Physiological and enzymatic responses of Chlorella vulgaris exposed to produced water and its potential for bioremediation.

In South America, Colombia is known as an important oil-producing country. However, the environmental impact of crude oil industry has not been studied deeply and few studies have been carried out for evaluating responses of algae and its adaptation under specific conditions. Enzymatic and physiological effects in Chlorella vulgaris and its potential for bioremediation after exposure to produced water (PW) were assessed using different PW concentrations (0, 25, 50, 75 and 100%) and crude oil. Variables such as cell density, growth rate (µ), percentage of growth inhibition (% I), chlorophyll a and b and cell diameter were evaluated during 5 days. Furthermore, enzymatic biomarkers such as superoxide dismutase (SOD) and catalase (CAT) were also measured. Results showed that the treatment with 100% PW had the highest cell density and µ; similarly, 25% PW treatment had a similar behaviour, being these two treatments with the highest growth. A dose-dependent response was seen for chlorophyll a and b and cell diameter, showing significant differences between treatments and the control. Different levels of SOD and CAT were observed in algae exposed to PW. At 24 h, an increase in SOD and CAT activity was observed, probably due to effects caused by xenobiotics. After 72 h, a decrease in the activity of both enzymes was observed. The results evidenced that C. vulgaris can adapt easily to PW, showing an increase on its growth and stabilisation in its antioxidant activity. Additionally, cell diameter results and decrease of hydrocarbons and phenols show the potential of these algae to degrade xenobiotics from PW.

Liver biomarkers response of the neotropical fish Aequidens metae to environmental stressors associated with the oil industry.

The Acacias River in Colombia receives large volumes of industrial effluents mostly derived from the oil industry. To contribute to the study of the possible effects of industrial wastewaters on the aquatic environment and particularly on fish populations, a native neotropical fish, Aequidens metae was used as a sentinel species. Wild specimens of A. metae were caught at three different places of the Acacias River taking as reference the point of discharge of an oil industry effluent; upstream, downstream, and at the vicinity of the discharge pipe. A fourth sampling site was chosen as a reference site away from urban settlements. Samplings were performed twice, during the rainy and dry seasons. After anesthesia animals were weighted and measured, and humanely sacrificed. Livers were extracted, frozen on site and transported to the laboratory. Condition indices were calculated. Total protein content and the detoxification 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity were estimated. Histopathological alterations were also evaluated. Water quality was estimated through the measurement of several variables. Results obtained evidenced that the highest induction in EROD activity and the strongest histological alterations in liver of the monitored fish appeared during the dry seasons at the discharge site and downstream to this point.

Effects of 17beta-trenbolone in male eelpout Zoarces viviparus exposed to ethinylestradiol.

To evaluate the interaction between 17beta-trenbolone (TB) and 17alpha-ethinylestradiol (EE2), male eelpout, Zoarces viviparus, was exposed for 21 days (April to May 2008) to 5 ng l(-1) EE2 and 5 or 20 ng l(-1) TB, separately or in combination in a flow-through SW system. The effects on hepatosomatic (HSI) and gonadosomatic index (GSI), plasma vitellogenin (Vtg) concentration, gonadal histology, hepatic and testicular Vtg mRNA and estrogen receptor (ERalpha) mRNA expression were investigated. No effects on HSI were observed. A significant decrease was observed in the GSI of all males exposed to EE2 (<0.7%) when compared to controls (1.4%). Histological alterations and immature stages were observed in the testis of all exposed males; however, males exposed to EE2 were the most affected. Increased tubule number and proportionally decreased tubule diameter were observed in the testis of all EE2 groups. No effects in Vtg mRNA expression were observed in the testis; however, a significant decrease in testis ERalpha mRNA was observed in males exposed to 20 ng l(-1) TB. The groups exposed to EE2 showed a significant increase in plasma Vtg (>300-fold), hepatic Vtg mRNA (>450-fold), and ERalpha mRNA (>100-fold) when compared to controls. This study shows that lower concentrations of 17beta-trenbolone are unable to counteract the EE2 estrogenic effects when the exposure is simultaneous.

Biochemical and histological alterations in Aequidens metae (Pisces, Cichlidae) and Astyanax gr. bimaculatus (Pisces, Characidae) as indicators of river pollution.

