Laicia Carneiro-Leite1,2, Lorena Pacheco da Silva1,3, Hellen Buzollo Pazzini4, Stella Indira Rocha Lobato1,2, Laís Pedroso Borges1,2, Yasmin dos Santos Reis1,3, Luciane Gomes-Silva1,2, Cristiéle da Silva Ribeiro5, Rosicleire Veríssimo-Silveira1 and Alexandre Ninhaus-Silveira1
The success of fish reproduction in captivity depends on the quality of the gametes. High-quality gametes are structurally well-formed, possess fertilization capacity, and generate living descendants (Valdebenito et al., 2015). In captivity, the production of these gametes can be controlled by environmental factors, such as photoperiod, water temperature, or spawning substrate; in addition, the nutritional and physiological conditions of the breeders, in particular, have a direct effect on the quality of the gametes and consequently on the performance of the fish’s reproductive system (Bobe, Labbé, 2010; Mylonas et al., 2010, 2017). Studies carried out by Izquierdo et al.(2001), Watanabe, Vassallo-Agius (2003), Ling et al. (2006), Hachero-Cruzado et al. (2009), and Norambuena et al. (2012) demonstrated that nutrition influences reproductive parameters such as gonadal development, the quantity and quality of oocytes and sperm, and the quality of larvae produced, as the availability of essential biochemical components for gametogenesis and reproduction control can be affected by the nutritional status of the reproductives (Izquierdo et al., 2001; Norambuena et al., 2012).
One of the two main nutritional factors that significantly affect reproductive performance in fish is the content of essential fatty acids (EFA) in the diet (Watanabe et al., 1984). Lipids, mainly long-chain polyunsaturated fatty acids (LC-PUFAs) that include arachidonic acid (20:4 n-6, ARA), eicosapentaenoic acid (20:5 n-3, EPA), and docosahexaenoic acid (22:6 n-3, DHA), are required in the diet because they are eicosanoid precursors that are involved in various physiological processes, including steroid production, gonadal development, and maintenance of membrane integrity (Jaya-Ram et al., 2008).
LC-PUFAs are also important for other functions, such as controlling the reproduction and development of fish embryos and/or larvae, as well as improving sperm quality (Izquierdo et al., 2001; Norambuena et al., 2012). LC-PUFAs can also be synthesized from two polyunsaturated fatty acids (PUFAs), linolenic acid (18:3 n3, ALA) and linoleic acid (18:2 n-6, LA), also known as omega-3 and omega-6, respectively. These fatty acids are considered EFA because of the inability of fish to synthesize them in the body and must be provided in the diet (Izquierdo et al., 2001; Norambuena et al., 2012).
The positive effects of the addition of lipids to the diet on aspects of fish reproduction, either in improving the quality of semen and oocytes or in increasing the fecundity rate, hatching rate, and larval survival, have already been reported for several species, such as Danio rerio (Hamilton, 1822) (Jaya-Ram et al., 2008), Oreochromis niloticus (Linnaeus, 1758) (Ng, Wang, 2011), Colisa fasciatus (=Trichogaster fasciata Bloch & Schneider, 1801) (Hossen et al., 2014), Oncorhynchus mykiss (Walbaum, 1792) (Hajiahmadian et al., 2016), Acipenser baerii Brandt, 1869 (Luo et al., 2017), Sparus aurata Linnaeus, 1758 (Ferosekhan et al., 2021), and Cyprinus carpio var. koi (Harshavardhan et al., 2021). In the evaluation of seminal characteristics, the following parameters must be analyzed: seminal color and volume, rate and duration of sperm motility, sperm morphology and sperm concentration (Solis-Murgas et al., 2011; Zhang et al., 2017). Knowledge of the physiology of reproduction, together with the numerous studies of fish biology, has allowed the determination of management procedures that allow the induction of gonadal maturation of fish in captivity and, consequently, the artificial fertilization of oocytes, enabling the production of fish on a large scale, thus allowing the growth of the fish farming industry (Zaniboni-Filho, Weingartner, 2007).
The yellowtail lambari, Astyanax lacustris (Lütken, 1875) (Garutti, Britski, 2000), is a rustic species of small size, with a fast life cycle and high productivity in intensive cultivation because of its ease of handling, artificial feeding, and high prolificity. It is considered a model species, in addition to presenting great economic importance in the market of live fish and for human consumption (Sabbag et al., 2011; Fonseca et al., 2017; Brambila-Souza et al., 2021). Therefore, this study aimed to evaluate whether the inclusion of omega-3 polyunsaturated fatty acids (PUFAs-ô3) in the diet of Astyanax lacustris would influence the seminal quality of breeders of the species.
