad de Adelaida (Australia) en oocitos de bovino adulto. Evaluamos el efecto de distintas concentraciones del péptido natriurético tipo C (CNP; 50, 100, 200 nM) y el 3-isobutyl-1-methylxanthine (IBMX; 500 µM) sobre el arresto meiótico durante 6 y 24 h. El cultivo de pre-MIV con CNP (100 nM) más IBMX durante 6 horas obtuvo el mayor efecto de parada meiótica (92% de oocitos en vesícula germinal; VG). A continuación, diseñamos un sistema bifásico utilizando este protocolo de pre-MIV, seguido de 20 h de MIV convencional, y comparamos con una MIV control (24 h). La MIV bifásica prolongó la comunicación entre células del cúmulo y oocito, evaluada mediante la densidad de proyecciones de transzona (TZP), mejoró la actividad mitocondrial y aumentó la producción de blastocistos (45.1% vs. 34.5%). En el segundo estudio, analizamos un sistema similar en oocitos de cabras de 1 a 2 meses de edad. Primero, evaluamos el efecto de distintas dosis de CNP (50, 100, 200 nM) con o sin la adición de 10 nM de estradiol. La pre-MIV con CNP (200 nM) más estradiol mantuvo un 74.7% de oocitos en VG y una alta densidad de TZP durante 6 h. En segundo lugar, cultivamos los oocitos con MIV bifásica (6 h pre-MIV más 24 h MIV) comparado con MIV control (24 h). La MIV bifásica incrementó el glutatión intra-oocitario y disminuyó los ROS, aumentó la expresión de DNA metiltranferasa 1 y proteína 6 estimuladora de TNF en complejos cúmulo-oocito (COCs), y mejoró la producción de blastocistos (30.2% vs. 17.2%). En conclusión, la MIV con melatonina y la pre-MIV con inhibidores meióticos son métodos que pueden mejorar sustancialmente la competencia de los oocitos de hembras muy jóvenes para producir embriones in vitro.Oocyte in vitro maturation (IVM) is a limiting step for the in vitro embryo production (IVEP). Conventional IVM can impair oocyte competence due to the damaging effect of reactive oxygen species (ROS) and the rapid and spontaneous meiotic resumption. These two factors are more intense and damaging for oocytes of juvenile goats (1-2 months old). In the present thesis, we hypothesized that two different strategies could improve IVM protocols in these oocytes: A) Supplementing the IVM medium with melatonin as a powerful antioxidant in order to reduce ROS; B) Designing a biphasic IVM system including a pre-maturation (pre-IVM) culture phase with meiotic inhibitors, followed by a conventional IVM phase. The effect of melatonin was evaluated in two studies. In the first study, various melatonin concentrations (10-11, 10-9, 10-7, 10-3 M) were tested in the IVM medium. The supplementation with 10-7 M of melatonin increased the blastocyst rate (28.9% vs. 11.7% in control) and decreased intra-oocyte ROS levels after IVM. In the second study, the mechanisms of action of melatonin (10-7 M) in the oocyte were assessed and the presence of melatonin receptor 1 (MT1) was detected in oocytes and cumulus cells by immunocytochemistry. Melatonin increased oocyte mitochondrial activity and ATP content, decreased intra-oocyte ROS levels, and improved blastocyst quality (55.8 vs. 30.4 cells in the inner cell mass), compared to control group without antioxidants. However, we could not determine if these effects were mediated by MT1 because IVM with melatonin plus luzindole (an inhibitor of MT1) showed no significant differences compared to melatonin and control groups. The strategy for improving oocyte competence during a pre-IVM culture with meiotic inhibitors was also developed in two studies. The first study was performed at the University of Adelaide (Australia) with oocytes from adult cow. We evaluated the effect of various concentrations of C-type natriuretic peptide (CNP; 50, 100, 200 nM) and 3-isobutyl-1-methylxanthine (IBMX; 500 µM) on oocyte meiotic arrest during 6 and 24 h. Pre-IVM culture with CNP (100 nM) plus IBMX for 6 h showed the greater effect on the meiotic arrest (92% of oocytes in germinal vesicle; GV). We developed a biphasic IVM system using this pre-IVM protocol followed by 20 h of conventional IVM, and compared to control IVM (24 h). Biphasic IVM prolonged cumulus-oocyte communication assessed by the density of transzonal projections (TZP), improved oocyte mitochondrial activity and increased blastocyst rate (45.1% vs. 34.5%). In the second study, a similar biphasic