Effect of dietary omega-3 polyunsaturated fatty acids supplementation of Astyanax lacustris males on semen quality

Abstract This study aimed to verify whether the inclusion of omega-3 polyunsaturated fatty acids (PUFAs-ô3) in the diet of Astyanax lacustris males improves seminal quality. Four hundred fish were arranged in 20 boxes of 180 L in the recirculation system. These were fed with isoprotein diets (32% CP), until apparent satiation, with four levels of marine fish oil rich in PUFAs-ô3 inclusion (In% = 0, 3, 6, and 9), for 105 days. After, males were hormonally induced to spermiation, and semen was collected after 226 ATUs. Parameters evaluated were osmolality, seminal volume and color, spermatic concentration, sperm morphology, membrane integrity and, sperm motility (total and progressive motility, velocity curvilinear, straight line and average path, linearity and rectilinearity coefficients, trajectory oscillation, head lateral displacement amplitude, and cross-beat frequency). Seminal volume was greater in the In-0 and In-3 groups. Inclusion of PUFAs-ô3 positively influenced the kinetic parameters, as treatment with 6% and 9% of inclusion fish oil resulted in higher values for most of these parameters and did not differ statistically from each other. Thus, it is concluded that the addition of PUFAs-ô3 to the feed of breeders significantly improved the seminal quality of A. lacustris males.


INTRODUCTION
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., 2010Mylonas et al., , 2017)).Studies carried out by Izquierdo et al. (2001), Watanabe, Vassallo-Agius (2003), Ling et al. (2006), Hachero-Cruzado et al. (2009), andNorambuena 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 Laicia Carneiro-Leite, Lorena P. Silva, Hellen B. Pazzini, Stella I. R. Lobato, Laís P. Borges, Yasmin S. Reis et al.
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), andCyprinus 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 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 1 st and 2 nd dry matter, percentage of crude protein by the Fatty acid on the Astyanax lacustris semen quality 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.

Ingredients (%)
In-0 In-  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.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).
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-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.

Silveira
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 nonparametric data.The statistical program R Studio was used for data analysis.To the boxes were considered as an experimental unit.

RESULTS
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).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.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).Fatty acid on the Astyanax lacustris semen quality

DISCUSSION
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.

FIGURE 1 |FIGURE 3 |
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).

TABLE 1 |
Composition of experimental feed formulated for Astyanax lacustris breeders.

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.

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