Material and methods
Experimental diets. An isoprotein diet (32% CP) was formulated (Tab. 1) and divided into four treatments with three levels of inclusion of purified marine fish oil (Campestre) containing high levels of PUFAs-ô3 (3, 6, and 9% in the diet) and a control diet without oil inclusion. To prepare the diets, the ingredients were ground, mixed, moistened and processed in an Exteec extruder, model Ex Micro, with a 3 mm die. The granules were dehydrated in a forced ventilation oven at 55 °C for 24 h. Afterward, the different amounts of marine fish oil were mixed in the feed. Before processing the feed, a bromatological analysis of all its ingredients was carried out (Laboratório de Bromatologia, UNESP/FEIS). The percentages of 1st and 2nd dry matter, percentage of crude protein by the Kjeldahl method, percentage of fat by the Soxhlet method and percentage of ash, in addition to the gross energy determined in a Parr calorimetric bomb, were determined according to the AOAC (2000). The fatty acid profile of marine fish oil (Tab. 2) and the four diets was carried out at the Laboratório de Estudos de Fisiologia Animal (LEFISA), UNESP/FEIS; Laboratório de Metabolismo e Reprodução de Organismos Aquáticos (LAMEROA-USP) (Tab. 3). Feed processing was carried out at the Instituto de Pesca, located in the administrative region of São José do Rio Preto, São Paulo, Brazil.
TABLE 1 | Composition of experimental feed formulated for Astyanax lacustris breeders. (1) Mineral mix (Premix Raguife; Santa Fé do Sul, SP, Brazil) [Fe 20 g/kg; Cu 3,500 mg/kg; Zn 24 g/kg; I 160 mg/kg; Mn 10 mg/kg; If 100 mg/kg; Co 80 mg/kg; vitamin A 2,400,000 IU/kg; vitamin D3 600,000 IU/kg; vitamin E 30,000 IU/kg; vitamin K3 3,000 mg/kg; vitamin C 60 g/kg; vitamin B1 4,000 mg/kg; vitamin B2 4,000 mg/kg; vitamin B6 3,500 mg/kg; vitamin B12 8,000 mcg/kg; inositol 25 g/kg; choline 100 g/kg; Pantothenic Ac 10 g/kg; biotin 200 mg/kg; B.C. Folic 1,200 mg/kg; niacin 20 g/kg; antioxidant *etc 5,000 mg/kg]. (2) Based on the analysis of ingredient composition. (3) Nitrogen-free extract (NFE) = dry matter – (crude protein + lipid + mineral matter + crude fiber). In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3.
Soybean-45% PB. bran
Fish (tilapia). flour
Marine fish oil
Dry Matter (%)
Crude Protein (%)
Ethereal Extract (%)
Mineral Matter (%)
Gross Fiber (%)
Non-Nitrogen Extractive3 (%)
Gross Energy (cal/g)
TABLE 2 | Fatty acid profile (% of total detected) of marine fish oil used in the composition of experimental diets for Astyanax lacustris breeders.
TABLE 3 | Fatty acid profile (%) detected in the four diets fed to Astyanax lacustris breeders. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3.
Experimental design. We used 400 males of A. lacustris two months old, with an average weight of 2.08 ± 0.44 g and average total length of 5.4 ± 0.39 cm. These were distributed in 20 polyethylene boxes of 180 L arranged in a recirculation system, with a density of 20 fish per box. The experiment was conducted in a completely randomized design with four treatments and five replications, totaling 20 experimental units. The treatments consisted of four levels of inclusion of PUFAs-ô3: In-0 (GC) = 0%, In-3 = 3%, In-6 = 6% and In-9 = 9%. Fish were fed twice a day (9 am and 5 pm) for 105 days until apparent satiation. During the entire experimental period, the following physicochemical variables of the water of the four treatments were monitored: pH, temperature, dissolved oxygen, and electrical conductivity measured every day in two periods in the morning and afternoon using a multiparameter sensor (ASKO – AK88v2). Total ammonia and nitrite levels were measured three days per week (Alcon Labcon Test Kit) (Tab. 4).
TABLE 4 | Water quality variables recorded during the 105-day experimental period. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3.
Dissolved oxygen (mg L-1)
Total ammonia (ppm)
Electric conductivity (µS cm-1)
Nitrite (µg L-1)
Semen collection. After 105 days, A. lacustris males were hormonally induced using Ovopel® (20 μg of GnRHa + 10 mg of metoclopramide [dopamine antagonist]) in a single dose (3 mg/kg of live fish) (Yasui et al., 2015). After 226 accumulated thermal units (226 ATUs) (Carneiro-Leite et al., 2020), the males were anesthetized with 1% benzocaine solution (Sigma – Aldrich E1501), and the semen was extruded using abdominal massage in the anteroposterior direction of the body and collected with the aid of micropipettes of 10–100 µL (Kasvi-K1-100B). Contamination of the semen with blood, feces and urine was carefully avoided through visual observation, for which a pre-assessment of the semen under microscopy was carried out to also verify if there was spermatic activation.