The water quality from rivers near to cities has decreased drastically over the decades due to the environmental pollution. The Ocoa River on its way through the Villavicencio city, Colombia, receives large volumes of domestic and industrial wastewater. In order to establish the effect of the contamination, biochemical and histopathology biomarkers were evaluated in the gills and the liver of two native fish species. Astyanax gr. bimaculatus and Aequidens metae were caught in three sites of the Ocoa River (S1, S2 and S3) and in a reference River during the rainy and dry season. A. metae showed to be more sensitive to water pollution. In general, the rainy season induced a greater negative impact in the fish monitored. At site S3 (after crossing the city where urban runoff and industrial effluents are discharge and close to a landfill), increased inhibition of the antioxidant response and lipid membranes damage were observed; in addition, in this site the histopathological alterations were greater in both fish species. The impact observed in this study on fish health provoked for the Ocoa River contamination demands the need to begin strategies to solve the problem of discharge of domestic and industrial wastewater in aquatic environments in Colombia.

Behavioural and gill histopathological effects of acute exposure to sodium chloride in moneda (Metynnis orinocensis).

To evaluate the toxicity of sodium chloride (NaCl), juveniles and adult Metynnis orinocensis were exposed for 96h to 0, 5, 10, 15, 20 or 40gL(-1) of salt. Food intake, behaviour, opercular frequency (OF), mortality, body weight and gill microscopic alterations were evaluated. Behavioural changes were observed in fish exposed to concentrations higher than 10gL(-1). Juveniles and adults showed a progressive decrease in the OF and body weight. Food intake decreased in concentrations below 15gL(-1). Juveniles and adults exposed to 15, 20 or 40gL(-1) had 100% mortality. Lamellar congestion, hyperplasia and fusion were the common microscopic alterations at higher concentrations. The gill congestion severity increased with salt concentration. The LC(50) for juveniles and adults were 10.5gL(-1) and 10.8gL(-1), respectively. These results suggest that salt concentrations lower than 5gL(-1) are safe for preventive and therapeutic practices in Metynnis orinocensis; whereas prolonged exposure higher than 10gL(-1) is deleterious in this species.

Evidence of small modulation of ethinylestradiol induced effects by concurrent exposure to trenbolone in male eelpout Zoarces viviparus.

The interaction of xenobiotics is common in aquatic ecosystems; therefore, we wanted to evaluate if trenbolone (TB) modulates the effects of 17α-ethinylestradiol (EE2). Male eelpout (Zoarces viviparus) were exposed to 5 ng L(-1) EE2 continuously for 19 d (EE2-C) or discontinuously (11 d, EE2-D) alone or in combination with low (50 ng L(-1), TBL) or high (500 ng L(-1), TBH) concentrations of TB (19 d). Exposure to EE2 caused reduced gonadosomatic index, increased plasma vitellogenin concentrations, up-regulated vtg and era mRNA expression and severe alterations in gonadal histology. TBL and TBH did not affect plasma vitellogenin, era or vtg mRNA expression. TBL and TBH did not counteract the EE2-induced increase in plasma vitellogenin and reduction in 11-ketotestosterone whereas TBH counteracted the EE2 induced increase in vtg and era mRNA expression. Exposure to TBH and EE2-C + TBH lead to severe gonadal histology alterations. TBL and EE2-D + TBH exposed fish showed less histopathological alterations.

Hábitos alimenticios de Leporinus friderici (Anostomidae: Teleostei) durante un ciclo hidrobiológico en el río Vaupés, Colombia / Feeding habits of Leporinus friderici (Anostomidae: Teleostei) during a hydrobiological cycle in Vaupés River, Colombia

