Vitrificación de ovocitos en pacientes oncológicas: experiencia en clínica ivi chile y revisión de la literatura / Vitrification of oocytes in oncological patients: experience in clinica ivi chile and review of literature

La sobrevida de pacientes con cáncer ha mejorado con el tiempo, especialmente en pacientes en edad fértil. La criopreservación de los ovocitos a través de la estimulación ovárica controlada (EOC) es la técnica más frecuente de preservación de la fertilidad. El objetivo del presente estudio es realizar un análisis descriptivo de los ciclos de pacientes que, previo al tratamiento de cáncer, realizaron un tratamiento de preservación de fertilidad. Se analizaron datos demográficos como edad, diagnóstico de ingreso y resultados clínicos, tales como tipo de protocolo de estimulación utilizado, número de ovocitos obtenidos, duración de la estimulación y momento de inicio en el ciclo. Resultados: La edad promedio fue 28.9 años. La duración media de la estimulación fue de 12 días, con un promedio de ovocitos obtenidos en total de 12. Se utilizaron 2 protocolos de estimulación ovárica, obteniendo mejores resultados con el esquema de antagonistas de GnRH asociado a letrozole y doble gatillante. Respecto al momento del ciclo en que se inició la estimulación ovárica, no hubo diferencias. Conclusiones: Es posible realizar preservación de la fertilidad previo a un tratamiento oncológico con buenos resultados en pacientes jóvenes, por lo que sugerimos realizarlo en todos los pacientes con diagnóstico oncológico antes el tratamiento del cáncer. Es recomendable comenzar la estimulación ovárica en cualquier fase del ciclo ya que se obtienen los mismos resultados y permite un pronto inicio de la terapia oncológica.

Survival of patients with cancer has been improving over time, especially in young patient with fertility intention. Cryopreservation of oocytes through controlled ovarian stimulation (EOC) is the most frequent technique of fertility preservation. We analyzed the data obtained from oncological patients who attended IVI Chile between January 2008 and May 2017 in search of fertility preservation. Demographic data were obtained: age, diagnosis of admission, type of stimulation protocol used, number of oocytes obtained, duration of stimulation and pregnancy rate. Results: The average age: 28,9 years; average duration of stimulation:12 days. Number of oocytes obtained in total: 12. Two ovarian stimulation protocols were used. The one with the best results was the protocol with GnRH antagonists associated with letrozole and double triggering. Regarding the moment of the cycle where to start ovarian stimulation, there were no differences. Conclusions: It is possible to carry out a fertility preservation treatment prior to an oncological treatment with good results in young patients, so we suggest the preservation of fertility in all patients with an oncological diagnosis before oncological treatment. It is recommended to start ovarian stimulation at any phase of the cycle since the same results are obtained.

Las agregaciones de desove de Tylosurus pacificus (Beloniformes: Belonidae) y su pesca en el Parque Nacional Natural Utría (Pacífico colombiano) / Spawning aggregations of Tylosurus pacificus (Beloniformes: Belonidae) and their fishery in Utría Park, Colombian Pacific

ResumenAlgunas especies de peces marinos tienen complejas relaciones con los ecosistemas costeros durante sus periodos reproductivos, tal es el caso del pez aguja Tylosurus pacificus, que conforma agregaciones de desove en playas de grava en el Parque Nacional Natural Utría. Se describe la actividad pesquera y algunos aspectos de la biología reproductiva de T. pacificus y se proponen algunas medidas para mitigar el impacto sobre este evento y los procesos ecológicos asociados. Se realizó un monitoreo pesquero entre abril 2008 y febrero 2009 en el Parque Nacional Natural Utría (PNNU), Pacífico colombiano. Se analizaron los volúmenes de captura, estructura por talla, proporción de sexos y fecundidad (n= 84). La longitud total osciló entre 60.5 cm y 104 cm con una media y desviación estándar de 85.35 ± 9.09 cm. La fecundidad promedio fue 189 685.56 ovocitos por gónada, con una moda de 8 mm de diámetro y un desarrollo gonadal asincrónico. La especie desova en forma colectiva en la playa cuando la marea alcanza el nivel más alto durante la luna llena y nueva, generalmente al anochecer. Pescadores que habitan los pueblos cercanos aprovechan este recurso, durante la agregación reproductiva de la especie. Es importante ampliar el estudio de la reproducción de T. pacificus y los procesos ecológicos asociados a su desove para poder dar un aprovechamiento adecuado y garantizar la sostenibilidad de su pesquería a largo plazo.

Abstract:Some species of marine fish have complex relationships with coastal ecosystems during their reproductive periods, as the needle fish Tylosurus pacificus that forms spawning aggregations in gravel beaches in Utría Park. We described fishery and some aspects of the reproductive biology of T. pacificus and proposed some strategies to mitigate the impact of this event and associated ecological processes. Fisheries monitoring were conducted between April 2008 and February 2009 in the Utría National Park, Colombian Pacific. Catch volumes, length structure, sex ratio and fecundity (n= 84) were analyzed. The total length ranged among 60.5 and 104 cm with a mean and standard deviation of 85.35 ± 9.09 cm. The average fertility of oocytes per gonad was 189 685.56, with a mode of 8 mm diameter and an asynchronous gonadal development. This species spawns collectively on the beach when the tide reaches the highest level during full and new moon, usually in the evening. Fishermen of a near town take advantage of the spawning aggregation of this species. The reproduction study of T. pacificus and ecological processes associated with their spawning, should be expanded to give adequate use, and ensure the sustainability of their fishery over the long term. Rev. Biol. Trop. 65 (1): 77-87. Epub 2017 March 01.