IVM system was tested in juvenile-goat IVEP. First, the effect of various CNP doses (50, 100, 200 nM) were tested with and without the addition of 10 nM estradiol. Pre-IVM with CNP (200 nM) plus estradiol sustained 74.7% of oocytes in GV and a high density of TZPs during 6 h. Second, oocytes were cultured with biphasic IVM (6 h pre-IVM plus 24 h IVM) compared to control IVM (24 h). Biphasic IVM increased intra-oocyte glutathione levels and decreased ROS, up-regulated the expression of DNA methyltransferase 1 and TNF-stimulated gene 6 protein in cumulus-oocyte complexes (COCs), and improved blastocyst rate (30.2% vs. 17.2%). In conclusion, IVM with melatonin and pre-IVM with meiotic inhibitors are promising methods that can considerably improve the oocyte developmental competence of very young females for producing embryos in vitro

Oocyte competence: Study of melatonin and meiotic inhibitors to improve in vitro embryo production in juvenile goats

La producción in vitro de embriones (PIVE) tiene su mayor limitación en la eficacia de la maduración in vitro (MIV) de los oocitos. La MIV altera la competencia de los oocitos debido al efecto perjudicial de los radicales de oxígeno (ROS) y a la reanudación rápida y espontanea de la meiosis. En oocitos de cabras de 1 a 2 meses de edad, estos dos factores son más intensos y perjudiciales. En esta tesis, se plantearon dos estrategias para mejorar los protocolos de MIV en estos oocitos: A) Suplementar el medio de MIV con melatonina como potente antioxidante para disminuir los ROS; B) Diseñar un sistema bifásico de MIV con una fase de pre-maduración (pre-MIV) con inhibidores de la meiosis, seguida por una segunda fase de MIV convencional. El efecto de la melatonina fue evaluado en dos estudios. En el primero, se testaron diferentes concentraciones de melatonina (10-11, 10-9, 10-7, 10-3 M) en el medio de MIV. La suplementación con 10-7 M de melatonina aumentó el porcentaje de blastocistos (28.9% vs. 11.7% en el control) y redujo el nivel intra-oocitario de ROS. En el segundo estudio, evaluamos los mecanismos de acción de la melatonina (10-7 M) en los oocitos y detectamos la presencia del receptor de melatonina 1 (MT1) en oocitos y células del cúmulo mediante inmunocitoquímica. La melatonina incrementó la actividad mitocondrial y el ATP de los oocitos, redujo los ROS y mejoró la calidad de los blastocistos producidos in vitro (55.8 vs. 30.4 células de la masa celular interna), comparado con el grupo control sin antioxidantes. No pudimos determinar si estos efectos son mediados por el MT1 porqué el grupo melatonina más lucindol (antagónico del MT1) no mostró diferencias significativas respeto los grupos melatonina y control. La estrategia para mejorar la competencia de los oocitos mediante una pre-MIV con inhibidores meióticos se desarrolló en dos estudios. El primer estudio se llevó a cabo en la Universidad de Adelaida (Australia) en oocitos de bovino adulto. Evaluamos el efecto de distintas concentraciones del péptido natriurético tipo C (CNP; 50, 100, 200 nM) y el 3-isobutyl-1-methylxanthine (IBMX; 500 µM) sobre el arresto meiótico durante 6 y 24 h. El cultivo de pre-MIV con CNP (100 nM) más IBMX durante 6 horas obtuvo el mayor efecto de parada meiótica (92% de oocitos en vesícula germinal; VG). A continuación, diseñamos un sistema bifásico utilizando este protocolo de pre-MIV, seguido de 20 h de MIV convencional, y comparamos con una MIV control (24 h). La MIV bifásica prolongó la comunicación entre células del cúmulo y oocito, evaluada mediante la densidad de proyecciones de transzona (TZP), mejoró la actividad mitocondrial y aumentó la producción de blastocistos (45.1% vs. 34.5%). En el segundo estudio, analizamos un sistema similar en oocitos de cabras de 1 a 2 meses de edad. Primero, evaluamos el efecto de distintas dosis de CNP (50, 100, 200 nM) con o sin la adición de 10 nM de estradiol. La pre-MIV con CNP (200 nM) más estradiol mantuvo un 74.7% de oocitos en VG y una alta densidad de TZP durante 6 h. En segundo lugar, cultivamos los oocitos con MIV bifásica (6 h pre-MIV más 24 h MIV) comparado con MIV control (24 h). La MIV bifásica incrementó el glutatión intra-oocitario y disminuyó los ROS, aumentó la expresión de DNA metiltranferasa 1 y proteína 6 estimuladora de TNF en complejos