ResumenLa especie más importante de la pesquería del río Vaupés es un pez conocido como Warakú tres puntos (Leporinus friderici), un carácido migratorio representativo del departamento del Vaupés, Colombia, de importancia para la seguridad alimentaria de comunidades indígenas. Con el propósito de contribuir en el conocimiento biológico básico de la especie, el presente estudio determinó los hábitos y preferencias alimenticias de Leporinus friderici durante un ciclo hidrobiológico (marzo 2014 a mayo 2015) en el río Vaupés, Colombia. Fueron muestreados tres lugares, dos a lo largo del río Vaupés (comunidades indígenas de Yacayacá y Santa Cruz) y uno en el río Cuduyarí afluente del río Vaupés (comunidad de Piracemo). Se analizaron 316 contenidos estomacales aplicando los métodos de frecuencia de ocurrencia (FO), índice gravimétrico (W), coeficiente de vacuidad (CV) e índice de importancia relativa (IIR). De igual modo, se determinaron parámetros físicos y químicos del agua superficial y profunda. La profundidad del río en época de aguas ascendentes fue de 3.7 ± 0.6 m, aguas altas de 5.9 ± 1.4 m, aguas descendentes 4.6 ± 1.3 m y en aguas bajas 2.4 ± 1.0 m; se registró mayor acidez y turbidez del agua en las ascendentes. El CV fue de 14.5 %; sin embargo, el CV alcanzó un valor del 40 % en aguas ascendentes. En general se observó una mayor FO de material vegetal en los estómagos de los individuos muestreados (44.4-66.7 %), seguido por los insectos (21.1-33.3 %, dietas secundarias). Los índices gravimétricos reflejaron que el material vegetal fue el ítem consumido en mayor cantidad, seguido de los insectos y en casi igual proporción invertebrados y material animal. Las diferencias en la composición de la dieta confirman la naturaleza oportunista de esta especie con predominio de hábitos omnívoros.Este estudio puede ser utilizado como parte integral del conocimiento de la ecología trófica de la especie, con el fin de crear estrategias para la protección del L. friderici en la región del Vaupés.

AbstractThe most important fish species of the Vaupes river is known as a Warakú tres puntos (Leporinus friderici), the most representative migratory Characidae from the Vaupés state, Colombia, with a significant importance in food security of indigenous communities. To contribute with the knowledge of its basic biology, we determined the habits and food preferences of Leporinus friderici, during one hydrobiological cycle (March 2014 to May 2015) in the Vaupés river, Colombia. Three sites were sampled, two of them along the Vaupés river (Yacayacá and Santa Cruz communities) and the other one in the Cuduyarí river, tributary of the Vaupés river (Piracemo community). Physical and chemical water parameters from the surface and depth level of the river were determined. Stomach contents from 316 fish were analyzed by frequency of occurrence (FO), gravimetric index, vacuity index (VI) and relative importance index (RII). Vaupés's average depth in ascending water period was of 3.7 ± 0.6 m, during rainy season of 5.9 ± 1.4 m, in descending water period of 4.6 ± 1.3 m and during dry season of 2.4 ± 1.0 m. The higher acidity and turbidity were observed during the ascending water period. The average VI was 14.5 % for the period, but it reached 40 % in descending waters. In general, a FO of plant material was the most frequent (44.4-66.7 %), followed by insects (21.1-33.3 %, secondary diets). The gravimetric indices showed that plant material was the most consumed item, followed by insects, and in almost in equal proportion, invertebrates and animal material. The differences in diet composition confirm the opportunistic nature of this species with a predominance of omnivorous habits. This study can be used as an integral part of the feeding ecology of L. friderici knowledge, in order to develop strategies for its protection in the Vaupés region because of its importance for local communities.

Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio).

Fibrates are pharmaceuticals commonly used to control hypercholesterolemia in humans and they are frequently detected in the freshwater environment. Since cholesterol is the precursor of all steroid hormones, it is suspected that low cholesterol levels will impact steroidogenesis. However, the effect of fibrates on fish reproductive endocrinology is not clear; therefore the aim of the present study was to evaluate the effect of bezafibrate (BZF) on gonadal steroidogenesis and spermatogenesis of zebrafish (Danio rerio). For this purpose, adult males were exposed orally to 1.7, 33 and 70 mg BZF/g food for 21 days. Blood and gonads were collected after 48 h, 7 days and 21 days to evaluate plasma cholesterol and plasma 11-ketotestosterone (11-KT). The expression of gonadal genes involved in the steroidogenesis was quantified to determine a potential mechanism of action, likewise the effect on spermatogenesis was evaluated by examining gonadal histopathology. A time dependent monotonic decrease in the plasma cholesterol concentration was observed in fish exposed to BZF. Plasma 11-KT decreased significantly after 21 days of exposure in fish exposed to the high concentration of BZF. Different gene expression patterns were observed: down-regulation in ppara and pparg mRNA levels was observed in fish exposed to the higher concentrations after 48 h; however, the expression of pparg increased after 21 days. After 21 days an increase in the star and cyp17a1 mRNA expression was observed in fish exposed to 70 mg BZF/g food. Sampling time and bezafibrate concentration explained 52.4% and 20%, respectively, of the gene expression variability. Gonadal histology revealed the presence of germ cell syncytia in the tubular lumen of fish exposed to bezafibrate and also an increased number of cysts containing spermatocytes, which indicate testicular degeneration. The study shows that bezafibrate exerts a hypocholesterolemic effect in adult male zebrafish and its potential as an endocrine disruptor due to its effect on the gonadal steroidogenesis and spermatogenesis.

