Ameliorative potential of dietary supplements, ZnO-K, citrus essential oil, and pumpkin seed oil, on sperm quality in Nile tilapia: Insights from CASA, DNA integrity, antioxidant enzymes, and gene expressions

Ameliorative potential of dietary supplements, ZnO-K, citrus essential oil, and pumpkin seed oil, on sperm quality in Nile tilapia: Insights from CASA, DNA integrity, antioxidant enzymes, and gene expressions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ameliorative potential of dietary supplements, ZnO-K, citrus essential oil, and pumpkin seed oil, on sperm quality in Nile tilapia: Insights from CASA, DNA integrity, antioxidant enzymes, and gene expressions Marwa M. Ali, Kamal Fathy Elboray, Engy T. Megahed, Hany T. Abu-Taleb, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6146222/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jun, 2025 Read the published version in Fish Physiology and Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract Sperm quality improvement is crucial to achieving the reproductive efficiency of Oreochromis niloticus . This study examined the effect of three dietary antioxidant supplements, kaolinite-doped zinc oxide (ZnO-K), citrus essential oil (CEO), and pumpkin seed oil (PSO), on sperm quality. Integrated sperm examination tools, including Computer Assisted Semen Analysis (CASA) parameters, spermatozoa DNA integrity, antioxidant enzyme bioassays, and gene expressions, were applied to validate sperm quality. One hundred and ninety-two adult males (mean weight 421.31 ± 6.26 g) were divided into four groups, each with three replicates. The first control group was fed on a diet without supplements. The second group was fed on ZnO- K-containing diet (0.06 g kg − 1 ); the third group was fed on a CEO-containing diet (10 g kg − 1 ); and the fourth group was fed on a PSO-containing diet (15 g kg − 1 ). ZnO-K supplementation significantly elevated milt volume (1.40 ± 0.10 ml) and sperm concentration (5.676 x 10 9 sperm ml − 1 ), as well as enhancing CASA parameters, including sperm motility, velocities, and DNA integrity. An increase in antioxidant activities of the enzymes, catalase, CAT, glutathione peroxidase, GPX, and superoxide dismutase, SOD, were observed in the ZnO-K-feeding group, recording 47.333 ± 1.452 U ml − 1 milt, 65.667 ± 5.547 mU ml − 1 milt and 60.667 ± 3.382 U ml − 1 milt, respectively. Notably, upregulation of the expressed genes, HSP70 , and CC chemokines was recorded in sperms from ZnO-K- and CEO-feeding groups, compared with gene expression suppression in the PSO-feeding group. All these findings suggest that ZnO-K and CEO are efficient in enhancing the quality of O. niloticus sperm, with the most pronounced effects shown by ZnO-K. Sperm quality O. niloticus antioxidant supplements CASA parameters DNA integrity enzyme bioassay gene expression Figures Figure 1 Figure 2 Figure 3 Introduction The sustainability of Oreochromis niloticus breeding programs relies heavily on sperm quality, a primary determinant of the success of fertilization and overall reproductive success. Oxidative stress, being a result of an imbalance between reactive oxygen species (ROS) and antioxidant mechanisms, detrimentally impacts sperm motility, viability, and DNA integrity (Cabrita et al. 2014 ). As such, improving sperm quality through dietary supplementation with antioxidant enrichment is essential for sustainable O. niloticus production (Sarmento et al. 2017 ; Bombardelli et al. 2023 ). Nutritional antioxidants exhibited a great potential in enhancing sperm motility, DNA stabilization, antioxidant enzyme activity, and stress resistance-related gene expression in aquatic organisms (Ciereszko and Dabrowski 2000 ; Gammanpila et al. 2007 ; Zhang et al. 2015 ; Mahanty et al. 2017 ). ZnO-K, having antioxidant activity, has been effective at 90 mg kg − 1 diet in improving biological parameters, reproduction indices, lipid profile, and antioxidant enzyme activities for O. niloticus (Soaudy et al. 2021 ). However, previous manual measures of the semen quality of Nile tilapia under the effect of ZnO-K had several limitations, including avoiding sperm velocity measurements, and sperm DNA integrity, the efficient sperm quality parameters (Soaudy et al. 2021 ). Although research on CEO in O. niloticus is scarce, essential oils from lemongrass have positively influenced tilapia growth and antioxidant status (Al-Sagheer et al. 2018 ), while citrus lemon extracts have improved sperm quality in mice (Rahayu and Hanizar 2021 ) and health in O. niloticus (Mohamed et al. 2021 ). PSO, with its high content of polyunsaturated fatty acids, flavonoids, and antioxidants, has shown efficacy in enhancing rat sperm quality by mitigating oxidative stress (Benalia et al. 2015 ; Aghaie et al. 2016 ). Although the impact of pumpkin seed oil has been studied on human sperm, there is a lack of knowledge of the effect of this oil on fish sperm quality. However, previous studies heightened the impact of pumpkin seeds on only fish growth performance (Dada and Ejete-Iroh, 2015 ; Greiling et al. 2018 ). Evaluating fish sperm quality involved several advanced methodologies. CASA provides precise measurements of motility parameters, such as curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP), across various fish species (Rurangwa et al. 2001 ; Caldeira et al. 2019 ). Although limited in O. niloticus , DNA integrity assays have found application in other fish species, like zebrafish and Arctic charr (Gosálvez et al. 2014 ; Jeuthe et al. 2022 ). CAT, GPX, and SOD are antioxidant enzymes that neutralize oxidative damage, ensuring sperm structural integrity and function (Drevet 2006 ; Agarwal et al. 2014 ; Perumal 2014 ). Gene expression analysis also offers an understanding of the molecular mechanisms that regulate sperm function and its reaction to stressors. Various limitations of sperm quality evaluation can be avoided by examining the molecular pathways of stress responses mediated through the genes HSP70 and CC chemokine (Caballero-Campo et al. 2014 ; Liu et al. 2019 ). HSP70 was identified as a significant protein kinase activity inhibitor accountable for protecting cells from apoptosis in the absence of similar stressors, indicating its crucial role in cellular resistance and adaptation response to stress (Ferraz et al. 2019 ). HSP70 expression level is significantly correlated with sperm motility and reproductive success (Solanki et al. 2023 ). The gene CC chemokine plays a role in the regulation of immune responses, implying its function in reproductive biology (Palomino and Marti 2015 ). The current research is a significant advancement in the molecular biology of fish reproduction as it is the first investigation to examine HSP70 and CC chemokine gene expressions in tilapia sperm. Recent studies have highlighted the positive impacts of antioxidant additives on sperm quality in O. niloticus (Sarmento et al. 2017 ; Hassona et al. 2020 ; Soaudy et al. 2021 ; Bombardelli et al. 2023 ). Nonetheless, these findings have not incorporated integrated advanced tools to comprehensively assess the multifaceted traits linked to enhancing sperm quality. This study aims to evaluate the potential of ZnO-K, CEO, and PSO as dietary supplements to enhance sperm quality in O. niloticus , utilizing a holistic approach that integrates advanced sperm quality assessment tools, including CASA parameters, DNA integrity, antioxidant enzyme assays, and gene expressions. These integrated examining tools could provide a robust profile for sperm quality characters for currently studied food additives. In addition, this study addressed the knowledge gap about the impact of dietary supplements on the expression of the genes, HSP70 and CC chemokines , in O. niloticus sperm. Materials and Methods Fish management and experimental design Wild brood O . niloticus males were collected from Lake Nasser, Egypt, one of the largest habitats for this species (Elsaied et al. 2019). A total of 192 adult males, with a mean total length of 27.15 ± 0.43 cm and a mean weight of 421.31 ± 6.26 g, were selected for the study. Males were assigned to four groups, with three replicates for each group, to enhance the results' robustness (Soaudy et al. 2021). The fish groups were evenly distributed among indoor concrete ponds, each measuring 4.50 m in length, 2.25 m in width, and 0.75 m in height. The water temperature was maintained between 25 °C and 27 °C throughout the experimental period, with dissolved oxygen at 6.0 ± 0.03 mg l -1 , total ammonia at 0.15 ± 0.02 mg l -1 , nitrite (NO 2 ) at 0.02 ± 0.004 mg l -1 and nitrate (NO 3 ) at 0.74 ± 0.06 mg l -1 . All physicochemical water parameters were monitored according to standard methods APHA (1989). Experimental diets Four experimental diets were formulated (Table 1). Diet 1 served as the basal control without supplementation. Diet 2 was supplemented with 0.06 g kg -1 ZnO-K (Soaudy et al. 2021), which was synthesized using a hydrothermal method according to Mohammady et al. (2021) and Hrenovic et al. (2012), with slight modifications. Citrus oil and pumpkin seed oil (100% pure) were