Semen analysis. To verify the influence of the inclusion of marine fish oil on the quality of A. lacustris semen, the following parameters were analyzed: duration of motility, considering the activation of spermatozoa to the observation of 10% motile spermatozoa; seminal volume, considering until the moment of ejaculation stop or observation of contamination with blood; and sperm concentration (spermatozoa/mL), measured using a Neubauer-type Hematimetric Chamber, for which the semen was diluted in formalin-saline solution in a proportion of 1:1000, respectively (Ninhaus-Silveira et al., 2006). The osmolality of the seminal plasma was also determined, for which the semen was centrifuged at 3,000 rpm for 15 min, and the supernatant was collected and analyzed in an osmometer (OSMOMAT model 030, Berlin, Germany). The semen was characterized in terms of transparency, viscosity and color, compared to clean water and color patterns, but these parameters were not used for qualitative comparison between treatments Motility parameters were evaluated using a CASA system (ISAS® Integrated Semen Analysis System, Proiser, Valencia, Spain) coupled to a UB200i phase contrast microscope (UOP/Proiser) with an objective of 10x negative phase contrast. The images were captured with an ISAS 782C camera (Proiser, Spain) and processed with CASA software using 50 frames per second (fps). Semen was activated by adding 30 µl of distilled water to 0.5 µl of semen in a Makler™ camera (Sefi Medical Instruments Ltd, Israel), and an analysis was performed after 10 s of activation. Total motility (MOT, %), Progressive motility (PRG, %), rapid sperm (SptzFast, %), curvilinear velocity (VCL, μm/s), straightlinear velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, %), straightness coefficient (STR, %), mean oscillation of the spatial trajectory (WOB, %), amplitude of lateral displacement of the head (ALH, μm), crossbeat frequency (BCF, Hz). Sperm with VAP < 10 μm/s were considered immobile, with velocity > 25 μm/s = medium sperm and velocity > 50 μm/s fast sperm. All analyses were performed for four treatments: In-0 (GC), In-3, In-6 and In-9.
The integrity of the sperm membrane was measured using the eosin-nigrosin staining method, for this analysis the spermatozoa were stained in the proportion 1:10:10 (semen:eosin:nigrosin). Using a light microscope, 200 spermatozoa per slide were considered alive when they remained colorless, indicating an intact membrane, or dead when stained pink, indicating a ruptured membrane, according to Lopes et al. (2018). Five animals were used per box per treatment.
In addition to the analyses mentioned above, the morphological normality of the spermatozoa was also evaluated. For this, the semen of five animals per treatment was fixed in formalin-saline solution at a proportion of 1:1000 (semen: fixative), stained with rose bengala, in the proportion of 1:10 (dye: semen), and 10 µL was deposited on a glass slide and covered with a coverslip (Streit-Junior et al., 2008). On each slide, 100 spermatozoa were analyzed, and the analysis was performed under optical microscopy (Zeiss/AXIOCAM-MRc5), at 1000X magnification. Spermatozoa were classified as normal or damaged, with damage classified as primary and secondary alterations, according to Miliorini et al. (2011).
Statistical analysis. Analysis of variance (p<0.05) was applied to the data, the Tukey test was applied for parametric data, and the Kruskal-Wallis test was applied for non-parametric data. The statistical program R Studio was used for data analysis. To the boxes were considered as an experimental unit.
The semen had a whitish to yellowish color and a slightly viscous appearance, regardless of the treatment considered. Regarding the seminal volume, the treatments with 6% and 9% of oil inclusion provided the smallest semen volumes, with a decrease of 40 and 45% respectively in relation to the control treatment (In-0). Sperm motility time for the In-9 treatment showed significantly lower values between treatments (Tab. 5). For seminal osmolarity, sperm concentration, and membrane integrity, no statistical differences were observed between the treatments (Tab. 5). No significant difference was found for the percentage of primary and secondary morphological alterations, but the In-0 (GC) treatment was the one that presented the highest values for both, in addition to having a smaller amount of normal spermatozoa, with the treatment In-6 being what showed a higher percentage of normal spermatozoa (Tab. 6).
TABLE 5 | Seminal parameters of lambaris (Astyanax lacustris) fed with the inclusion of omega-3 polyunsaturated fatty acids (PUFAs-ô3) in their diet, for 105 days. Different letters indicate statistical difference between treatments (Kurskal-Wallis, Tukey, p<0.05). In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3.
Seminal Volume (µl)
Motility Duration (s)
Sperm Concentration (sptz/µl)
Membrane Integrity (%)
TABLE 6 | Analysis of the morphological normality of spermatozoa from fresh semen of Astyanax lacustris fed with the inclusion of omega-3 polyunsaturated fatty acids (PUFAs-ô3) in their diet, for 105 days. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3.
Total Defects (%)
For the MOT and PRG, a significant increase was observed with the inclusion of PUFAs-ô3, with an emphasis on 9% of oil inclusion, which presented higher values that differed significantly from these two parameters of In-0 (Fig. 1; Tab. 7). For the parameters VCL, VSL, VAP, number of fast sperm, LIN, STR, and WOB, treatments In-6 and In-9 provided significantly higher values than In-0 and In-3 (Figs. 2–3; Tab. 7). The treatments with greater amounts of oil inclusion (In-6 and In-9) resulted in higher values for the ALH and BCF, which statistically differed from the In-0 and In-3 treatments. (Figs. 4A–B).
FIGURE 1| Results obtained for total (MOT, %) and progressive (PRG, %) sperm motility after feeding with the inclusion of PUFAs-ô3 in the diet of Astyanax lacustris for a period of 105 days. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3. Different capital letters indicate statistical difference between treatments for MOT. Different lowercase letters indicate statistical difference between treatments for PRG (Kruskal-Wallis, p<0.05).