cúmulo-oocito (COCs), y mejoró la producción de blastocistos (30.2% vs. 17.2%). En conclusión, la MIV con melatonina y la pre-MIV con inhibidores meióticos son métodos que pueden mejorar sustancialmente la competencia de los oocitos de hembras muy jóvenes para producir embriones in vitro.Oocyte in vitro maturation (IVM) is a limiting step for the in vitro embryo production (IVEP). Conventional IVM can impair oocyte competence due to the damaging effect of reactive oxygen species (ROS) and the rapid and spontaneous meiotic resumption. These two factors are more intense and damaging for oocytes of juvenile goats (1-2 months old). In the present thesis, we hypothesized that two different strategies could improve IVM protocols in these oocytes: A) Supplementing the IVM medium with melatonin as a powerful antioxidant in order to reduce ROS; B) Designing a biphasic IVM system including a pre-maturation (pre-IVM) culture phase with meiotic inhibitors, followed by a conventional IVM phase. The effect of melatonin was evaluated in two studies. In the first study, various melatonin concentrations (10-11, 10-9, 10-7, 10-3 M) were tested in the IVM medium. The supplementation with 10-7 M of melatonin increased the blastocyst rate (28.9% vs. 11.7% in control) and decreased intra-oocyte ROS levels after IVM. In the second study, the mechanisms of action of melatonin (10-7 M) in the oocyte were assessed and the presence of melatonin receptor 1 (MT1) was detected in oocytes and cumulus cells by immunocytochemistry. Melatonin increased oocyte mitochondrial activity and ATP content, decreased intra-oocyte ROS levels, and improved blastocyst quality (55.8 vs. 30.4 cells in the inner cell mass), compared to control group without antioxidants. However, we could not determine if these effects were mediated by MT1 because IVM with melatonin plus luzindole (an inhibitor of MT1) showed no significant differences compared to melatonin and control groups. The strategy for improving oocyte competence during a pre-IVM culture with meiotic inhibitors was also developed in two studies. The first study was performed at the University of Adelaide (Australia) with oocytes from adult cow. We evaluated the effect of various concentrations of C-type natriuretic peptide (CNP; 50, 100, 200 nM) and 3-isobutyl-1-methylxanthine (IBMX; 500 µM) on oocyte meiotic arrest during 6 and 24 h. Pre-IVM culture with CNP (100 nM) plus IBMX for 6 h showed the greater effect on the meiotic arrest (92% of oocytes in germinal vesicle; GV). We developed a biphasic IVM system using this pre-IVM protocol followed by 20 h of conventional IVM, and compared to control IVM (24 h). Biphasic IVM prolonged cumulus-oocyte communication assessed by the density of transzonal projections (TZP), improved oocyte mitochondrial activity and increased blastocyst rate (45.1% vs. 34.5%). In the second study, a similar biphasic IVM system was tested in juvenile-goat IVEP. First, the effect of various CNP doses (50, 100, 200 nM) were tested with and without the addition of 10 nM estradiol. Pre-IVM with CNP (200 nM) plus estradiol sustained 74.7% of oocytes in GV and a high density of TZPs during 6 h. Second, oocytes were cultured with biphasic IVM (6 h pre-IVM plus 24 h IVM) compared to control IVM (24 h). Biphasic IVM increased intra-oocyte glutathione levels and decreased ROS, up-regulated the expression of DNA methyltransferase 1 and TNF-stimulated gene 6 protein in cumulus-oocyte complexes (COCs), and improved blastocyst rate (30.2% vs. 17.2%). In conclusion, IVM with melatonin and pre-IVM with meiotic inhibitors are promising methods that can considerably improve the oocyte developmental competence of very young females for producing embryos in vitro

Oocyte competence : study of melatonin and meiotic inhibitors to improve in vitro embryo production in juvenile goats

