, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, FARIAS VEGA, CRISTIAN ALEX, Beltran, Jorge F., Belén, Lisandra Herrera, Lee-Estevez, Manuel, Villalobos, Elías Figueroa, Dumorné, Kelly, Farias, Jorge G.

Ca2+ is a key element in the sperm activation process of Salmo salar. However, the molecular mechanisms by which this ion enters the sperm cell have been poorly studied. In this study, we examined, for the first time, the role of the voltage-gated T-type Ca2+ channel in the activation of sperm motility of Salmo salar. Using an in vitro inhibition assay, a significant decrease in total and progressive motility (P < 0.0001) was observed in Salmo salar sperm when they were treated with NNC-55-0396, a highly selective blocker. The in silico analysis showed that this blocker is docked with a strong affinity for the pore of the voltage-gated T-type calcium channel suggesting the blocking of Ca2+ ions. The results show that the T-type voltage-gated Ca2+ channel is key to sperm motility in Salmo salar. © 2020 Elsevier B.V., All rights reserved.

, DANTAGNAN DANTAGNAN, HERMAN PATRICIO, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, Villasante, Alejandro, Villalobos, Elías Figueroa, Godoy, Karina Alejandra, Dantagnan, Patricio P., Lopez-Polo, Johana, Opazo, Rafael, Romero, Jaime

Serum proteins are essential for maintaining osmotic pressure, regulating pH, transporting metabolites, and supporting immune responses. Studying the effects of plant-based diets on these proteins is crucial to understanding their impact on fish health and immune function. Methods: This study was conducted in a recirculation system, with rainbow trout distributed across nine tanks and fed three diets: fishmeal (control), medium plant meal (MPM; 40% of fishmeal replacement), and high plant meal (HPM; 80% of fishmeal replacement). Plasma protein and plasma glucose levels were measured at the initial and final stages, under both fasting (24 h after fast) and postprandial (6 h after fed) conditions, to assess dietary impacts. Additionally, the hepatosomatic index (HSI) was calculated at the end of the experiment to evaluate potential liver adaptations to the diets. Results: The initial protein and glucose levels were similar across all groups. However, by the end of the treatment, the fishmeal-fed group showed significantly higher total protein and globulin levels, while the albumin levels remained consistent across diet types. A significant interaction between sampling time and dietary treatment in fish reduced the clarity of dietary effects on postprandial plasma glucose levels. Furthermore, the HSI was significantly lower in fish fed the high plant meal (HPM) diet compared to the fishmeal and medium plant meal (MPM) groups, suggesting potential metabolic adaptation in response to plant-based diets. Conclusions: Replacing fishmeal with plant-based proteins in rainbow trout diets reduces total globulin and protein concentrations but leaves albumin unaffected, alongside reductions in fasting and postprandial glucose levels. The low growth and different HSI in fish fed plant-based diets highlights potential liver stress, emphasizing the need for further research to optimize sustainable aquaculture feeds while maintaining fish health and performance. © 2025 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, 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, 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, Merino, Osvaldo, Risopatron, Jennie, Valdebenito, Ivan, Figueroa, Elias, Farias, Jorge G.

Motility is a key in spermatozoon function, determining semen quality and fertilizing capacity. Motility in fish spermatozoa occurs when they are diluted in a swimming solution, the adequacy of which is determined by factors which vary by species. Spermatozoon motility rate and velocity, as well as the duration of the motility period, are influenced by the temperature of the swimming medium. Increasing the temperature of the swimming medium has a significant negative effect on spermatozoa motility kinematics. It increases the metabolism rate of cells, causing a mismatch of energy resources and promoting changes in sperm movement. It also generates an increase in the production of reactive oxygen species (ROS) and thus oxidative stress, and moreover results in an inadequate capacity of antioxidant enzymes to protect the cell against the effects of oxidative stress induced by higher temperatures. The aim of this review is to update present knowledge about: mitochondria main source of ATP; the protein phosphorylation related to motility; epigenetic regulation of the temperature of the activation and the role of ion channels in regulation motility. These mechanisms involved in sperm motility are of vital importance in regulating fertilization, all of which can be influenced by the environment. We emphasize the importance of investigating spermatozoon mechanisms in depth, especially in the context of climate change which results in changing water temperatures. Our findings provide a better understanding of fish sperm physiology, and a biological foundation for the further development of spermatozoon motility investigation, and reproduction technologies.

, 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, 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.

, DANTAGNAN DANTAGNAN, HERMAN PATRICIO, HERNANDEZ ARIAS, ADRIAN JESUS, FIGUEROA VILLALOBOS, ELIAS GUSTAVO, Villasante, Alejandro, Rodríguez, Héctor, Dantagnan, Patricio P., Hernández Arias, Adrián J., Villalobos, Elías Figueroa, Romero, Jaime

Atlantic salmon (Salmo salar) fed a carbohydrate-rich diet exhibit suboptimal growth performance, along with other metabolic disturbances. It is well known that gut microbes play a pivotal role in influencing metabolism of the host, and these microbes can be modified by the diet. The main goal of the present study was to determine the effect of feeding graded levels of digestible carbohydrates to Atlantic salmon on the distal intestine digesta microbiota at 3 sampling times (i.e., weeks 4, 8 and 12), during a 12-week trial. A low carbohydrate-to-high protein diet (LC/HP, 0% wheat starch), a medium carbohydrate-to-medium protein diet (MC/MP, 15% wheat starch) or a high carbohydrate-to-low protein diet (HC/LP, 30% wheat starch) was fed to triplicate fish tanks (27 to 28 fish per tank). We performed an in-depth characterization of the distal intestine digesta microbiota. Further, growth parameters, liver histology and the expression of genes involved in hepatic neolipogenesis in fish were measured. Fish fed a HC/LP diet showed greater hepatosomatic and viscerosomatic indexes (P = 0.026 and P = 0.018, respectively), lower final weight (P = 0.005), weight gain (P = 0.003), feed efficiency (P = 0.033) and growth rate (P = 0.003) compared with fish fed the LC/HP diet. Further, feeding salmon a high digestible carbohydrate diet caused greater lipid vacuolization, steatosis index (P = 0.007) and expression of fatty acid synthase (fas) and delta-6 fatty acyl desaturase (d6fad) (P = 0.001 and P = 0.001, respectively) in the liver compared with fish fed the LC/HP diet. Although, the major impact of feeding a carbohydrate-rich diet to Atlantic salmon in beta diversity of distal intestine digesta microbiota was observed at week 4 (HC/LP vs MC/MP and HC/LP vs LC/HP; P = 0.007 and P = 0.008, respectively) and week 8 (HC/LP vs MC/MP; P = 0.04), no differences between experimental groups were detected after 12 weeks of feeding. Finally, at the end of the trial, there was a negative correlation between lactic acid bacteria (LAB) members, including Leuconostoc and Lactobacillus, with hepatic steatosis level, the hepatosomatic and viscerosomatic indexes as well as the expression of fas and d6fad. Weissella showed negative correlation with hepatic steatosis level and the hepatosomatic index. Finally, further research to explore the potential use of LAB as probiotics to improve liver health in carnivorous fish fed fatty liver-induced diet is warranted. © 2022 Elsevier B.V., All rights reserved.

, 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.