FIGURE 2| Results obtained for A. Percentage of fast (SptzRápido), medium (SptzMedium) and slow (SptzSlow), B. Curvilinear velocity (VCL, µm/s), C. Linear velocity (VSL, µm/ s), D. Mean velocity (VAP, µm/s%) after feeding with the inclusion of PUFAs-ô3 in the diet of Astyanax lacustris for a period of 105 days. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3. Different capital letters indicate statistical difference between treatments for SptzFast. Different lowercase letters indicate statistical difference between treatments for SptzMedium, SptzSlow percentage, VCL, VSL and VAP (Kruskal-Wallis, p<0.05).
FIGURE 3| Results obtained for A. Linearity (LIN, %), B. Straightness coefficient (STR, %) and C. Mean oscillation of the spatial trajectory (WOB, %), after feeding with the inclusion of PUFAs-ô3 in the diet of Astyanax lacustris for a period of 105 days. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3. Different lowercase letters indicate statistical difference between treatments for LIN, STR and WOB (Kruskal-Wallis, p<0.05).
TABLE 7 | Variation of sperm kinetic parameters of broodstock treated with marine fish oil in relation to control. Total motility – MOT; Progressive motility – PRG; Rapid sperm – SptzFast; Curvilinear velocity – VCL; Linear velocity – VSL; Average velocity – VAP. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3; I – percentage increase compared to control.
FIGURE 4| Results obtained for A. Head lateral displacement amplitude (ALH, µm) and B. Crossbeat frequency (BCF, Hz), after feeding with the inclusion of PUFAs-ô3 in the diet of Astyanax lacustris for a period of 105 days. In-0 – diet without inclusion of PUFAs-ô3; In-3 – feed with inclusion of 3% PUFAs-ô3; In-6 – feed with inclusion of 6% of PUFAs-ô3; In-9 – feed with inclusion of 9% PUFAs-ô3. Different lowercase letters indicate statistical difference between treatments for ALH and BCF (Kruskal-Wallis, p<0.05).
The quality of gametes is essential for obtaining high rates of fertilization and hatching in the reproductive process and is considered a limiting factor for reproductive success (Gallego et al., 2013; Köprücü et al., 2015). Sperm motility can be evaluated and measured using parameters related to the fertilizing capacity of sperm, including total and progressive sperm motility, speed of sperm movement, sperm concentration, presence of abnormalities, morphological changes in gametes, and integrity of the plasma membrane (Fauvel et al., 2010; Gallego et al., 2013).
Studies have shown that introducing higher amounts of PUFAs into fish diets improves semen quality (Köprücü et al., 2015; Yonar et al., 2020). According to Lahnsteiner et al. (2009), lipids are the main energy resources of the sperm in salmonids and are important for maintaining sperm viability. This is noticeable in our work with Astyanax lacustris, as many of the parameters used to evaluate seminal quality showed a positive increase with the introduction of marine fish oil into the diet of breeders.
In the present study, treatments with higher oil inclusion (In-6 and In-9) had lower seminal volume when compared to the control group (In–0) and In-3; however, for the species Dicentrarchus labrax (Linnaeus, 1758) (Asturiano et al., 2001) and Oncorhynchus mykiss (Köprücü et al., 2015), the supplementation with PUFAs-ô3 resulted in greater seminal volume when compared to the control, which can be considered a result of the difference in the physiology of the referred species; these have different reproductive cycles and live in colder environments, as well as the need for a refinement of studies to determine the most adequate nutritional conditions for the inclusion of PUFAs-ô3.
As observed in Oncorhynchus mykiss (Köprücü et al., 2015) and Sparus aurata (Ferosekhan et al., 2021), the duration of sperm motility was increased by the inclusion of PUFAs-ô3 in the diet of breeders of these species, corroborating the data obtained in our experiment with A. lacustris. However, in the present study, the results showed that for A. lacustris, the inclusion of limit oil was 6% because, with the highest inclusion (9%), the duration of motility was significantly reduced. Regarding the parameters of sperm concentration and integrity of the sperm plasma membrane, the inclusion of marine fish oil had no positive or negative influence, since the number of spermatozoa per volume of semen is a species-specific characteristic not connected to nutritional supplementation and, in this case, the high level of sperm membrane integrity must be related to the good genetics of the specimens used.
As for the presence of gametes without formation deformities, there was no significant change considering the inclusion or absence of PUFAs-ô3 in the diet. This result was also observed for Rhamdia quelen (Quoy & Gaimard, 1824) (Rodrigues et al., 2022) with dietary supplementation with different sources of PUFAs. What can be highlighted is that, although no statistical difference was observed, GC (0%) provided a lower percentage of normal spermatozoa, which can be conjectured that despite not having a significant improvement, PUFAs-ô3 influenced cell formation.
The effect of PUFAs on motility was also observed in Oncorhynchus mykiss (Köprücü et al., 2015), where the use of a diet rich in n-3 polyunsaturated fatty acids increased motility compared to controls (no addition of PUFAs-ô3). Butts et al. (2015) found a similar effect for Anguilla anguilla (Linnaeus, 1758) semen; the addition of polyunsaturated fatty acids (EPA, DHA, and ARA) in the feed of breeders resulted in a significant increase in sperm motility. With a more sensitive motility analysis methodology, we corroborated the above reports with other fish species in which the inclusion of PUFAs-ô3 provided an increase in sperm motility and, more specifically, in our experiment with A. lacustris MOT and PRG.