La producción in vitro de embriones (PIVE) tiene su mayor limitación en la eficacia de la maduración in vitro (MIV) de los oocitos. La MIV altera la competencia de los oocitos debido al efecto perjudicial de los radicales de oxígeno (ROS) y a la reanudación rápida y espontanea de la meiosis. En oocitos de cabras de 1 a 2 meses de edad, estos dos factores son más intensos y perjudiciales. En esta tesis, se plantearon dos estrategias para mejorar los protocolos de MIV en estos oocitos: A) Suplementar el medio de MIV con melatonina como potente antioxidante para disminuir los ROS; B) Diseñar un sistema bifásico de MIV con una fase de pre-maduración (pre-MIV) con inhibidores de la meiosis, seguida por una segunda fase de MIV convencional. El efecto de la melatonina fue evaluado en dos estudios. En el primero, se testaron diferentes concentraciones de melatonina (10-11, 10-9, 10-7, 10-3 M) en el medio de MIV. La suplementación con 10-7 M de melatonina aumentó el porcentaje de blastocistos (28.9% vs. 11.7% en el control) y redujo el nivel intra-oocitario de ROS. En el segundo estudio, evaluamos los mecanismos de acción de la melatonina (10-7 M) en los oocitos y detectamos la presencia del receptor de melatonina 1 (MT1) en oocitos y células del cúmulo mediante inmunocitoquímica. La melatonina incrementó la actividad mitocondrial y el ATP de los oocitos, redujo los ROS y mejoró la calidad de los blastocistos producidos in vitro (55.8 vs. 30.4 células de la masa celular interna), comparado con el grupo control sin antioxidantes. No pudimos determinar si estos efectos son mediados por el MT1 porqué el grupo melatonina más lucindol (antagónico del MT1) no mostró diferencias significativas respeto los grupos melatonina y control. La estrategia para mejorar la competencia de los oocitos mediante una pre-MIV con inhibidores meióticos se desarrolló en dos estudios. El primer estudio se llevó a cabo en la Universidad de Adelaida (Australia) en oocitos de bovino adulto. Evaluamos el efecto de distintas concentraciones del péptido natriurético tipo C (CNP; 50, 100, 200 nM) y el 3-isobutyl-1-methylxanthine (IBMX; 500 µM) sobre el arresto meiótico durante 6 y 24 h. El cultivo de pre-MIV con CNP (100 nM) más IBMX durante 6 horas obtuvo el mayor efecto de parada meiótica (92% de oocitos en vesícula germinal; VG). A continuación, diseñamos un sistema bifásico utilizando este protocolo de pre-MIV, seguido de 20 h de MIV convencional, y comparamos con una MIV control (24 h). La MIV bifásica prolongó la comunicación entre células del cúmulo y oocito, evaluada mediante la densidad de proyecciones de transzona (TZP), mejoró la actividad mitocondrial y aumentó la producción de blastocistos (45.1% vs. 34.5%). En el segundo estudio, analizamos un sistema similar en oocitos de cabras de 1 a 2 meses de edad. Primero, evaluamos el efecto de distintas dosis de CNP (50, 100, 200 nM) con o sin la adición de 10 nM de estradiol. La pre-MIV con CNP (200 nM) más estradiol mantuvo un 74.7% de oocitos en VG y una alta densidad de TZP durante 6 h. En segundo lugar, cultivamos los oocitos con MIV bifásica (6 h pre-MIV más 24 h MIV) comparado con MIV control (24 h). La MIV bifásica incrementó el glutatión intra-oocitario y disminuyó los ROS, aumentó la expresión de DNA metiltranferasa 1 y proteína 6 estimuladora de TNF en complejos cúmulo-oocito (COCs), y mejoró la producción de blastocistos (30.2% vs. 17.2%). En conclusión, la MIV con melatonina y la pre-MIV con inhibidores meióticos son métodos que pueden mejorar sustancialmente la competencia de los oocitos de hembras muy jóvenes para producir embriones in vitro.Oocyte in vitro maturation (IVM) is a limiting step for the in vitro embryo production (IVEP). Conventional IVM can impair oocyte competence due to the damaging effect of reactive oxygen species (ROS) and the rapid and spontaneous meiotic resumption. These two factors are more intense and damaging for oocytes of juvenile goats (1-2 months old). In the present thesis, we hypothesized that two different strategies could improve IVM protocols in these oocytes: A) Supplementing the IVM medium with melatonin as a powerful antioxidant in order to reduce ROS; B) Designing a biphasic IVM system including a pre-maturation (pre-IVM) culture phase with meiotic inhibitors, followed by a conventional IVM phase. The effect of melatonin was evaluated in two studies. In the first study, various melatonin concentrations (10-11, 10-9, 10-7, 10-3 M) were tested in the IVM medium. The supplementation with 10-7 M of melatonin increased the blastocyst rate (28.9% vs. 11.7% in control) and decreased intra-oocyte ROS levels after IVM. In the second study, the mechanisms of action of melatonin (10-7 M) in the oocyte were assessed and the presence