Parámetros biológicos básicos para la conservación de Pseudocurimata lineopunctata (Characiformes, Curimatidae) en el Río Anchicayá, Valle del Cauca, Colombia / Basic biological parameters for Pseudocurimata lineopunctata (Characiformes, Curimatidae) conservation in the Anchicayá River of the Cauca Valley, Colombia

ResumenPseudocurimata lineopunctata representa un importante aporte nutricional para las comunidades locales en Colombia y Ecuador. A pesar que su captura anual es baja (590 kg/año), no hay restricciones sobre el tamaño mínimo, y esta especie está catalogada como vulnerable en riesgo de extinción moderada por la Corporación Autónoma Regional del Valle del Cauca (Colombia). Para apoyar los programas de conservación, se requiere de datos biológicos de las especie. Por lo tanto, los objetivos de este estudio fueron describir la proporción de sexos, la distribución, el tamaño en relación al peso, las fases macroscópicas de la madurez reproductiva, fecundidad y talla de primera madurez sexual para P. lineopunctata. Las muestras de peces fueron capturados con redes por nueve meses (Febrero-Octubre) en 2007. La longitud total (Lt) de los peces capturados varió entre 10.7 y 16.5 cm, con peso total (Pt) entre 25.0 y 67.5 g. Las hembras representaron el 52.6 % del grupo de muestra mientras que los machos el 47.4 %, y se observaron diferencias en la proporción de sexos en relación con el tamaño del pez. Durante todos los meses muestreados se capturaron ejemplares en estado de madurez avanzado. Con base en el análisis de los índices gonadosómatico, (IGS), gonádico (IG) y de condición (K), se postula que habría dos máximos de desove, uno de junio-julio y otro en septiembre-octubre. La talla de primera madurez sexual para las hembras fue determinada en 9.2 cm y para los machos en 10.1 cm de Lt. La fecundidad absoluta fue estimada en 3 598 ovocitos/♀, con una fecundidad relativa de 84 ovocitos/g♀. Se hace necesario realizar investigaciones adicionales que aumenten la información sobre la reproducción de esta especie, con la finalidad de apoyar futuros programas de repoblamiento.

Abstract:Pseudocurimata lineopunctata represents an important nutritional source for local human communities in Colombia and Ecuador. Although the yearly catch of this fish is low (590 kg/year), there are no restrictions on minimum size, and this species is categorized as vulnerable with moderate extinction risk by the Regional Autonomous Corporation of the Cauca Valley (Colombia). To support conservation programs, biological data of the target species are required. Therefore, the aims of this study were to describe the sex ratio, distribution, size to weight ratio, macroscopic stages of reproductive maturity, fecundity, and size at first sexual maturity for P. lineopunctata. For this, fish samples were captured with nets over nine months (February-October) in 2007. The total length (Lt) of the captured fish ranged between 10.7 and 16.5 cm, with total weight (Pt) between 25.0 and 67.5 g. Females represented 52.6 % of the sample group while males represented 47.4 %, and differences in sex ratio were observed in relation to fish size. For all sampled months, the fish captured showed an advanced maturity state. Based on analyses of the gonadosomatic index (IGS), gonadic index (IG), and Fulton's condition factor (K), and two spawning peaks were determined, one in June-July, and the other in September-October. The size at first sexual maturity was 9.2 cm for females and 10.1 cm for males. Absolute fecundity was estimated as 3 598 oocytes/ g♀. It is necessary to carry out additional investigations on the reproduction of this species, to support future repopulation programs. Rev. Biol. Trop. 65 (1): 239-253. Epub 2017 March 01.

Oocyte distribution within and between ovary lobes is largely homogeneous in Lachnolaimus maximus (Perciformes: Labridae) / La distribución de los ovocitos en y entre los lóbulos ováricos de Lachnolaimus maximus (Perciformes: Labridae) es generalmente homogénea

Abstract:In fish reproduction, previous information of ovary oocyte distribution is necessary, when oocytes quantitative estimates are required to estimate batch or annual fecundity. Heterogeneous oocyte distribution requires a standardized sampling protocol to prevent bias in estimates, whereas homogeneous distribution, allows sampling of any portion of gonads with no risk of bias. We studied gonad oocyte distribution patterns in the hogfish Lachnolaimus maximus population from Southern Gulf of Mexico. For this, 23 mature females were selected from a total of 47 individuals exhibiting visible oocytes in the ovaries. These females were classified by reproductive phase and sub-phase (early developing-ED, developing-D, spawning capable-SC and actively spawning-AS). Six histological sections were taken from the anterior, middle and posterior regions of the left and right ovary lobes of each individual. Digital image processing (AxioVision and Image ProPlus programs) was used to estimate oocyte density per unit area, and for different development stages. Contingency tables were used to analyze oocyte distribution frequencies between the regions of each lobe, and between the lobes of each ovary. This was supported with a Pearson's χ2 test for goodness-of-fit and a replicated G test to confirm distribution heterogeneity. Oocyte stage distribution was homogeneous in almost all 23 females regardless of the ovary lobe. In the left ovary lobe, oocyte distribution was uniform in all three regions sampled regardless the female phase or sub-phase. In the right ovary lobe, oocyte frequencies were similarly uniform for the ED, D and AS phase and sub-phases; nevertheless, during SC phase, some heterogeneity was observed in tertiary vitellogenesis-Vtg3 oocytes, especially in samples from middle and posterior regions of this lobe. Females in AS sub-phase are normally used to estimate batch fecundity in fish such as L. maximus, which has shown to have asynchronous oocyte development and batch spawning. Given the homogeneous oocyte distribution pattern within and between the ovary lobes in females in AS sub-phase, no systematization is required of the gonad histological sampling protocol to estimate species batch fecundity. Nevertheless, due to the heterogeneous Vtg3 oocytes distribution in the right ovary lobe of females in the SC phase, it is best to systematically take sections of any region in the left ovary lobe when conducting a study encompassing all of a species' reproductive aspects. Rev. Biol. Trop. 65 (1): 293-303. Epub 2017 March 01.