Endosulfan affects health variables in adult zebrafish (Danio rerio) and induces alterations in larvae development.

Adult zebrafish (Danio rerio) were exposed to 0 (control), 0.16 or 0.48µg/L of the insecticide, endosulfan, for 28days. Haematology, whole body ions, thiobarbituric acid reactive substances (TBARS), Na(+)K(+)-ATPase, organ histology and reproduction were assessed in adults. The resulting offspring were examined for latent effects on development (heart rate and morphometrics). On day 14, adult fish exposed to 0.16µg/L endosulfan showed significantly lower red blood cell counts than those exposed to 0.48µg/L endosulfan; adult fish exposed to 0.16 ug/L also showed elevated TBARS compared to controls. Both concentrations of endosulfan caused a 4.0 fold increase in Na(+)K(+)-ATPase activity compared to controls (ANOVA, p<0.05). On day 14, the livers of fish exposed to endosulfan had fewer, enlarged hepatocytes, with cell diameters greater than the controls (ANOVA, p<0.05). Morphological alterations in the progeny of fish exposed to endosulfan were observed. Heart beat frequency was significantly lower in larvae from exposed adults to 0.16 µg/L compared to the control (ANOVA, p<0.05). These findings show that sublethal exposure to endosulfan causes adverse sublethal effects in adult D. rerio, and effects on the development of their offspring.

Functional characterization of water transport and cellular localization of three aquaporin paralogs in the salmonid intestine.

Intestinal water absorption is greatly enhanced in salmonids upon acclimation from freshwater (FW) to seawater (SW); however, the molecular mechanism for water transport is unknown. We conducted a pharmacological characterization of water absorption in the rainbow trout intestine along with an investigation of the distribution and cellular localization of three aquaporins (Aqp1aa, -1ab, and -8ab) in pyloric caeca, middle (M), and posterior (P) intestine of the Atlantic salmon. In vitro iso-osmotic water absorption (J(v)) was higher in SW than FW-trout and was inhibited by (mmol L(-1)): 0.1 KCN (41%), 0.1 ouabain (72%), and 0.1 bumetanide (82%) suggesting that active transport, Na(+), K(+)-ATPase and Na(+), K(+), 2Cl(-)-co-transport are involved in establishing the driving gradient for water transport. J(v) was also inhibited by 1 mmol L(-1) HgCl(2), serosally (23% in M and 44% in P), mucosally (27% in M), or both (61% in M and 58% in P), suggesting involvement of both apical and basolateral aquaporins in water transport. The inhibition was antagonized by 5 mmol L(-1) mercaptoethanol. By comparison, 10 mmol L(-1) mucosal tetraethylammonium, an inhibitor of certain aquaporins, inhibited J(v) by 20%. In the presence of glucose, mucosal addition of phloridzin inhibited water transport by 20%, suggesting that water transport is partially linked to the Na(+)-glucose co-transporter. Using polyclonal antibodies against salmon Aqp1aa, -1ab, and -8ab, we detected Aqp1aa, and -1ab immunoreactivity in the brush border and sub-apical region of enterocytes in all intestinal segments. The Aqp8ab antibody showed a particularly strong immunoreaction in the brush border and sub-apical region of enterocytes throughout the intestine and also stained lateral membranes and peri-nuclear regions though at lower intensity. The present localization of three aquaporins in both apical and lateral membranes of salmonid enterocytes facilitates a model for transcellular water transport in the intestine of SW-acclimated salmonids.

Gonadal alterations in male eelpout (Zoarces viviparus) exposed to ethinylestradiol and trenbolone separately or in combination.