of melatonin receptor 1 (MT1) was detected in oocytes and cumulus cells by immunocytochemistry. Melatonin increased oocyte mitochondrial activity and ATP content, decreased intra-oocyte ROS levels, and improved blastocyst quality (55.8 vs. 30.4 cells in the inner cell mass), compared to control group without antioxidants. However, we could not determine if these effects were mediated by MT1 because IVM with melatonin plus luzindole (an inhibitor of MT1) showed no significant differences compared to melatonin and control groups. The strategy for improving oocyte competence during a pre-IVM culture with meiotic inhibitors was also developed in two studies. The first study was performed at the University of Adelaide (Australia) with oocytes from adult cow. We evaluated the effect of various concentrations of C-type natriuretic peptide (CNP; 50, 100, 200 nM) and 3-isobutyl-1-methylxanthine (IBMX; 500 µM) on oocyte meiotic arrest during 6 and 24 h. Pre-IVM culture with CNP (100 nM) plus IBMX for 6 h showed the greater effect on the meiotic arrest (92% of oocytes in germinal vesicle; GV). We developed a biphasic IVM system using this pre-IVM protocol followed by 20 h of conventional IVM, and compared to control IVM (24 h). Biphasic IVM prolonged cumulus-oocyte communication assessed by the density of transzonal projections (TZP), improved oocyte mitochondrial activity and increased blastocyst rate (45.1% vs. 34.5%). In the second study, a similar biphasic IVM system was tested in juvenile-goat IVEP. First, the effect of various CNP doses (50, 100, 200 nM) were tested with and without the addition of 10 nM estradiol. Pre-IVM with CNP (200 nM) plus estradiol sustained 74.7% of oocytes in GV and a high density of TZPs during 6 h. Second, oocytes were cultured with biphasic IVM (6 h pre-IVM plus 24 h IVM) compared to control IVM (24 h). Biphasic IVM increased intra-oocyte glutathione levels and decreased ROS, up-regulated the expression of DNA methyltransferase 1 and TNF-stimulated gene 6 protein in cumulus-oocyte complexes (COCs), and improved blastocyst rate (30.2% vs. 17.2%). In conclusion, IVM with melatonin and pre-IVM with meiotic inhibitors are promising methods that can considerably improve the oocyte developmental competence of very young females for producing embryos in vitro

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulusoocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats

Resveratrol supplementation during in vitro maturation improves embryo development of prepubertal goat oocytes selected by brilliant cresyl blue staining

This study aimed to investigate the effect of resveratrol supplementation in maturation medium on the developmental ability and bioenergetic\oxidative status of prepubertal goat oocytes selected by brilliant cresyl blue (BCB). Oocytes collected from slaughterhouse-derived ovaries were selected by 13 µM BCB staining and classified as grown BCB+ and growing BCB- oocytes. All oocytes were matured in vitro in our conventional maturation medium and supplemented with 1 µM (BCB+R and BCB-R) and without (Control groups: BCB+C and BCB-C) resveratrol. After 24 h, IVM-oocytes were fertilized with fresh semen and presumptive zygotes were in vitro cultured for 8 days. Oocytes were assessed for blastocyst development and quality, mitochondrial activity and distribution, and levels of GSH, ROS, and ATP. BCB+R (28.3%) oocytes matured with resveratrol presented significantly higher blastocyst development than BCB+C (13.0%) and BCB- groups (BCB-R: 8.3% and BCB-C: 4.7%). Resveratrol improved blastocyst development of BCB-R oocytes at the same rate as BCB+C oocytes. No differences were observed in blastocyst quality among groups. GSH levels were significantly higher in resveratrol groups (BCB+R: 36554.6; BCB-R: 34946.7 pixels/oocyte) than in control groups (BCB+C: 27624.0; BCB-C: 27655.4 pixels/oocyte). No differences were found in mitochondrial activity, ROS level, and ATP content among the groups. Resveratrol-treated oocytes had a higher proportion of clustered active mitochondria in both BCB groups (BCB+R: 73.07%; BCB-R: 79.16%) than control groups (BCB+C: 19.35%; BCB-C: 40%). In conclusion, resveratrol increased blastocyst production from oocytes of prepubertal goats, particularly in better quality oocytes (BCB+)