ResumenPara el estudio de la reproducción de los peces es necesario, un conocimiento previo sobre la distribución de los ovocitos en el ovario, así como estimaciones cuantitativas del número de ovocitos para estimar la fecundidad por lote o anual. Una distribución heterogénea exige tener un protocolo de muestreo estandarizado para prevenir sesgos en las estimaciones, mientras que una distribución homogénea permite utilizar muestras de cualquier parte de las gónadas sin riesgo de sesgo. Nosotros estudiamos el patrón de distribución de los ovocitos de la población de la doncella de pluma Lachnolaimus maximus del sur del Golfo de México. Para este propósito, 23 hembras maduras fueron seleccionadas de un total de 47 individuos que presentaron ovocitos observables a simple vista en los ovarios. Estas hembras fueron clasificadas según su fase o sub-fase reproductiva (desarrollo temprano-DT, desarrollo-D, aptitud para desovar-AD y desove activo-DA). Seis secciones histológicas fueron realizadas de las regiones anterior, media y posterior de los lóbulos derecho e izquierdo del ovario de cada individuo. Un procesamiento digital de imágenes (AxioVision e Image ProPlus) fue utilizado para estimar la densidad de los ovocitos, en diferentes estadios de desarrollo, por unidad de área. Las frecuencias de distribución de los ovocitos fueron analizadas entre regiones de un mismo lóbulo y entre lóbulos de cada ovario por medio de tablas de contingencia. Este análisis involucró la aplicación de la prueba de bondad de ajuste del χ2 de Pearson y de la prueba de G replicada en el caso de observar una distribución heterogénea. La mayoría de las 23 hembras analizadas presentó una distribución homogénea de los diferentes estadios de ovocito, en cualquier lóbulo considerado. En el lóbulo ovárico izquierdo, la distribución de los ovocitos fue similar en las tres regiones muestreadas, en cualquier fase o sub-fase de las hembras. En el lóbulo ovárico derecho, las frecuencias de los ovocitos fueron semejantes para las hembras en fase y sub-fases de DT, D y DA; sin embargo, durante la sub-fase de AD, una heterogeneidad en el desarrollo de los ovocitos en vitelogénesis terciaria-Vtg3 fue observada, especialmente en las muestras de las regiones media y posterior de este lóbulo. Las hembras en sub-fase de DA son usualmente utilizadas para estimar la fecundidad por lote en las especies de peces como L. maximus, la cual presenta un desarrollo asincrónico de los ovocitos y realiza desoves sucesivos por lote. Debido al patrón de distribución homogéneo de los ovocitos en y entre los lóbulos ováricos de las hembras en sub-fase de DA, no se requiere estandarizar un protocolo de muestreo histológico de las gónadas para estimar la fecundidad por lote de la especie. Sin embargo, debido a la distribución heterogénea de los ovocitos en Vtg3 en el lóbulo ovárico derecho de las hembras en fase de DA, es preferible tomar sistemáticamente secciones de cualquier región del lóbulo ovárico izquierdo cuando se realiza un estudio que incluye todo los aspectos reproductivos de la especie.

Reproductive biology of Sodhiana iranica (Brachyura: Gecarcinucidae) from Southern Iran / Biología reproductiva del cangrejo de agua dulce Sodhiana iranica (Brachyura: Gecarcinidae) del sur de Irán

Abstract:Freshwater crab, Sodhiana iranica, is an endemic gecarcinucid crab that has been recently reported from Southern Iran. This research examined some reproductive aspects of S. iranica from Eelood freshwater spring, Southern Iran. Crabs were haphazardly sampled from April 2012 to April 2013, on a bimonthly basis. Measurements of Gonado-Somatic Index (GSI), Hepato-Somatic Index (HSI), oocyte diameter, and other aspects such as carapace width (CW) and total body weight (TW) were made in the captured specimens. Results showed a single seasonal reproductive cycle. Maturation and spawning occurred from December 2012 to April 2013 during the study period. The peaks of HSI were observed in April 2012 and February 2013. The oocyte diameter showed its most significant increase between August 2012 and February 2013. Considering the single seasonal breeding of S. iranica, a correct management, during the reproductive cycle, is necessary to support a healthy stock of this crab. Rev. Biol. Trop. 65 (1): 365-373. Epub 2017 March 01.

ResumenEl cangrejo agua dulce Sodhiana iranica es un cangrejo gecarcinucido que ha sido recientemente encontrado en el sur de Irán. Este trabajo examina algunos aspectos de la reproducción del cangrejo de agua dulce S. Iranica en el manantial Eelood del sur de Irán. Los cangrejos se muestrearon al azar entre abril 2012 y abril 2013 cada dos meses. Las mediciones del índice gonadosomático, el hepatosomático (HSI), el diámetro de los ovocitos y las observaciones de las etapas de maduración de los especímenes capturados, revelaron un único ciclo reproductivo estacional. La maduración y el desove se produjeron entre Diciembre 2012 y Abril 2013. El pico del índice hepatosomático se observó en Abril 2012 y Febrero 2013, respectivamente, y resultó más significativo el incremento en la media del diámetro de los ovocitos, que se produjo entre Agosto 2012 y Febrero 2013. En este estudio, se encontró que S. iranica se reproduce una vez al año estacionalmente. Por lo anterior, la gestión correcta durante el ciclo reproductivo de esta especie es necesaria para mantener la naturaleza y salud del stock de cangrejo.

Semen bovino sexado congelado-descongelado en producción de embriones in vitro / In vitro embryo production with frozen-thawed bovine semen sexed

El objetivo de este trabajo fue evaluar la fertilidad in vitro del semen bovino sexado (SX) vs. no sexado (NS) congelado-descongelado de dos toros Holstein, cada uno de la misma partida. Determinar el sexo de las crías significa un avance importante para la producción. El citómetro de flujo separa los espermatozoides X e Y por diferencia de ADN (4 % mayor en X), con 90 % de efectividad. Los complejos ovocito-cúmulus (COC) se obtuvieron de folículos de 2 a 8 mm de ovarios de frigorífico, se cultivaron para maduración 22 h en TCM-199 + 5 % de SFB + 10 % licor folicular bovino (LFB), en gotas de 100 µl, cubiertos con aceite mineral, en incubadora (38,5 C, 5 % CO2 y 95 % de humedad). Posmaduración, se formaron al azar 4 grupos de COC los cuales fueron inseminados con NS y SX de los toros 1 y 2. Los COC se incluyeron en gotas de 100 ml a razón de 10 COC por gota de semen capacitado a una concentración de 2x106 espermatozoides/ml en todos los grupos, incubados durante 6 h. Posteriormente se cultivaron en CR1aa + 5 % SFB, en incubadora. A las 48 h se evaluó el clivaje y al día 7 el desarrollo embrionario. Los resultados fueron analizados con el Test de c2. Se encontró diferencias significativas en el toro 1 en el desarrollo embrionario a favor del NS (p<0,05). En el toro 2 no se encontró diferencias significativas en el clivaje ni en el desarrollo (p<0,05).

The objective of this study was to evaluate the in vitro fertility of sexed (SX) vs. non-sexed (NS), frozen-thawed bovine semen from two Holstein bull, from the same batch each one. Offspring sexing represents an important advance for livestock production. Flow cytometry separates X and Y spermatozoa by difference in DNA (4 % greater in X) with 90 % effectiveness. Cumulus-oocytes complexes (COC) were obtained from follicles measuring between 2 and 8 mm collected from slaughterhouse ovaries; they were then cultured 22 h for maturation in TCM-199 + 5 % BFS + 10 % bovine follicular fluid (BFF) in 100 µl drops with mineral oil, in incubator (38.5 C, 5% CO2 and 95 % humidity). Postmaturation, 4 groups were randomly formed and inseminated with NS and SX of the 1 and 2 bulls, including them in 100 µl drops at 10 COC per drop of capacitated semen diluted to a concentration of 2x106 sperms/ml in all groups, incubated during 6 h. They were then cultured in CR1aa + 5% BFS in an incubator. At 48 h cleavage and at day 7 embryonic development, were assessed. Results were analyzed with c2 square Test. There were significant differences (P<0.05) in the embryonic development in bull 1, grater in NS. In bull 2 there were not significant differences in cleavage neither in embryo development.

Vitrificación de ovocitos bovinos parcialmente madurados de la raza frisón rojo chileno / Vitrification of partially matured bovine oocytes of the Chilean red friesian breed

Se evaluó la tasa de maduración in vitro post descongelación de ovocitos bovinos, de la raza Frisón Rojo Chileno, parcialmente madurados y vitrificados. Complejos Cúmulus-ovocito fueron obtenidos por aspiración folicular, clasificados morfológicamente y aleatoriamente cultivados in vitro en TCM199 (10 % Suero Fetal Bovino (SFB), 50 mg/mL gentamicina, 0,2 mM piruvato de sodio, 0,08 µg/mL FSH, 1 µg/mL LH y 1 µg/mL estradiol) en los grupos: a) control (n= 137), madurados por 24 h a 38,5 C, 5 % CO2 y 99 % humedad y, b) tratamiento (n= 156), madurados por 6 h, parcialmente denudados, e incubados hasta completar 20 h, para luego ser vitrificados por el método Open Pulled Straws (OPS). Los ovocitos fueron expuestos a la solución de vitrificación uno (SV1) (Buffer Fosfato Salino (PBS), 10 % Etilenglicol (EG), 10% DMSO) por 30 s, posteriormente traspasados a la SV2 (PBS, 20 % EG, 20 % DMSO) por 25 s. Inmediatamente los ovocitos fueron cargados en pajuelas francesas estiradas (OPS) y sumergidos en nitrógeno líquido. Las ovocitos fueron descongelados introduciéndolos en una secuencia de soluciones con concentraciones decrecientes de sucrosa (0,3; 0,15 y 0 M respectivamente). Finalmente, los ovocitos continuaron con la maduración por 4 h adicionales. Posterior al periodo de maduración, los ovocitos de ambos grupos fueron fijados, teñidos y evaluados. Las proporciones de ovocitos en Metafase I (MI), Metafase II (MII) y degenerados fueron comparadas mediante el test de Chi cuadrado. La vitrificación aumentó (p 0,05) el porcentaje de pérdida y de ovocitos dañados en comparación al control. Además, aumentó (p 0,05) la tasa de ovocitos en MI y el número de ovocitos degenerados, y redujo el porcentaje de ovocitos MII, en comparación al control. Por tanto, la vitrificación por el método Open Pulled Straw de ovocitos parcialmente madurados in vitro es una alternativa viable para la conservación de material genético de hembras Frisón Rojo Chileno.

Post thawing in vitro maturation rate was evaluated for partially matured vitrified oocytes from Chilean Red Friesian cattle. Cumulus-Oocytes Complexes were obtained by follicular aspiration, classified by morphology and randomly in vitro matured in TCM199 (10 % Bovine Fetal Serum (BFS), 50 mg/mL gentamicine, 0.2 mM sodium piruvate, 0.08 µg/ml FSH, 1 µg/mL LH and 1 µg/mL estradiol) in the following groups: a) control (n= 137), matured for 24 h at 38.5 C, 5 % CO2 y 99 % humidity, and b) treatment (n= 156), matured for 6 h, partially denuded, and incubated until completion of 20 h. Then, oocytes were vitrified by the Open Pulled Straws (OPS) method. Oocytes were exposed to vitrification solution one (VS1) (Phosphate Buffered Saline (PBS), 10 % Ethylen glycol (EG), 10 % DMSO) for 30 s, then they were exposed to VS2 (PBS, 20% EG, 20% DMSO) for 25 s. Afterwards, oocytes were loaded into open pulled straws and submerged into liquid nitrogen. Oocytes were thawed by exposure to sequential solutions with decreasing concentrations of sucrose (0.3; 0.15 y 0 M respectively). Finally, oocytes continued the in vitro maturation for 4 additional hours. After completion of maturation period oocytes from both groups were fixated, stained and evaluated. The proportion of lost and damaged, MI, MII, and degenerate oocytes were compared between groups by Chi square test. Vitrification procedure increased (p 0.05) the percentage of oocytes lost and damaged when compared to control group. Additionally, vitrification increased (p 0.05) the proportion of MI and degenerated oocytes, and decreased the proportion of MII oocytes. Therefore, vitrification by the OPS method of partially matured bovine oocytes is a reliable alternative for the conservation of germinal cells from Chilean Red Friesian females.

In vitro fertilization of porcine oocytes is affected by spermatic coincubation time / A fertilização in vitro de ovócitos suínos é afetada pelo tempo de coincubação espermático

The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF), and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte). The sperm and oocytes were coincubated according to the following treatments (T): T1 = oocytes exposed to spermatozoa for one hour (173 oocytes), T2 = oocytes exposed to spermatozoa for two hours (170 oocytes), and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes). After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium) to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm). Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P<0.05) after two hours of coincubation time than it was for one or three hours. Furthermore, 68.60% of the ova coincubated with the spermatozoa for two hours were monospermic. The oocytes exposed to spermatozoa for one hour (T1) presented a higher (P<0.01) rate of polyspermy than those in T2 and T3. Fertilization performance (%) did not differ (P>0.05) between oocytes exposed to spermatozoa for one (T1) and three hours (T3). However, optimum (P=0.048) results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P>0.05) between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.(AU)

Esse estudo foi realizado para avaliar os efeitos de diferentes tempos de coincubação dos gametas sobre a fertilização in vitro (FIV) de suínos e se a eficiência dessa técnica poderia ser melhorada pela redução no período que os ovócitos são expostos aos espermatozoides durante a FIV. Um total de 508 (em grupos de 50) complexos cumulus-ovócito (COCs) imaturos foram maturados no meio NCSU-37. Os COCs foram cultivados por 40-44 horas e então inseminados com sêmen in natura (2.000 espermatozoides/ovócito). Os espermatozoides e ovócitos foram coincubados de acordo com os seguintes tratamentos (T): T1 = ovócitos expostos aos espermatozoides por uma hora (173 ovócitos); T2 = ovócitos expostos aos espermatozoides por duas horas (170 ovócitos) e T3 = ovócitos expostos aos espermatozoides por três horas (165 ovócitos). Após esses períodos de coincubação, os ovócitos foram lavados em meio de fertilização (meio TALP) para remoção dos espermazoides não ligados a zona pelúcida e cultivados em outro mesmo meio (não contendo espermatozoides). Após 18-20 horas de fertilização, os prováveis zigotos foram corados com Hoechst-33342 para avaliação dos resultados da FIV. A taxa de penetração foi maior (P<0,05) após o tempo de coincubação de duas horas em comparação a uma e três horas. Além disso, 68,60% dos ovócitos coincubados com os espermatozoides por duas horas foram monospérmicos. Os ovócitos expostos aos espermatozoides por uma hora (T1) apresentaram elevada (P<0,01) taxa de polispermia em comparação com o T2 e T3. A eficiência da fertilização (%) não diferiu (P>0,05) entre os ovócitos expostos aos espermatozoides por uma (T1) e três horas (T3). Entretanto, ótimos (P=0,048) resultados foram obtidos após duas horas de coincubação, quando a taxa da eficiência da fertilização foi 50,16 ± 8,52%. O número de espermatozoides penetrados por ovócito e a formação de pro-núcleo masculino não diferiu (P>0,05) entre os tratamentos avaliados. Sob as condições de ensaio realizadas, especialmente em relação à concentração espermática utilizada, a coincubação dos gametas por um período de duas horas parece ser ótima para as taxas de monospermia e eficiência da fertilização. Portanto, um tempo provavelmente ótimo para obter um elevado número de embriões suínos capazes de ter um desenvolvimento normal.(AU)

Random-start controlled ovarian stimulation for emergency fertility preservation in a patient with myelodysplastic syndrome: a case report

This study reports a case of a gonadotropin-releasing hormone agonist trigger in a young female with myelodysplastic syndrome (MDS) who underwent fertility preservation using random-start controlled ovarian stimulation. This method involves the stimulation of the ovary regardless of a patient's menstrual-cycle phase. A review of the related literature is also provided. A 17-year-old patient was diagnosed with MDS and required initiation of peripheral blood stem cell transplantation within a maximum of 3 weeks and was in the luteal phase of the menstrual cycle when the possibility of attempting preservation of fertility was presented to her. She opted for a random-start controlled ovarian stimulation with gonadotropins. With successful hemorrhagic prophylaxis, 17 oocytes were retrieved including 10 mature and 7 immature oocytes. Of the immature oocytes, 3 were successfully matured in vitro and a vitrification protocol was used to freeze the 13 mature oocytes.