The sperm kinetic parameters VCL, VSL, and VAP were also positively affected by the diet with 6% marine fish oil, corroborated by Luo et al. (2017), who found that diets containing higher amounts of PUFAs resulted in increased sperm kinetics in Acipenser baerii. This is an important effect because these kinetic parameters have been used as indicators of sperm quality and are highly correlated with fertilization capacity because they allow sperm to find and penetrate the micropyle faster (Figueroa et al., 2016; Gallego et al., 2017; Leite et al., 2018). In addition, spermatozoa need energy to move, and the improvement of these parameters in treatments with greater oil inclusion may be related to greater mitochondrial β-oxidation for energy generation, due to the greater availability of PUFAs in these treatments, which can be used for the production of ATP through oxidative phosphorylation (Mansour et al., 2003; Díaz et al., 2021).
According to Beirão et al. (2011), spermatozoa from fish with a more linear trajectory had the highest correlation with fertilization rate, demonstrating that this characteristic is one of the important parameters to be taken into account in the determination of seminal quality in fish. In our experiment, treatments with higher oil inclusion (In-6 and In-9) increased the percentage of fast sperm and the kinetic parameters LIN, STR, WOB, ALH, and BCF, further corroborating the idea that PUFAs are beneficial for the improvement of A. lacustris sperm quality.
Another interesting kinetic parameter to consider is ALH, considered a good indicator of sperm maturation (Kowalski et al., 2006; Król et al., 2009). In our experiment, treatments In-6 and In-9 provided significantly higher values for ALH, which may indicate that the sperm of these treatments will provide greater seminal quality and, consequently, are more suitable for fertilization.
Thus, we can conclude that the inclusion of purified marine fish oil containing high levels of PUFAs-ô3 brought benefits to the seminal quality of A. lacustris. The In-6 and In-9 treatments improved the evaluated seminal parameters most efficiently. However, considering that the differences between the evaluated parameters are subtle between the two treatments, as well as the volume of oil to be used, we consider that In-6 is the best treatment for improving the seminal quality of A. lacustris.
The authors would like to thank the financial support for this research to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Proc. Universal 4342832018–5 and Proc. PQ 306084/2018–0) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Proc. 2015/10115–5) and, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their scholarship granted (Proc. 88887.605023/2021–00) and the Instituto de Pesca, São José de Rio Preto, São Paulo, in the person of researcher Giovani S. Gonçalves, who made the feed used in this work. The authors would like to thank the Laboratório de Bromatologia, Laboratório de Estudos de Fisiologia Animal (LEFISA), Laboratório de Ictiologia Neotropical (LINEO), UNESP/FEIS, and Laboratório de Metabolismo e Reprodução de Organismos Aquáticos (LAMEROA), USP/Campus São Paulo, for their support scientific.
Association of Official Analytical Chemists (AOAC). Official methods of analysis of the Association of Agriculture Chmists. 17th ed. Arlington: AOAC; 2000.
Asturiano JF, Sorbera LA, Carrillo M, Zanuy S, Ramos J, Navarro JC, Bromage N. Reproductive performance in male European sea bass (Dicentrarchus labrax, L.) fed two PUFA-enriched experimental diets: a comparison with males fed a wet diet. Aquaculture. 2001; 194(1–2):173–90. https://doi.org/10.1016/S0044-8486(00)00515-9
Beirão J, Cabrita E, Pérez-Cerezales S, Martínez-Páramo S, Herráez MP. Effect of cryopreservation on fish sperm subpopulations. Cryobiology. 2011; 62(1):22–31. https://doi.org/10.1016/j.cryobiol.2010.11.005
Bobe J, Labbé C. Egg and sperm quality in fish. Gen Comp Endocrinol. 2010; 165(3):535–48. https://doi.org/10.1016/j.ygcen.2009.02.011
Brambila-Souza G, Mylonas CC, Mello PH, Kuradomi RY, Batlouni S.R, Tolussi CE, Moreira RG. GnRHa implants and temperature modulate Lambari-do-rabo-amarelo, Astyanax altiparanae (Characiformes: Characidae) induced reproduction out of the reproductive season. Aquac Res. 2021; 52(11):5595–605. https://doi.org/10.1111/are.15435
Butts IAE, Baeza R, Stottrup JG, Krüger-Johnsen M, Jacobsen C, Pérez L, Asturiano JF, Tomkiewicz J. Impact of dietary fatty acids on muscle composition, liver lipids, milt composition and sperm performance in European eel. Comp Biochem Physiol A Mol Integr Physiol. 2015; 183:87–96. https://doi.org/10.1016/j.cbpa.2015.01.015
Carneiro-Leite L, Bashiyo-Silva C, Oliveira YAA, Borges LP, Sanchez MP, Silva LG, Lobato SIL, Rodrigues-Lisoni FC, Veríssimo-Silveira R, Ninhaus-Silveira A. Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol. Neotrop Ichthyol. 2020; 18(3):e200039. https://doi.org/10.1590/1982-0224-2020-0039
Díaz R, Quiñones J, Short S, Contreras P, Ulloa-Rodríguez P, Cancino-Baier D, Sepúlveda N, Valdebenito I, Farías JG. Effect of exogenous lipids on cryotolerance of Atlantic salmon (Salmo salar) spermatozoa. Cryobiology. 2021; 98:25–32. https://doi.org/10.1016/j.cryobiol.2021.01.004
