, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Risopatron, J., Merino, O., Cheuqueman, C., Figueroa, E., Sanchez, R., Farias, J. G., Valdebenito, I.

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 aquaculture.

, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Effer, Brian R., Sanchez, Ruben R., Ubilla, Andrea M., Figueroa, Elias V., Valdebenito, Ivan I.

For Salmo salar, there is a lack of information on the morphology of the first blastomeres formed during embryonic development and which could be used as a diagnostic tool for the first stages of development. The purpose of this investigation, therefore, was to characterize morphometrically the first blastomeres of S. salar. From a pool of eggs incubated at 7.5 degrees C, 100 microphotographs of blastodiscs were extracted and analyzed at different incubation periods: 12, 14, 16, 20 or 24 h. Blastodiscs were characterized morphologically after 16, 20 or 24 h incubation, and classified into symmetric or asymmetric groups according to their morphology. The ratio of length (L) versus width (W) of each blastomere was determined, to establish its symmetry. In addition, 20 microphotographs of blastodiscs of normal appearance were analysed morphologically (control blastodisc: CB) for comparison (20 or 24 h). Results show that the first cleavage ends after 16 h of development. Seven categories were established during blastomere characterization: 47% normal (G1); 27% with dispersed margins (G2); 10% unequal (G3); 9% 'pie-shaped' (G4); 3% amorphous (G5); 2% three equal blastomeres and one different one (G6); and 2% with eccentric cleavage (G7). Although the incidence of abnormal cleavage in S. salar is uncertain, there is a potential for some asymmetries to be corrected during embryogenesis to generate viable individuals. More studies are necessary to correlate these abnormal cleavage patterns with indicators of quality in the later stages of embryogenesis in this species, to establish a quality assessment tool for gametes and/or embryos in salmonid species.

, 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, 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, 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.

, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, VALDEBENITO ISLER, NEMESIO IVAN, Figueroa, E., Valdebenito, I., Merino, O., Ubilla, A., Risopatron, J., Farias, J. G.

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium+1.3M dimethyl sulphoxide+0.3M glucose+2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 05ml of sperm suspension were frozen in 4cm of N2L. They were thawed in a thermoregulated bath (40 degrees C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick-end labelling (TUNEL)], plasma membrane integrity (SYBR-14/PI) and mitochondrial membrane potential (MMit, JC-1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 15x10(7) spermatozoa oocyte(-1), by observation of the first cleavages after 16h incubation at 10 degrees C. In the cryopreserved semen (T), the mean +/- s.d. DNA fragmentation was 48 +/- 25%; plasma membrane integrity 752 +/- 63%; mitochondrial membrane potential 517 +/- 36%; motility 585 +/- 53%; curved line velocity (V-CL) 612 +/- 174 mu ms(-1); average-path velocity (V-AP) 501 +/- 173 mu ms(-1); straight-line velocity (V-SL) 591 +/- 184 mu ms(-1); fertilization rate 816 +/- 19%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, V-CL, V-AP and V-SL compared with the controls (P<005). Also the mitochondrial membrane potential correlated with motility, fertilization rate, V-CL and V-SL (r=075; r=059; r=077 and r=079, respectively; P<005); and the fertilization rate correlated with V-CL and V-SL (r=059 and r=055, respectively).

, 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.