Viabilidad de Ovocitos Vitrificados y Madurados in Vitro de Gata Doméstica Adulta (Felis catus) en Estación Reproductiva / Vitrified and in Vitro Matured Oocytes Viability from Adult Housecat (Felis catus) in Reproductive Season

La vitrificación de ovocitos de mamíferos es considerada una técnica en experimentación. La supervivencia de los ovocitos es extremadamente variable, según las técnicas utilizadas e influida por una serie de condiciones. El objetivo de éste trabajo fue determinar los efectos de la vitrificación en ovocitos de gata domestica adulta en estación reproductiva y madurados in vitro. Se obtuvieron 33 ovarios correspondientes a hembras de más de un año en buen estado nutricional y sin tratamiento hormonal, ovariectomizadas en domicilio del propietario por profesional veterinario. Los ovarios fueron trasladados al laboratorio y se fragmentaron por microdisección bajo microscopio estereoscópico en caja de Petri con solución de buffer fosfato salino modificado con suero de ternero inactivado y antibióticos para la obtención, evaluación y selección de los complejos cúmulo-ovocitos (CCOs) de buena calidad. Se vitrificaron 506 CCOs en pajuelas de 0.25 ml con 10-12 ovocitos cada una y almacenados por 30 días en nitrógeno líquido (N2) a -196 C. Posteriormente se desvitrificaron las pajuelas, recuperándose 320 CCOs, los que fueron madurados in vitro. Transcurrido ese tiempo se evaluaron los CCOs, descartándose 50 por presentar signos de degeneración. En los 270 restantes se observó buena expansión del cúmulo, citoplasma uniforme y oscuro e integridad de la zona pelúcida. A estos últimos se los dividió en dos grupos similares para evaluar la viabilidad, a uno se lo coloreó con metil tiazol tetrazolio y al otro con Azul Tripán. En ambos se constató un resultado positivo para la viabilidad de los CCOs. El análisis de los resultados nos permite concluir que la vitrificación comprometió la integridad de un importante número de CCOs aunque más del 50% respondió en cultivo favorablemente, mostrando signos de viabilidad esperados. Estos resultados hacen necesarios la profundización en el mejoramiento del protocolo para incrementar el porcentaje de viabilidad.

Vitrification of mammalian oocytes is a technique considered in experimentation. Oocyte survival is extremely variable, according to the techniques used and influenced by a number of conditions. The objective of this study was to determine the effects of adult domestic cat oocyte matured in vitro and vitrified in breeding season. We obtained 33 ovaries from housecats in good nutritional status without hormonal treatment, ovariectomized by a veterinary professional at home. The ovaries were transported to the laboratory and fragmented by microdissection under a stereoscopic microscope in a petri dish with modified buffer saline phosphate solution, inactivated calf serum and antibiotics to the collection, evaluation and selection of good quality cumulus-oocyte complexes (COCs). 506 COCs were vitrified in 0.25 ml straws each with 10­12 oocytes and stored for 30 days in liquid nitrogen (N2) at -196 °C. Then the straws were thawed, recovering 320 CCOs, which were matured in vitro. After this time the COCs were evaluated, discarding 50 which showed signs of degeneration. In the remaining 270 COCs good cumulus expansion was observed and also uniformly dark cytoplasm and integrity of the zona pellucida. The latter were divided into two similar groups to assess the feasibility; one was stained with Methylthiazblyl tetrazolium and the other with Trypan Blue. Both tested positive for the viability of the CCOs was found. The analysis of the results allow us to conclude that vitrification compromised the integrity of a large number of CCOs although more than 50% responded favorably in culture, showing signs of expected viability. According to these results it is necessary to further improve the protocol to increase the percentage of viability.

Efecto de la vitrificación de ovocitos humanos sobre la capacidad de unión y el estado acrosomal de espermatozoides humanos / Effect of the vitrification of human oocytes on the binding capacity and acrosome status of human sperm

Conocer los aspectos moleculares que acontecen en el proceso de unión de los espermatozoides humanos a la zona pelúcida (ZP) humana es uno de los grandes retos de la biología de la Reproducción. Por otra parte conocer si el proceso de fecundación puede verse afectado por la criopreservación de los gametos femeninos sigue siendo otra cuestión debatida en la literatura. En base a esto, el objetivo principal de este trabajo fue conocer si la vitrificación ovocitaria puede alterar la interacción de los espermatozoides con el glicocáliz de la ZP y demostrar si la ZP de estos ovocitos pierde la capacidad de inducir la reacción acrosómica en los espermatozoides. Según nuestros resultados el método de vitrificación ovocitaria cerrado (S3) no altera la capacidad de unión de los espermatozoides a la zona pelúcida, ni la capacidad de ésta para inducir la reacción acrosómica.

To know the molecular aspects that occur in the process of human sperm binding to the human zona pellucida (ZP) is one of the great challenges of reproduction biology. Moreover knowing if the fertilization process may be affected by cryopreservation of female gametes is still another issue discussed in the literature. Based on this, the main objective of this study was to determine whether the oocyte vitrification may alter the interaction of sperm with the glycocalyx of ZP and show whether these oocytes lost the ability to induce the acrosome reaction in sperm. According to our results the oocyte closed vitrification method (S3) does not alter the ability of the sperm binding to the zona pellucida, and their ability to induce the acrosome reaction.

Oocyte quality of tambaqui (Colossoma macropomum) during the reproductive season / Qualidade ovocitária de tambaqui (Colossoma macropomum) ao longo da estação reprodutiva

The study aimed to analyze the Colossoma macropomum reproductive behavior and quality of the female gametes throughout the reproductive season. The experiment was carried out in Pimenta Bueno - Rondônia State (Northern Brazil) during the reproductive season (2010-2011) using 36 females. Each sampling was performed on a 15 ± 5 days interval. Female gametes were collected by stripping and the following analyses were performed: weight of oocytes released (g); productivity index, fertilization and hatching rate. During the sampling period was verified effect (p < 0.05) of collecting time into the season for oocytes weight, productivity index and fertilization rate. Although the period 3 (December) did not differ significantly from other periods, it showed better parameters for the quality of C. macropomum oocytes.