Fauvel C, Suquet M, Cosson J. Evaluation of fish sperm quality. J Appl Ichthyol. 2010; 26(5):636–43. https://doi.org/10.1111/j.1439-0426.2010.01529.x
Ferosekhan S, Turkmen S, Pérez-García C, Xu H, Gómez A, Shamna N, Afonso JM, Rosenlund G, Fontanillas R, Gracia A, Izquierdo M, Kaushik S. Influence of genetic selection for growth and broodstock diet n-3 LC-PUFA levels on reproductive performance of gilthead seabream, Sparus aurata. Animals. 2021; 11(2):519. https://doi.org/10.3390/ani11020519
Figueroa E, Valdebenito I, Merino O, Ubilla A, Risopatrón J, Farias JG. Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology. J Fish Biol. 2016; 89(3):1537–50. https://doi.org/10.1111/jfb.13052
Fonseca T, Costa-Pierce BA, Valenti WC. Lambari aquaculture as a means for the sustainable development of rural communities in Brazil. Rev Fish Sci Aquac. 2017; 25(4):316–30. https://doi.org/10.1080/23308249.2017.1320647
Gallego V, Cavalcante SS, Fujimoto RY, Carneiro PCF, Azevedo HC, Maria AN. Fish sperm subpopulations: Changes after cryopreservation process and relationship with fertilization success in tambaqui (Colossoma macropomum). Theriogenology. 2017; 87:16–24. https://doi.org/10.1016/j.theriogenology.2016.08.001
Gallego V, Pérez L, Asturiano JF, Yoshida M. Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles). Aquaculture. 2013; 416–17:238–43. https://doi.org/10.1016/j.aquaculture.2013.08.035
Garutti V, Britski HA. Descrição de uma espécie nova de Astyanax (Teleostei: Characidae) da bacia do alto rio Paraná e considerações sobre as demais espécies do gênero na bacia. Comun Mus Ciênc Tecnol PUCRS, Sér. Zool. 2000; 13:65–88.
Hachero-Cruzado I, Olmo P, Sánchez B, Herrera M, Domingues P. The effects of an artificial and a natural diet on growth, survival and reproductive performance of wild caught and reared brill (Scophthalmus rhombus). Aquaculture. 2009; 291(1–2):82–88. https://doi.org/10.1016/j.aquaculture.2009.03.004
Hajiahmadian M, Moghanlou KS, Agh N, Ardabili FF. Semen characteristics of rainbow trout (Oncorhynchus mykiss) following diets containing different vegetable fatty acid levels. Reprod Domest Anim. 2016; 51(6):979–84. https://doi.org/10.1111/rda.12776
Harshavardhan MA, Aanand S, Kumar JSS, Senthilkumar V. Comparative evaluation of commercial vegetable oil, fish oil, palm oil and groundnut oil as a lipid source in maturation and reproductive performance of fancy koi, Cyprinus carpio var. koi. Aquaculture. 2021; 545:737248. https://doi.org/10.1016/j.aquaculture.2021.737248
Hossen MS, Reza AHMM, Rakhi SF, Takahashi K, Hossain Z. Effects of polyunsaturated fatty acids (PUFAs) on gonadal maturation and spawning of striped gourami, Colisa fasciatus. Int Aquat Res. 2014; 6(65). https://doi.org/10.1007/s40071-014-0065-7
Izquierdo MS, Fernandez-Palacios H, Tacon AGJ. Effect of broodstock nutrition on reproductive performance of fish. Aquaculture. 2001; 197(1–4):25–42. https://doi.org/10.1016/S0044-8486(01)00581-6
Jaya-Ram A, Kuah M-K, Lim P-S, Kolkovski S, Shu-Chien AC. Influence of dietary HUFA levels on reproductive performance, tissue fatty acid profile and desaturase and elongase mRNAs expression in female zebrafish Danio rerio. Aquaculture. 2008; 277(3–4):275–81. https://doi.org/10.1016/j.aquaculture.2008.02.027
Köprücü K, Yonar ME, Özcan S. Effect of dietary n-3 polyunsaturated fatty acids on antioxidant defense and sperm quality in rainbow trout (Oncorhynchus mykiss) under regular stripping conditions. Anim Reprod Sci. 2015; 163:135–43. https://doi.org/10.1016/j.anireprosci.2015.10.008
Kowalski RK, Hliwa P, Andronowska A, Król J, Dietrich GJ, Wojtczak M, Stabiński R, Ciereszko A. Semen biology and stimulation of milt production in the European smelt (Osmerus eperlanus L.). Aquaculture. 2006; 261(2):760–70. https://doi.org/10.1016/j.aquaculture.2006.08.038
Król J, Kowalski RK, Hliwa P, Dietrich GJ, Stabiński R, Ciereszko A. The effects of commercial preparations containing two different GnRH analogues and dopamine antagonists on spermiation and sperm characteristics in the European smelt Osmerus eperlanus (L.). Aquaculture. 2009; 286(3–4):328–31. https://doi.org/10.1016/j.aquaculture.2008.09.034
Lahnsteiner F, Mansour N, McNiven MA, Richardson GF. Fatty acids of rainbow trout (Oncorhynchus mykiss) semen: Composition and effects on sperm functionality. Aquaculture. 2009; 298(1–2):118–24. https://doi.org/10.1016/j.aquaculture.2009.08.034
Leite JS, Oliveira-Araújo MS, Almeida-Monteiro PS, Campello CC, Campos ACN, Salmito-Vanderley CSB. Seasonal variation in seminal quality in Brazilian bocachico (Teleostei, Characiformes). Rev Caatinga. 2018; 31(3):759–66. https://doi.org/10.1590/1983-21252018v31n326rc
Ling S, Kuah MK, Muhammad TST, Kolkovski S, Shu-Chien AC. Effect of dietary HUFA on reproductive performance, tissue fatty acid profile and desaturase and elongase mRNAs in female swordtail Xiphophorus helleri. Aquaculture. 2006; 261(1):204–14. https://doi.org/10.1016/j.aquaculture.2006.06.045
Lopes JT, Oliveira-Araújo MS, Nascimento RV, Ferreira YM, Montenegro AR, Salmito-Vanderley CSB. Efeito de vitaminas e aminoácidos como suplementação da solução crioprotetora para a criopreservação do sêmen de tambaqui (Colossoma macropomum). Acta Sci Vet. 2018; 46:1593.
Luo L, Ai L, Liang X, Hu H, Xue M, Wu X. n-3 Long-chain polyunsaturated fatty acids improve the sperm, egg, and offspring quality of Siberian sturgeon (Acipenser baerii). Aquaculture. 2017; 473:266–71. https://doi.org/10.1016/j.aquaculture.2017.02.021
Mansour N, Lahnsteiner F, Berger B. Metabolism of intratesticular spermatozoa of a tropical teleost fish (Clarias gariepinus). Comp Biochem Physiol B: Biochem Mol Biol B. 2003; 135(2):285–96. https://doi.org/10.1016/s1096-4959(03)00083-6
Miliorini AB, Murgas LDS, Rosa PV, Oberlender G, Pereira GJM, Costa DV. A morphological classification proposal for curimba (Prochilodus lineatus) sperm damages after cryopreservation. Aquac Res. 2011; 42(2):177–87. https://doi.org/10.1111/j.1365-2109.2010.02575.x
Mylonas CC, Ducan NJ, Asturiano JF. Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality. Aquaculture. 2017; 472:21–44. https://doi.org/10.1016/j.aquaculture.2016.04.021
Mylonas CC, Fostier A, Zanuy S. Broodstock management and hormonal manipulations of fish reproduction. Gen Comp Endocrinol. 2010; 165(3):516–34. https://doi.org/10.1016/j.ygcen.2009.03.007
Ng W-K, Wang Y. Inclusion of crude palm oil in the broodstock diets of female Nile tilapia, Oreochromis niloticus, resulted in enhanced reproductive performance compared to broodfish fed diets with added fish oil or linseed oil. Aquaculture. 2011; 314(1–4):122–31. https://doi.org/10.1016/j.aquaculture.2011.01.034
Ninhaus-Silveira A, Foresti F, Veríssimo-Silveira R, Senhorini JA. Seminal analysis, cryogenic preservation, and fertility in matrinxã fish, Brycon cephalus (Günther, 1869). Braz Arch Biol Technol. 2006; 49(4):651–59. https://doi.org/10.1590/S1516-89132006000500015
Norambuena F, Estevez A, Bell G, Carazo I, Duncan N. Proximate and fatty acid compositions in muscle, liver and gonads of wild versus cultured broodstock of Senegalese sole (Solea senegalensis). Aquaculture. 2012; 356–57:176–85. https://doi.org/10.1016/j.aquaculture.2012.05.018
Rodrigues ML, Barcellos LJG, Moro EB, Sosa BS, Gomes RLM, Bittencourt F, Sanches EA, Signor A. Gonad development and sperm characteristics of male silver catfish (Rhamdia quelen) fed diets with different oil sources. Braz J Dev. 2022; 8(4):24032–51. https://doi.org/10.34117/bjdv8n4-086
Sabbag OJ, Takahashi LS, Silveira NA, Aranha AS. Custos e viabilidade econômica da produção de lambari-do-rabo amarelo em Monte Castelo/ SP: um estudo de caso. Bol Inst Pesca. 2011; 37(3):307–15.
Solis-Murgas LD, Felizardo VO, Ferreira MR, Andrade ES, Veras GC. Importância da avaliação dos parâmetros reprodutivos em peixes nativos. Rev Bras Reprod Anim. 2011; 35(2):186–91.