purchased from local markets in Egypt. Diet 3 contained 10 g kg -1 citrus essential oil, CEO (Kesbiç et al. 2020). Diet 4 was supplemented with 15 g kg -1 pumpkin seed oil, PSO (Dada and Ejete-Iroh 2015). Each of ZnO-K, CEO, and PSO was mixed thoroughly with other ingredients in the basal diet (350 g kg -1 crude protein) (Table 1), using a feed mixer (Hobart Corporation, Troy, OH, USA) and blended with soybean oil. Distilled water was added to the premixed ingredients to form a dough, which was then extruded through a hand-operated noodle maker. The pellets were dried at room temperature and stored and sealed in cellophane bags at −4°C until use. Gross energy was calculated based on Brett (1973) and proximate composition was analyzed according to AOAC (2012). Fish males were fed at 3% of their body weight, twice daily, for 60 days (Vilela et al. 2003). Collection of fish milt Milt samples were collected from all males in each replicate by gently applying pressure to the abdomen, avoiding contamination with water, feces, or urine, according to Abascal et al., 2007. Milt was collected in sterile, pre-chilled Eppendorf tubes on ice, and the milt analysis parameters were measured immediately after sampling. Computer Assisted Semen Analysis (CASA) parameters Sperm motility and velocity were assessed using a CASA system (Spermolyzer, Mira Lab, https://mira-lab.com/product/spermolyzer/) equipped with a high-speed digital camera and software tailored for fish sperm analysis. The CASA system was adjusted for capturing 30 frames per second for individual sperm cells (400–700 per sample), and parameters were set according to the method of Alcántar‐Vázquez et al. (2022). Sperm motility and velocity measurements A sample of 20 µl milt, three replicates from each group, was diluted (1:20 v/v) in Hank’s balanced salt solution. For the analysis, 5 µL of diluted semen solution was dropped on a CASA slide, which was placed on the microscope stage at 25 O C. Sperm motility was activated by adding pond-filtered water and the sperm movements were captured, under 200× magnification, within a minute of activation. In total, six tracks were captured, four tracks from the corners and 2 tracks from the center of the slide, to quantitatively measure spermatozoa motility (Alcántar‐Vázquez et al 2022). The velocity parameters assessed with CASA included curvilinear velocity (VCL, µm s −1 ), measuring the average velocity of sperm along their actual curvilinear path; straight line velocity (VSL, µm s −1 ), the time-average velocity of a sperm head along the straight line between its first and last detected positions; and average path velocity (VAP, µm s −1 ), the velocity along the average path of the spermatozoon (Boyers et al. 1989; Kime et al. 2001). Sperm vitality assessment Sperm vitality was assessed via plasma membrane integrity using eosin-nigrosin staining (Kledmanee et al. 2013). Each of the fresh milt samples, three replicates from each group, was diluted at a ratio of 1:49 (v/v) with 0.9% NaCl saline solution, and 5 µl of diluted milt were mixed quickly with 5 µl of 0.5% Eosin Y stain, for 30 sec, followed by 10% Nigrosin stain. The integrity of the spermatozoa plasma membrane was determined for 200 sperm cells in each microscopic field, under magnification 400x. The spermatozoa with intact plasma membranes appeared colorless and differentiated from the stained dead sperm cells, which lacked membrane structural integrity. The number and percentage of live and dead sperms were calculated by CASA. Spermatozoa DNA integrity Sperm DNA integrity was evaluated using three milt replicates from each group, using the sperm chromatin dispersion (SCD) method, the Halo Test, according to the kit (BASO Biotech Co. Ltd., Lot M22606, Taiwan) manual instruction with modifications. Fresh milt was diluted in phosphate-buffered saline buffer (pH 7.4), according to Bombardelli et al. (2023). About 7 μL of diluted milt aliquot was mixed with 120 μL of low melting agarose (0.5%) at 37 ◦ C and evenly spread on glass slides pre-coated with 1.5% agarose. The embedded sperms were then treated with a lysis solution containing high salt and detergents to remove nuclear proteins, allowing DNA relaxation (Bombardelli et al. 2023). The slides were then exposed to a denaturation solution, NaOH (300 mM)/EDTA (200 mM), to induce DNA denaturation in fragmented sperm. Finally, the slides were washed, dehydrated, and stained with DNA-specific dyes in the kit. DNA integrity in sperm cells was visualized under 400x magnification and analyzed using CASA system. