Effect of the age of broodstock males on sperm function during cold storage in the trout (Oncorhynchus mykiss)

The knowledge of sperm quality in the broodstock males of different ages is a prerequisite to identify the reproductive ability of cultivated fish for the hatchery management. Thus, in this work, we analysed sperm function of the semen stored of broodstock males of rainbow trout (Oncorhychus mykiss) in different reproductive ages (2, 3 and 4years old). Sperm samples of each reproductive age were stored in Storfish((R)) during 10days at 4 degrees C, and then, motility, viability, mitochondrial function (MMP), superoxide anion (O-2(-)) level and DNA fragmentation (DNA(frag)) were assessed. The results demonstrated that sperm function parameters were affected significantly by the age of the males and the time of storage. Motility, viability and MMP significantly decreased, and DNA(frag) and O-2(-) level increased with the age increment and the time of storage. In conclusion, sperm quality of 2 and 3years old were superior to those of 4years old, based on higher quality of various sperm functions such as motility, viability, MMP, DNA integrity and level O-2(-) during short-term storage. This information must be considered for optimum utilization of broodstock males in aquacultur

Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: Stability of mitochondrion as criterion of effectiveness

The aim of the present investigations was to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryoinjuries. Spermatozoa were isolated and vitrified using five different mediums: Group 1: standard buffer for fish spermatozoa, Cortland®-medium (CM, control); Group 2: CM+1% bovine serum albumin (BSA); Group 3: CM+1% BSA+0.125M sucrose; Group 4: CM+1% BSA+40% seminal plasma; and Group 5: CM+1% BSA+40% seminal plasma+0.125M sucrose. For cooling, 20μL suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM+1% BSA at 37°C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility, cytoplasmic membrane integrity (SYBR-14/propidium iodide staining technique), and mitochondrial membrane integrity (JC-1 staining). Motility (86%, 71%, 80%, 81%, and 82%, for Groups 1, 2, 3, 4, and 5, respectively) and cytoplasmic membrane integrity (90%, 82%, 83%, 84%, and 87%, respectively) of spermatozoa in all the 5 groups were not decreased significantly. All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility and cytoplasmic membrane integrity. However, mitochondrial membrane potentials of the spermatozoa in Groups 1, 2, 3, 4, and 5 were changed significantly (6%, 50%, 37%, 55%, and 34%, respectively) (P1,2,3,4,5<0.001; P2,3,4,5 <0.01)(P3-5>0.1). This rate was maximal in Group 4 (CM+1% BSA+40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA+40% seminal plasma without significant loss of important physiological parameters. © 2011 Elsevier B.V

Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (Salmo salar) spermatozoa: Relation with post-thaw motility

Sperm motility in fish with external fertilization is critical for reproductive efficiency in aquaculture, especially in salmonids. Gamete preservation techniques, such as cryopreservation, however, reduce sperm motility and fertilizing capacity. Very few studies have addressed cryodamage from energetic and cell signalling approaches. In this study, cAMP-dependent protein kinase (PKA) and AMP-activated kinase (AMPK) activities were quantified in fresh and cryopreserved spermatozoa of Atlantic salmon (Salmo salar); and the relation with motility was analysed. Results indicate there was a decrease in membrane integrity and motility in post-thawed spermatozoa compared to fresh samples, however, there was about 30% of cells with intact plasma membrane but incapable of motility. The PKA and AMPK activities were less after cryopreservation, indicating that loss of motility may be related to alteration of these key enzymes. Furthermore, PKA and AMPK activities were positively correlated with each other and with motility; and inhibition decreased motility, indicating there is a functional relationship between PKA and AMPK. The PKA inhibition also decreased AMPK activity, but results from protein-protein docking analyses indicated AMPK activation directly by PKA is unlikely, thus an indirect mechanism may exist. There have been no previous reports of these kinase actions in fish spermatozoa, making these findings worthy of assessment when there are future studies being planned, and may serve as base knowledge for optimization of cryopreservation procedures and development of biotechnologies to improve reproduction efficiency in the aquaculture industr

Effects of Diet-Induced Obesity and Deficient in Vitamin D on Spermatozoa Function and DNA Integrity in Sprague-Dawley Rats

Obesity has adverse effects on male fertility and usually is diagnosed with a prevalence of vitamin D deficiency (VD-). Discussion on the impact of obesity/VD- on sperm function has been limited. This study analyzed the effects of diet-induced obesity/VD- on viability and plasma membrane integrity (PMI), superoxide anion (O2-) level, and DNA fragmentation (DNAfrag) in sperm Sprague-Dawley rats. The males were randomized into four groups and fed for a period of 12 weeks: G1: control diet with vitamin D (C/VD+), G2: control diet without vitamin D (C/VD-), G3: high-fat diet with vitamin D (HF/VD+), and G4: high-fat diet without vitamin D (HF/VD-). Sperm function parameters were analyzed by flow cytometry. PMI percentages and O2- levels were not affected by any of the diets. DNA fragmentation was increasing significantly (p<0.05) in the spermatozoa of animals with diets vitamin D deficient (G2) and diet-induced obesity (G4). Our results allow us to point out that diet-induced obesity and VD- produce greater damage in DNA sperm of rats. The use of nutraceuticals containing vitamin D could be reducing the risk of fragmentation of DNA in spermatozoa