To evaluate the interaction between 17ß-trenbolone (TB) and 17α-ethinylestradiol (EE2) in relevant environmental concentrations, male eelpout Zoarces viviparus were exposed in a flow-through seawater-system for 21 days to 5 ng l⁻¹ EE2, 5 ng l⁻¹ or 20 ng l⁻¹ TB or to combinations of both compounds. The effects on hepatosomatic index (HSI), gonadosomatic index (GSI) and gonadal histology were studied. No significant effects on HSI were observed in any treatment; in contrast, decreased GSI was observed in males exposed to EE2 alone or in combination with TB compared to controls (p<0.05). The histology revealed that the males were in the beginning of spermatogenesis. Males from the control group and some from the TB groups showed tubules with cysts containing spermatogonia, spermatocytes and spermatids; however, some testes of males exposed to TB showed slight to moderate interstitial fibrosis. Nevertheless, the most severely affected were males exposed to EE2 showing marked interstitial fibrosis, necrosis of germinal cells and reduced number of spermatocytes and spermatogonia in the cyst. Likewise, increased tubule number and proportionally decreased tubule diameter were observed in the testis of all EE2 exposed groups (p<0.05). Finally, a similar tubule number was observed in males exposed to EE2+20 ng l⁻¹ TB compared to control (p>0.05). This study shows that EE2 dramatically disrupts the spermatogenesis and low doses of 17ß-trenbolone are unable to effectively counteract the morphological effects of EE2.

Effect of culture medium on growth and protein content of Chlorella vulgaris / Efecto del medio de cultivo sobre el crecimiento y el contenido proteico de Chlorella vulgaris / Efeito do diferentes meios de cultura sobre o crescimento e o conteúdo proteico em Chlorella vulgaris

The use of live food in larviculture and fingerling stages has become an excellent option in pisciculture to reduce mortality and production costs. Live food is cost-effective, nutritious, and it has enzymes necessary for fish development. Objective: this study evaluated the effect of several culture media (chu10 , NPK complex fertilizer, worm humus, and equine manure) on growth and protein content of Chlorella vulgaris. Methods: 3 L volumes with constant light and aeration were used for algae culture. Six replicates were used per each treatment. One mL of C. vulgaris was added to each experimental unit to obtain 8.3 x 10(6) cel/mL as initial density. Algae counts were conducted every two days using the Neubauer chamber to determine cell density, parameters such as temperature, dissolved oxygen, pH, nitrates, and phosphates were measured. Crude protein was determined with the Kjeldahl method. The assay lasted 82 days. Results: microalgae reached the highest density with complex fertilizer NPK (10.9 ± 1.6 x 10(6) cel/mL) on day 22, followed by worm humus (5.3 ± 1.1 x 10(6) cel/mL) on day 48, equine manure (4.9 ± 0.9 x 10(6) cel/mL) on day 18, and the last was chu10 (2.2 ± 0.6 x 10(6) cel/mL) on day 12. The highest protein content was found in algae grown in worm humus (56.8%), and the equine manure (32.5%), in contrast, complex fertilizer NPK had the lowest value (16.8%). Conclusion: this study showed that organic media are a good choice for the culture of C. vulgaris, providing proper growth and high protein content of microalgae.

En piscicultura, el uso de alimento vivo durante las fases de larvicultura y alevinaje se ha convertido en una opción para reducir la tasa de mortalidad y los costos de producción, debido a que es económico, nutritivo y posee enzimas necesarias para el desarrollo de las larvas de peces. Objetivo: el presente trabajo evaluó el efecto de diferentes medios de cultivo (chu10, fertilizante complejo NPK, humus de lombriz y equinaza) sobre el crecimiento y el contenido proteico en Chlorella vulgaris. Métodos: para su cultivo se utilizaron volúmenes de 3 L, con luz y aireación constante. De cada tratamiento se realizaron seis réplicas y en cada unidad experimental se adicionó 1 mL de C. vulgaris con una densidad de 8.3 x 10(6) cel/mL. Para determinar la densidad celular se realizaron conteos cada dos días haciendo uso de cámara de Neubauer. Se midió temperatura, oxígeno disuelto, pH, nitratos y fosfatos. La proteína cruda se determinó por el método de Kjeldahl. La duración total del ensayo fue de 82 días. Resultados: la microalga alcanzó su mayor densidad con fertilizante complejo NPK (10.9 ± 1.6 x 10(6) cel/mL) en el día 22, seguido del humus de lombriz (5.3 x 106 ± 1.1 x 10(6) cel/mL) en el día 48, equinaza (4.9 ± 0.9 x 10(6) cel/mL) en el día 18, y por último con el chu10 (2.2 ± 0.6 x 10(6) cel/mL) en el día 12. Se encontró mayor contenido proteico en las células cultivadas en humus de lombriz (56.8%) y equinaza (32.5%); a diferencia, el del fertilizante complejo NPK fue muy bajo (16.8%). Conclusiones: este estudio permite concluir que los medios orgánicos son una buena opción para cultivar C. vulgaris, otorgando un adecuado crecimiento y un alto contenido proteico de la microalga.

Na piscicultura, o uso de alimento vivo durante as fases da cultura de larvas e juvenis têm sido convertidos em uma opção para reduzir a taxa de mortalidade e os custos de produção, porque é econômico, nutritivo e possui as enzimas necessárias para o desenvolvimento das larvas de peixe. Objetivo: o objetivo desta pesquisa foi avaliar a efeito de diferentes meios de cultura (chu10, fertilizante mineral complexo NPK, vermicomposto e esterco equino) sobre o crescimento e o conteúdo proteico em Chlorella vulgaris. Métodos: para a cultura, foram utilizados volumes de 3 L, com luz e aeração permanente. De cada tratamento, realizaram-se seis repetições e em cada unidade experimental, adicionou-se 1 mL de C. vulgaris com uma densidade de 8.3 x 10(6) cel/mL. Para determinar a densidade celular, realizaram-se contagens a cada dois dias usando a câmara de Neubauer. Aferiu-se a temperatura, oxigênio dissolvido, pH, nitratos e fosfatos. A proteína bruta foi determinada por o método de Kjeldahl. A duração total do ensaio foi de 82 dias. Resultados: os resultados mostraram que a microalga alcançou sua maior densidade com o fertilizante complexo NPK (10.9 ± 1.6 x 10(6) cel/mL) no dia 22, seguido do vermicomposto (5.3 ± 1.1 x 10(6) cel/mL) no dia 48, o esterco equino (4.9 ± 0.9 x 10(6) cel/mL) no dia 18 e por último com o chu10 (2.2 ± 0.6 x 10(6) cel/mL) no dia 12. Foi encontrado maior conteúdo proteico nas células cultivadas em vermicomposto (56.8%) e esterco equino (32.5%); a diferença, do fertilizante complexo NPK que foi muito baixo (16.8%). Conclusões: este estudo permitiu concluir que os meios orgânicos são uma boa opção para cultivar C. vulgaris, obtendo um crescimento adequado e um alto conteúdo proteico da microalga.

Protocolo para la crioconservación de semen de yamú (Brycon amazonicus Spix & Agassiz 1829) / Protocol for cryopreservation of yamú (Brycon amazonicus) semen

La crioconservación es una herramienta importante para el almacenamiento de gametos por tiempo indefinido, especialmente de especies con hábitats y poblaciones amenazadas. En peces, es además una alternativa para optimizar los procesos reproductivos en cautiverio de especies con maduración gonadal asincrónica y ciclos reproductivos estacionales como el yamú (Brycon amazonicus - Spix & Agassiz 1829), un carácido distribuido en las cuencas de los ríos Amazonas, Orinoco y Essequibo. Este reporte presenta el protocolo para la crioconservación de semen en esta especie, obtenido después de evaluar diferentes sustancias crioprotectoras, curvas de congelación y volúmenes de empaque. Su eficiencia ha sido demostrada en varios experimentos a nivel de laboratorio y de granjas comerciales, lográndose tasas de fertilidad cercanas a aquellas con semen fresco, constituyéndose en una importante herramienta metodológica para la crioconservación seminal de la especie.

Evaluación de diferentes crioprotectores para la crioconservación de espermatozoides de yamú (Brycon amazonicus) / Evaluation of different cryoprotectors for cryopreservation of yamú (Brycon amazonicus) spermatozoa