O estudo foi conduzido com o objetivo de analisar o comportamento reprodutivo da espécie Colossoma macropomum, quanto à qualidade de seus gametas femininos ao longo da estação reprodutiva. O experimento foi executado em Pimenta Bueno-Rondônia durante a estação reprodutiva do C. macropomum. Utilizaram 36 fêmeas durante a estação de 2010-2011. Cada coleta apresentou um intervalo de 15±5 dias. Através da extrusão foram coletados os gametas femininos e realizadas as seguintes análises ao longo da estação: peso de oócitos liberados (g); índice de produtividade; taxa de fertilização e eclosão. Durante a estação 2010-2011 foi verificado efeito (p < 0,05) de período (coleta) dentro da estação para peso de oócitos, índice de produtividade e taxa de fertilização. Apesar do período 3 (coleta – mês de dezembro) não ter diferenciado significativamente de alguns períodos, foi o que apresentou os melhores parâmetros estabelecidos para a qualidade dos oócitos de C. macropomum.

A Correlative Study of Oocytes Morphology with Fertilization, Cleavage, Embryo Quality and Implantation Rates after Intra Cytoplasmic Sperm Injection.

ntroduction: Non-invasive selection of developmentally human oocytes may increase the overall effi ciency of human assisted reproduction. Morphologic abnormalities in the oocyte are relevant for determining its developmental fate. Th e objective is to evaluate the infl uence of MII oocyte morphology on intra cytoplasmic sperm injection (ICSI) outcomes. Material and Methods: 132 patients undergoing ICSI cycles and having female factors of infertility and unexplained infertility. Couples having male factors of infertility were excluded. A total of 1200 oocytes were retrieved from 132 ICSI cycles, of which 1056 MII oocytes were evaluated. Th e criteria for morphological evaluations were: (i) Normal MII oocytes showing clear cytoplasm with uniform texture and homogenous fi ne granularity, a round or ovoid fi rst polar body with a smooth surface, and perivitelline space of normal size. (ii) MII oocytes with extra cytoplasmic abnormalities (fi rst polar body and perivitelline space abnormalities). (iii) MII oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, inclusion body and presents of vacuoles). (iv) MII oocytes with combined abnormalities. Result: From 1056 MII oocytes, 180 (17.04%) had normal morphology while 876 (82.95%) had at least one demonstrable morphological abnormality. Cytoplasmic abnormalities were observed in 516 (58.9%) of the oocytes. Extra cytoplasmic abnormalities were observed in 104 (11.87%) while combined abnormalities were responsible for the remaining 256 (29.22%). Th ere were no signifi cant diff erences in fertilization, cleavage, and embryo quality between the groups but there was a highly signifi cant diff erence in implantation rate which was higher in the group of normal oocytes morphology than abnormal oocytes morphology, oocytes with cytoplasmic, extracytoplasmic and combined abnormality 11.11%, 7.33%, 9.03%, 2.3%, and 4.34% respectively. Conclusion: MII oocyte morphology did not aff ect fertilization, cleavage, and embryo quality, but aff ecting implantation rate.

Effect of gonadotrophins, oestradiol and insulin on cumulus expansion of Nili Ravi buffalo oocytes

The objective of the present study was to investigate the cumulus expansions of Nili Ravi buffalo oocytes during cultured in TCM -199 supplemented with 2 micro g/ml oestradiol [E[2]], 0.05 IU/ml recombinant human follicle stimulating hormone [rhFSH], 2IU/ml human chorionic gonadotrophin [hCG], and 0.12 IU/ml insulin [I]. The cumulus oocytes complexes [COCs] were collected from 2-8mm follicles from local abattoir ovaries. Supplementation of medium with single hormones showed significant [P<0.0001] increase in mean diameter of COCs with rhFSH except E[2], hCG and insulin after 24 hours compared to the increase in the mean diameter of COCs matured in TCM-199 without any hormonal supplementation. With rhFSH even at 8th hour, significant increase [P<0.001] in cumulus expansion was observed. In combination of hormones the significant [P<0.0001] cumulus expansion was achieved in E[2]+rhFSH treatment group. The non significant [P>0.05] cumulus expansion was observed in treatment groups viz. E[2]+hCG, E[2]+Insulin, rhFSH+hCG, rhFSH+Insulin, hCG+Insulin, E[2]+rhFSH+hCG and E[2]+rhFSH+hCG+Insulin after 24 hours. In conclusion, supplementation of rhFSH alone and in combination with E2in TCM-199 has highly significant effect on cumulus expansion

Signaling Pathways in Insulin- and IGF-I Mediated Oocyte Maturation in Lower Vertebrates.

The endocrine control of oocyte maturation in fish and amphibians has proved to be a valuable model for investigating the rapid and non-genomic steroid actions at the cell surface. Considerable progress has made over the last decade in elucidating signaling pathways in steroid-induced oocyte maturation. In addition to steroids, various growth factors have also been reported to be involved in this process and progress being made to elucidate their mechanism of actions. Exposure of fully-grown oocytes to steroids or growth factors (insulin/IGFs) initiates various signaling cascade, leading to formation and activation of maturation-promoting factor (MPF), a key enzyme that catalyzes entry into M-phase of meiosis I and II. Whereas the function of MPF in promoting oocyte maturation is ubiquitous, there are differences in signaling pathways between steroids- and growth factors-induced oocyte maturation in amphibian and fish. Here, we have reviewed the recent advances on the signaling pathways in insulin- and IGF-I-induced oocyte maturation in these two groups of non-mammalian vertebrates. New findings demonstrating the involvement of PI3 kinase and MAP kinase in induction of oocyte maturation by insulin and IGF-I are presented.

Cambios morfológicos en gametos del barbo tigre Puntius tetrazona (Cypriniformes: Cyprinidae) e implementación de la fertilización in vitro / Morphological changes in gametes of tiger barb Puntius tetrazona (Cypriniformes: Cyprinidae) and the implementation of in vitro fertilization

The production of ornamental fishes represents an economic activity of a growing number of Mexican families. Nevertheless, the reproduction of fish in captivity is one of the complications faced by farmers. This study was set up to: (i) evaluate the morphological and functional changes induced by hydration in the gametes of fish tiger barb (Puntius tetrazona; 240 samples) at tree times after hydration (10, 20 and 30s) with classic spermograms (volume, sperm concentration, viability, motility, and normal morphology); and (ii) evaluate the implementation of in vitro fertilization based on the ovulation rate, the percentage of fertilization and hatching, and the larval numbers obtained after 72 hours. The average volume of milt was 3.0±0.7μL, and the minimum, maximum and average concentration of sperm was 44.4x10(6) spz/mL, 52.3x10(6)spz/mL, and 48.1±5.9x10(6)spz/mL, respectively. The viability and motility of the sperm was 84.6±3.2% and 81.5±2.2%, respectively. The diameter of the sperm with/without water contact was 2.1±0.6μm and 3.8±1.0μm (p<0.05); the largest diameter was recorded 30 seconds after the contact with water. For oocytes, the smaller and larger diameters were recorded at 10 and 30s, respectively (both with/without water contact); the oocytes diameters after 10 and 30 seconds of contact with water were 1.11 and 1.55mm, respectively. A higher ovulation rate was recorded using the in vitro fertilization: 250±50 oocytes versus 28±09 oocytes (during natural fertilization; p<0.05). Nevertheless, fertilization and hatching rates were higher for the natural fertilization (80 and 60%, respectively). Considering the number of larvae obtained after 72 hours, our results showed a higher value for the in vitro fertilization (75±18 compared to 13.4±12 of the natural fertilization; p<0.05). We propose this fish as a model for other ornamental fishes of commercial interest. Our results demonstrate that the in vitro fertilization is a very high viable option to optimize and maximize resources; besides, the reproduction management optimization under controlled conditions may enhance wild fish stocks preservation. Rev. Biol. Trop. 62 (4): 1353-1363. Epub 2014 December 01.

El objetivo del presente estudio fue conocer las características de los gametos de Puntius tetrazona (n=240), los cambios morfológicos a partir de su activación mediante espermogramas cásicos y por otro lado, se evaluó la implementación de la fertilización in vitro a partir de la tasa ovulatoria, el % de fertilización y eclosión y el número de larvas vivas a las 72h. El volumen promedio de semen fue de 3.0±0.7µL. La concentración espermática mínima, máxima y promedio fue 44.48x10(6)spz/mL, 52.3x10(6)spz/mL y 48.1±5.9x10(6)spz/mL, respectivamente. La viabilidad promedio fue de 84.68±3.27%. La motilidad promedio fue 81.53±2.28%. El diámetro de los espermatozoides fluctuó entre 2.16±0.2 y 2.79±0.3µm; 3.84±0.3 y 4.86±0.31µm sin y con contacto con el agua respectivamente, con diferencias significativas. El diámetro mayor fue a los 30s en contacto con el agua. Los ovocitos de menor y mayor diámetro se registraron a los diez y 30s sin y con contacto con el agua respectivamente. Los diámetros de los ovocitos en diez y 30s en contacto con el agua fluctuaron entre 1.11 y 1.55mm respectivamente. La mayor tasa ovulatoria fue en la fertilización in vitro con 250±50 ovocitos frente a 28±09 de la natural, con diferencias significativas. Los porcentajes de fertilización y eclosión fueron más elevados en la fertilización natural con 80% y 60% respectivamente. Se registraron 75±18 larvas a las 72 horas en el grupo in vitro comparado con 13.4±12 larvas de la fertilización natural. Con lo anterior, la técnica que permitió mayor cantidad de larvas fue la de fertilización in vitro.

An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes.

Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 μM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

Histological description of oogenesis in chiton virgulatus (Mollusca: Polyplacophora) / Descripción histológica de la ovogénesis de chiton virgulatus (Mollusca: Polyplacophora)

This paper describes the oogenesis of Chiton virgulatus, based on histological observations under transmission and scanning electron microscopy. Three oocyte types were identified: i) previtellogenic oocytes with a mean diameter of 50±20.5 µm, surrounded by elongated follicular cells of approximately 5 µm, ii) immature vitellogenic oocytes with a mean diameter of 113±15.3 µm and small cytoplasmic projections denoting the onset of the oocyte hull development; adjacent to each projection are pores approximately 0.7 µm in diameter, and iii) mature vitellogenic oocytes with a mean diameter of 146±24.8 µm; the oocyte cytoplasmic projections grow and its apical zone becomes trident-shaped; follicular cells adopt a bulbous shape due to the growth of the elongation and can reach up to 20 µm in length. The morphology and ultrastructure of the projections of the mature vitellogenic oocyte, as well as the size of pores at their base, are specific to C. virgulatus; therefore, these features could be used in taxonomic or fertilization studies.

En el presente trabajo se describe la ovogénesis de Chiton virgulatus, utilizando histología y las técnicas de microscopía electrónica de barrido y de transmisión. Se identificaron tres tipos de ovocitos: i) ovocitos previtelogénicos con un diámetro promedio de 50±20,5 µm, rodeados por células foliculares de forma alargada y un tamaño de aproximadamente 5 µm, ii) ovocitos vitelogénicos inmaduros con un diámetro promedio de 113±15,3 µm, este tipo de ovocitos presentan pequeñas proyecciones citoplasmáticas, que indican el inicio del desarrollo del casco del ovocito. Adyacentes a cada prolongación se presentan poros con un diámetro aproximado de 0,7 µm y iii) ovocitos vitelogénicos maduros con un diámetro promedio de 146±24,8 µm, las proyecciones citoplasmáticas del casco del ovocito crecen y en su parte apical adquieren la forma de un tridente, las células foliculares, dado el crecimiento de la prolongación toman el aspecto bulboso y llegan a medir hasta 20 µm de longitud. La morfología y la ultraestructura de las proyecciones del casco del ovocito vitelogénico maduro, así como el tamaño del poro en la base de las proyecciones son particulares para C. virgulatus, dichas características podrían ser utilizadas en trabajos de taxonomía y fertilización.

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.