, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Dumorné, Kelly, Valdebenito, Iván, Contreras, Pablo, Rodríguez, Patricio Ulloa, Risopatron, Jennie, Villalobos, Elías Figueroa, Estevez, Manuel Lee, Díaz, Rommy, Farias, Jorge G.

In this research we evaluated simple aspects of the sperm biology of Genypterus blacodes, in particular assessing the effects of pH (6, 7, 8 and 9), temperature (4, 8 and 16 °C) and osmolalities 100% sea water (1010 mOsm/kg, Control), 75% sea water (774 mOsm/kg, T1), 50% sea water (488 mOsm/kg, T2) and distilled water (0 mOsm/kg, T3)) on the motility of Genypterus blacodes intratesticular spermatozoa. In addition, we determined the fertilization rate. Our results show that G. blacodes spermatozoa have a sperm density of 5.35 ± 0.16 × 109 spermatozoa/mL. Sperm motility is initiated on contact with a hyperosmotic swimming medium under normal conditions (1010 mOsm/kg, pH 8 and 8 °C). The longest motility duration (432.48 ± 8.89 s) was recorded at 4 °C. The maximum percentage of motile cells was recorded at 8 °C (65.66 ± 4.95) at osmolality 1010 mOsm/kg, whereas an optimum was observed at pH 8. The fertility rate was 73.9 ± 17%. This is the first report on sperm motility of G. blacodes spermatozoa. In conclusion, the results of this study permit a baseline to be established for further research and protocols for artificial reproduction of this species to be developed and optimized. In addition, the information gathered in this research will be useful for developing the biotechnology of Genypterus blacodes. © 2018 Elsevier B.V., All rights reserved.

, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Villalobos, Elías Figueroa, Lee-Estevez, Manuel, Valdebenito, Iván, Watanabe, Ii Sei, Pinheiro de Souza Oliveira, Ricardo Pinheiro S., Romero, Jaime, Castillo, Rodrigo L., Farias, Jorge G.

The development of cryopreservation techniques has led to important changes in animal reproductive biotechnology. However, these techniques are associated with cellular and molecular damage, affecting the mitochondrial function and quality of spermatozoa; moreover studies in fish are limited. In this work, the effects of cryopreservation on ultrastructure, mitochondrial function and antioxidant defences in Atlantic salmon (Salmo salar) spermatozoa were assessed, along with intracellular calcium (Ca2+)i, mitochondrial DNA sequence and sperm function (motility and fertilization rate). Significant ultrastructure alterations of the middle piece and mitochondria were observed in cryopreserved spermatozoa as compared to controls. Oxygen consumption and ATP were also significantly different in cryopreserved spermatozoa, and in spermatozoa incubated with electron transport chain (ETC) uncouplers and inhibitors. Mitochondrial membrane potential, motility, fertilization rate and Ca2+ i in cryopreserved spermatozoa displayed significant reductions compared to fresh spermatozoa. Mitochondrial potential correlated significantly with motility and fertilization rate. A redox imbalance was observed in frozen spermatozoa due to increased lipid peroxidation and superoxide anion production as compared to fresh spermatozoa. Likewise, increased activity of glutathione peroxidase and total glutathione (GSH/GSSG), as well as reduced catalase activity, were observed in comparison with fresh spermatozoa. Our results contribute to a better understanding of cryodamage to mitochondrial functions in fish spermatozoa, and enabled us to establish potential quality assessment indicators. The data suggest that cryopreservation induces a reduction in overall sperm quality and functionality through disruption of the mitochondrial ultrastructure and function, leading to energy depletion and increased oxidative stress. This knowledge may also lead to the identification of a potential biotechnological tool for improving reproductive efficiency in species of commercial and biological interest. © 2019 Elsevier B.V., All rights reserved.

, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Valdebenito, Iván, Villalobos, Elías Figueroa, Valdebenito, Matías, Paiva, Luis

The chorion is the primary envelop that protects the fish embryo against mechanical actions, pathogens, and abrupt changes in physical and chemicals conditions of the incubation medium. During embryo development, chorion alterations are not rare, but the occurrence of these is scarcely reported. Increased frequency of chorion alterations can result in increased embryo mortality and thus decreased reproductive performance and losses for fish farms. In this study, we characterize different chorion alterations observed in samples collected over 14 years from 12 salmon and trout farms located in the region of La Araucanía in southern Chile, which sent live eyed-stage embryos ( eyed-eggs ) for quality analysis to our laboratory. We found soft chorion as the most common alteration observed, being present in the whole 14-year series analyzed in Atlantic Salmon (Salmo salar) and affecting up to 35.0% of the samples examined in a year. This alteration also affected up to 20.0 and 5.7% of Coho Salmon (Oncorhynchus kisutch) and Rainbow Trout (Oncorhynchus mykiss) samples analyzed in a year, respectively. We also found an increase of other chorion alterations, including perforated and white-spotted chorion in Atlantic and Coho Salmon, in the last 8 years. Among the three species, Rainbow Trout exhibited fewer chorion alterations. As the embryonated eggs analyzed here were obtained from broodstocks maintained under standard industrial conditions, these alterations might be linked to changes in environmental conditions affecting the incubation water that need to be further investigated. © 2021 Elsevier B.V., All rights reserved.

, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Dumorné, Kelly, Valdebenito, Iván, Risopatron, Jennie, Villalobos, Elías Figueroa, Díaz, Rommy, Farias, Jorge G.

In this study, the morphology and ultrastructure of Genypterus blacodes spermatozoa were characterized through scanning and transmission electron microscopy. Findings revealed that the G. blacodes spermatozoa can be differentiated into three major parts: a spherical head without an acrosome (typical for externally fertilizing fish), a short mid-piece, and a long flagellum. The mean length of the spermatozoa was 57.6 ± 6.08 ?m, with flagella accounting for 56.2 ± 7.2 ?m. The head was 1.47 ± 0.2 ?m long, and 0.89 ± 0.06 ?m wide. The mid-piece had a total dimension of 0.72 ± 0.16 ?m, and was 0.31 ± 0.02 ?m in length and 0.6 ± 0.05 ?m in width. It was located lateral to the nucleus and contained 4 or 5 spherical mitochondria. The mitochondria were separated from the axoneme by a cytoplasmic canal. The main piece of the flagellum had short irregular side-fins, and the axoneme was composed of the typical 9 + 2 microtubular doublet structure enclosed by a cell membrane. The present study reveals that G. blacodes sperm can be categorized as a primitive type. This study is the first to provide comprehensive details on the morphology and ultrastructure of spermatozoa in G. blacodes. © 2018 Elsevier B.V., All rights reserved.

, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Valdebenito, Iván, Villalobos, Elías Figueroa, Sandoval-Vargas, Leydy Y., Contreras-Mellado, Pablo, Sánchez-Caamaño, Juan Carlos, Farias, Jorge G., Risopatron, Jennie

There is no information about the characteristics of early cleavage in the Patagonian blennie (Eleginops maclovinus), which can be used as a diagnostic tool for embryo quality. The purpose of this investigation, therefore, was to characterize the first blastomeres of E. maclovinus morphologically. Of a 'pool' of incubated eggs at 10.7 ± 0.5°C, 100 microphotographs of blastodiscs were extracted at different incubation periods from 0.25 to 5 h after fertilization and analyzed. Blastodiscs taken at 3.5, 4.0 and 5.0 h were characterized and classified into symmetric or asymmetric groups according to their morphology. The proportions of length (L) and width (W) of each blastomere were determined to establish its symmetry. Additionally, 20 microphotographs of blastodiscs of normal appearance were analyzed morphologically (control blastodisc: CB) and compared other blastodiscs (4.0 and 5.0 h). The results showed that before fertilization oocytes presented a somehow turgid aspect (maximum average diameter of 987 ± 41 Am) and after fertilization and hydration, their diameter increased to 1001.5 ± 11 Am (but not statistically significant) and presented a spherical shape. First cleavage ends after 3.5 h of development, forming two blastomeres 467 ± 45 ?m length (L) and 328 ± 21 ?m width (W) with a L/W ratio of 1.43 ± 0.19. The second cleavage ends after development at 4.5 h forming four blastomeres 238 ± 65 ?m length and 227 ± 65 ?m width with a ratio L/W of 1.06 ± 0.09. Five categories were identified during the blastomere characterization: 70% normal or symmetric; 8% with odd numbers of blastomeres; 6% unequal; 6% 'pie shaped' and 10% amorphous. © 2019 Elsevier B.V., All rights reserved.