El uso de dimetilsulfoxido (DMSO), etilenglicol (ETG), propilenglicol (PPG) y metanol (MET) para la crioconservación de semen de yamú (Brycon amazonicus - Spix & Agassiz 1829) fue evaluadocon el objetivo de determinar la calidad post-descongelación y la fertilidad in vitro del semen. Para este propósito, machos y hembras, sexualmente maduros fueron inducidos a la maduración final de lasgónadas por medio de extracto de hipófisis de carpa. El semen obtenido fue evaluado y diluido (1:4) en una solución a base de yema de huevo (12 por ciento) y glucosa (5.5 por ciento). Se utilizó cada crioprotector aconcentraciones de: 3.75, 5, 7.5, 10, 11.25 y 15 por ciento para un total de 24 tratamientos. El semen diluido fue empacado en pajillas de 0.5 mL para ser congelado en vapores de nitrógeno líquido (NL) a -194 ºC durante 30 min y luego transferidas y almacenadas hasta por 10 días en un termo de NL para su evaluación. Para las pruebas de fertilidad, las pajillas (n= 6) de cada uno de los tratamientos fueron descongeladas a 35 ºC por 60 seg y posteriormente evaluadas. Se encontró que los tratamientos DMSO-5 por ciento, ETG-3.75 por ciento, PPG-3.75 por ciento y MET-3.75 por ciento presentaron los mayores porcentajes de movilidad (PM) y tiempos de activación (TA), siendo DMSO-5 por ciento el más alto (PM 76 ± 2.4; TA 88.2 ± 6.1). De otro lado, los tratamientos PPG-3.75 por ciento y MET-3.75 por ciento mostraron los mayores porcentajes de fertilidad (50 ± 1.4 y 47.8 ± 1.4, respectivamente), pero fueron inferiores (p<0.05) cuando se compararon con el semen fresco (64.7 ± 1.7). En general se observó que a medida que aumentó la concentración del crioprotector, la calidad seminal postdescongelación disminuyó, sin observarse relación directa entre la movilidad espermática y la fertilidad.

Efectos del sistema de conservación sobre la fertilidad de oocitos de yamú (Brycon amazonicus) durante cortos períodos de almacenamiento / Effects of the conservation system on eggs fertility of yamú (Brycon amazonicus) during shortterm storage

El objetivo fue evaluar los efectos del sistema de conservación sobre la fertilidad de oocitos de yamú (Brycon amazonicus) durante cortos períodos de almacenamiento. Se utilizaron hembras y machosadultos tratados con extracto de hipófisis de carpa para estimular la ovulación y aumentar el volumen seminal. Oocitos obtenidos de la misma hembra fueron sometidos a tres sistemas de conservación: in situ (IS, mantenidos dentro de la cavidad ovárica), ex situ a temperatura ambiente (T°A) y ex situ a temperatura de refrigeración (T°R). El diámetro ovocitario (DO) y la viabilidad fueron evaluados a los 0 (control), 15, 30, 60 y 120 min de almacenamiento. El DO fue medido con reglilla acoplada al ocular del estéreomicroscopio y la viabilidad mediante prueba de fertilidad, seminando 3 g de oocitos con 250 mL de semen fresco. También fueron medidos la longitud total y el volumen del saco vitelino de las larvas. Bajo los tres sistemas de conservación, DO aumentó con el tiempo, siendo mayor (p<0.001) que el control a partir de los 15 min de almacenamiento. Los mayores DO fueron observados en oocitos conservados a T°R (p<0.05). Hasta 60 min de almacenamiento, en los sistemas de conservación IS y a T°A, la fertilidad no varió (p>0.05) con respecto al control. A los 120 min de almacenamiento, todos los sistemas de conservación mostraron fertilidad menor que el control; sin embargo, en el sistema T°R disminuyó más rápidamente, siendo menor (p<0.05) que el control a los 30 min. Aunque las larvas no presentaron evidentes alteraciones morfológicas, las del control fueron más largas (p<0.01) que aquellas de oocitos conservados. En conclusión, oocitos ovulados de yamú pueden mantenerse viables hasta por 60 min, conservándolos en la cavidad ovárica o a temperatura ambiente. En contraste, la refrigeración deterioró rápidamente la fertilidad de oocitos ovulados.

Determinación del espermatocrito y efecto del volumen de la dosis seminante sobre la fertilidad en yamú (Brycon amazonicus) / Determination of spermatocrit and effect of volume of insemination dose on fertility in yamú (Brycon amazonicus)