Streit-Junior DP, Sirol RN, Ribeiro RP, Moraes GV, Vargas LDM, Watanabe AL. Qualitative parameters of the piapara sêmen (Leporinus elongatus Valenciennes, 1850). Braz J Biol. 2008; 68(2):373–77. https://doi.org/10.1590/S1519-69842008000200019
Valdebenito II, Gallegos PT, Effer BR. Gamete quality in fish: evaluation parameters and determining factors. Zygote. 2015; 23(2):177–97. https://doi.org/10.1017/S0967199413000506
Watanabe T, Arakawa T, Kitajima C, Fujita S. Effect of nutritional quality of broodstock diets on reproduction of red sea bream. Nippon Suisan Gakkaishi. 1984; 50(3):495–501. https://doi.org/10.2331/suisan.50.495
Watanabe T, Vassallo-Agius R. Broodstock nutrition research on marine finfish in Japan. Aquaculture. 2003;227(1–4):35–61. https://doi.org/10.1016/S0044-8486(03)00494-0
Yasui GS, Senhorini JA, Shimoda E, Pereira-Santos M, Nakaghi LSO, Fujimoto T, Arias-Rodriguez L, Silva LA. Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish. Animal. 2015; 9(3):464–70. https://doi.org/10.1017/S1751731114002511
Yonar SM, Köprücü K, Özcan S. Dietary profile of n-3 series LC-PUFAs in rainbow trout under regular stripping condition: Semen production and quality, hepato-somatic index, haemato-immunologic values, oxidative stress and fatty acid composition of liver, muscle and sêmen. Aquac Res. 2020; 51(1):370–78. https://doi.org/10.1111/are.14384
Zaniboni-Filho E, Weingartner M. Técnicas de indução da reprodução de peixes migradores. Rev Bras Reprod Anim. 2007; 31(3):367–73.
Zhang J, Ma W, Xie B, Gui J-F, Mei J. Beneficial effect and potential molecular mechanism of chloroquine on sperm motility and fertilizing ability in yellow catfish. Aquaculture. 2017; 468:307–13. https://doi.org/10.1016/j.aquaculture.2016.10.028
Laicia Carneiro-Leite1,2, Lorena Pacheco da Silva1,3, Hellen Buzollo Pazzini4, Stella Indira Rocha Lobato1,2, Laís Pedroso Borges1,2, Yasmin dos Santos Reis1,3, Luciane Gomes-Silva1,2, Cristiéle da Silva Ribeiro5, Rosicleire Veríssimo-Silveira1 and Alexandre Ninhaus-Silveira1
 Universidade Estadual Paulista “Júlio de Mesquita Filho”, Faculdade de Engenharia, Campus de Ilha Solteira (UNESP/FEIS),Laboratório de Ictiologia Neotropical (LINEO), Departamento de Biologia e Zootecnia, Rua Monção, 226, 15385-000 Ilha Solteira,SP, Brazil. (LCL) firstname.lastname@example.org, (LPS) email@example.com, (SIRL) firstname.lastname@example.org, (LPB) email@example.com,(YSR) firstname.lastname@example.org, (LGS) email@example.com, (RVS) firstname.lastname@example.org, (ANS) email@example.com (corresponding author).
 Programa de Pós-Graduação em Ciências Biológicas (Zoologia), Instituto de Biociências de Botucatu (IBB/UNESP), Rua Prof. Dr. Antônio Celso Wagner Zanin, 250, 18618-689 Botucatu, SP, Brazil.
 Programa de Pós-Graduação em Ciência e Tecnologia Animal (PPCTA), UNESP/FEIS, 15385-000 Ilha Solteira, SP, Brazil.
Laicia Carneiro-Leite: Conceptualization, Data curation, Investigation, Methodology, Writing-original draft, Writing-review and editing.
Lorena Pacheco da Silva: Formal analysis, Methodology.
Hellen Buzollo Pazzini: Conceptualization, Formal analysis, Methodology.
Stella Indira Rocha Lobato: Investigation, Methodology
Laís Pedroso Borges: Investigation, Methodology
Yasmin dos Santos Reis: Investigation, Methodology.
Luciane Gomes-Silva: Investigation, Methodology
Cristiéle da Silva Ribeiro: Data curation, Investigation, Formal analysis, Methodology.
Rosicleire Veríssimo-Silveira: Methodology, Resources, Visualization.
Alexandre Ninhaus-Silveira: Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Writing-review and editing.
The experiment was carried out at Universidade Estadual Paulista “Júlio de Mesquita Filho,” Campus de Ilha Solteira, at the Laboratório de Ictiologia Neotropical (LINEO). All technical procedures used in this study were approved by the Ethics Committee on the Use of Animals (CEUA) of the Faculdade de Engenharia – Campus de Ilha Solteira in the process CEUA – 12/2019 FEIS/UNESP, the animals used in this project were not collected from the wild, so there was no need to obtain a license from SISBIO.
The author declares no competing interests.
How to cite this article
Carneiro-Leite L, Silva LP, Pazzini HB, Lobato SIR, Borges LP, Reis YS, Gomes-Silva L, Ribeiro CS, Veríssimo-Silveira R, Ninhaus-Silveira A. Effect of dietary omega-3 polyunsaturated fatty acids supplementation of Astyanax lacustris males on semen quality. Neotrop Ichthyol. 2023; 21(3):e230077. https://doi.org/10.1590/1982-0224-2023-0077
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Creative Commons CC-BY 4.0
© 2023 The Authors.
Diversity and Distributions Published by SBI
Accepted September 8, 2023 by Bernardo Baldisserotto
Submitted July 23, 2023
Epub October 20, 2023