VITRIFICATION OF MAMMALIAN SPERMATOZOA

La criopreservación de espermatozoides y su utilización en inseminación artificial ha causado gran impacto sobre la reproducción de especies comerciales y en humanos. Sin embargo, a pesar del gran avance en el tema, constantemente se modifican los procedimientos de congelamiento, a fin de obtener mejores resultados de viabilidad y movilidad espermática, para lograr de esta forma mayores porcentajes de fecundaciones exitosas. No obstante, el proceso de criopreservación induce perdida de la función espermática que se refleja en alteraciones de la membrana plasmática, daño a nivel mitocondrial, deterioro en la integridad del ADN, aumento en la producción de las especies reactivas de oxígeno (ROS) y alteraciones del citoesqueleto, lo que finalmente tiene efectos negativos en la viabilidad y motilidad espermática, elementos esenciales para la fecundación. En la búsqueda de métodos que induzcan el menor daño celular, es que ha desarrollado en los últimos años los métodos de vitrificación espermática. Estos han permitido no solo superar en cuanto a la mantención de la función espermática a los métodos convencionales de congelación, sino también que lo hace más rápido, seguro y de menor costo. Esto último al eliminar los equipos necesarios para la congelación lenta y también en algunos casos la no utilización de nitrógeno líquido para el almacenamiento, bastando un congelador de - 80°C. Su aplicación en mamíferos, ha sido reportada exitosamente en humanos con nacimientos de niños sanos, tanto en técnicas de fecundación in vitro como inseminación intrauterina y recientemente con el nacimiento de los primeros felinos (gatos) con técnicas de inseminación intrauterina

Assessment of Sperm Function Parameters and DNA Fragmentation in Ejaculated Alpaca Sperm (Lama Pacos) by Flow Cytometry

Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14/PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin/PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = -0.41) and with plasma membrane integrity (p = 0.01; r = -0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry.Fil: Cheuquemán, C.. Universidad de la Frontera; Chile;Fil: Merino, O.. Universidad de la Frontera; Chile;Fil: Giojalas, Laura Cecilia. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Investigaciones Biologicas y Tecnologicas, Argentina; ArgentinaFil: Von Baer, A.. Universidad de la Frontera; Chile;Fil: Sánchez, R.. Universidad de la Frontera; Chile;Fil: Risopatrón, J.. Universidad de la Frontera; Chile

Effect of the addition of two superoxide dismutase analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation

This research was supported by the National Science and Technology Council (CONCYTEC) of Peru, Research Superior Council of San Marcos University (CSI-UNMSM), and Center of Biotechnology in Reproduction (CEBIOR) of La Frontera University. The authors thank Juan Olazábal and other professionals and students of IVITA-Maranganí for assistance during collection of alpaca semen samples.Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica - Concyte

El inicio de la función ovárica postparto en vacas lecheras primíparas y multíparas

Se añalizó el inicio de la función ovárica después del parto en vacas lecheras primíparas y multíparas. Se seleccionaron dos grupos de hembras de raza Frisona negra; uno (n=15) de primíparas (VP) y otro (n=15) de multíparas de segundo y tercer parto (VM). Se calcularon los siguientes índices: intervalo parto-actividad ovárica (PAO), primera ovulación postparto (PPO) y el intervalo desde el parto hasta el primer celo detectado (PPC). Los resultados mostraron diferencias para el peso vivo y la condición corporal al parto entre ambos grupos de animales (p<0,05). El inicio de la función ovárica postparto fue mas tardío en las VP (PAO = 40 vs 23 días; PPO= 49 vs 33 días y PPC=76 vs 65 días). La producción de leche acumulada a los 100 días de lactancia también fue menor (p<0,05) en éstas, no encontrándose relación entre producción de leche y el reinicio de actividad ovárica. El peso y la CC al parto presentaron una correlación negativa respecto al reinicio de la actividad ovárica postparto en las VP

Chorion in fish: Synthesis, functions and factors associated with its malformations

The chorion is an acellular envelope surrounding the oocyte. In fish, this envelope plays a pivotal role during fertilization and protects the developing embryo against environmental and mechanical factors until the moment of hatching. The chorion comprises a diverse number of glycoproteins called choriogenins, for which synthesis is mediated by the plasma concentrations of estradiol-17β hormone. In fish, its synthesis can occur in the liver, the oocyte or both, depending on the species. The quality of this envelope, as well as other intrinsic characteristics of the egg (its genes, its maternal mRNA transcripts, and the composition of the yolk) can be affected by environmental and/or nutritional factors and, therefore, the quality and/or embryo survival. The analysis of the studies carried out on the chorion and the factors associated with its quality are required in identifying practical solutions for the aquaculture industry, especially for those dedicated to producing and selling embryos, considering that the presence of these malformations can lead to economic losses. Thus, this review analyzed some reports on fish chorion malformations and highlights the need for specific studies on the factors that influence these alterations, especially those related to the diets and nutritional status of reproductive females. Although there are studies that allow us to infer how environmental or nutritional factors can affect the biology of the chorion, there is an evident need for other studies that directly relate the molecular machinery of choriogenesis with the occurrence of malformations. This review summarizes the knowledge of the genesis of the chorion and gives an approach to the effect of environmental and/or nutritional factors on its quality and embryo survival to establish perspectives for future studies