El yamú (Brycon amazonicus . Spix & Agassiz 1829) es un pez omnívoro de rápido crecimiento y carne de alta calidad y aceptación comercial. Para estandarizar el espermatocrito (SPCTO), 23 muestras de semen fueron centrifugadas a 14000 g durante 1, 3, 5, 7 ó 10 min. El tiempo de centrifugación tuvo un efecto significativo sobre el valor del espermatocrito (SPCTO) (p<0.05). Para conocer la concentración espermática (CE) durante la época reproductiva y su relación con el SPCTO, la espermiación de 40 machos fue inducida con extracto de hipófisis de carpa (4.0 mg.kg -1). La CE fue evaluada mediante cámara de Neubauer (dilución 1:1200) y el SPCTO por centrifugación a 14000 g durante 5 min. La relación entre SPCTO y CE fue positiva y altamente significativa (y= 1.69 + 0.59x; r2= 0.74; p<0.0001), pero no se observaron diferencias estadísticas (p>0.05) entre los meses evaluados (marzo, abril y mayo). Finalmente, para determinar el efecto del volumen de la dosis seminante sobre la fertilidad, 2 g de oocitos fueron seminados con 3, 15, 45, 90 o 180 mL de semen (n= 12), cuya CE fue previamente ajustada para contener 50000 espermatozoides (sptz)/oocito, utilizando como diluyente plasma seminal homólogo. Los porcentajes de fertilidad fueron 83, 86, 86, 85 y 87 por ciento, respectivamente (p>0.05).

Aspectos generales de la crioconservación espermática en peces teleósteos / General aspects of the spermatic cryopreservation in teleosts

Los protocolos actuales para la crioconservación de semen de peces aún muestran muchas deficiencias para lograr óptimas tasas de fertilidad, observándose gran variación dentro y entre especies. Pocos eventos celulares y moleculares han sido descubiertos a partir de los resultados encontrados por los investigadores. Los procesos de formación de cristales de hielo a nivel intracelular, el efecto tóxico de los crioprotectores y la recristalización se muestran como los aspectos de mayor relevancia en el criodaño. Hipótesis como la transición de fasesde los lípidos en la membrana plasmática y la ruptura de la misma por el influjo rápido del agua, han sido propuestas para explicar el daño celular durante el proceso de crioconservación.

Evaluación de cinco protectores para la crioconservación de semen de Cachama Blanca (Piaractus brachypomus / Evaluation of five cryoprotectant for cryopreservation of Cachama Blanca (Piaractus brachypomus) milt

La Cachama Blanca (Piaractus brachypomus) es un pez de escama de los Llanos Orientales colombianos, considerada la especie íctica nativa más importante de la acuicultura colombiana. La utilización de semen criopreservado para la reproducción de esta especie puede traer grandes posibilidades de mejoramiento genético; sin embargo, a pesar que han sido realizados esfuerzos para lograr la crioconservación de semen, los índices de fertilidad aún son inferiores a los observadosutilizando semen fresco. El objetivo del presente trabajo fue evaluar la calidad y fertilidad de semende cachama blanca, crioconservado con cinco diferentes sustancias crioprotectoras: glicerol (GLY), dimetil sulfóxido (DMSO), metanol (MET), propilen glicol (PPG) y etilen glicol (ETG). Se utilizó semen de 15 reproductores adultos, 4.8 ± 1.1 kg de peso corporal, tratados con 4.0 mg/kg de extracto de hipófisis de carpa (EPC) vía IM. El semen fue recolectado mediante estrujamiento, recibido en tubos graduados y mantenido a temperatura ambiente (26°C) hasta su evaluación. Los resultados están expresados en promedio ± error estándar. Antes de la dilución la movilidad masal fue de 92 ± 1 por ciento y la concentración de 17.7 x 109 ± 1.8 x109 espermatozoides por mL. Se utilizaron dos diluyentes: D1 (glucosa [C6H12O6] 5,5 g; yema de huevo 7.0 mL y agua destilada c.s.p. 100 mL) y D2 (leche enteraen polvo 15 g, agua destilada c.s.p. 100 mL). Para D1 se utilizó como crioprotector 5 por ciento (D1-DMSO 5o D1-GLY 5), 10 por ciento (D1-DMSO 10 o D1-GLY 10) o 15 por ciento (D1-DMSO 15 o D1-GLY 15) de dimetil sulfóxido o de glicerol, respectivamente. Para el D2, 5 por ciento (D2-MET 5, D2-PPG 5 o D2-ETG 5), 10 por ciento (D2-MET 10, D2-PPG 10 o D2-ETG 10) o 15 por ciento (D2-MET 15, D2-PPG 15 o D2-ETG 15) de metanol, propilen glicol o etilen glicol, respectivamente. Los mayores porcentajes de movilidad masal posdescongelación fueron observados cuando se utilizó D2-PPG 5 (64±2.4 por ciento) y D2-MET 10....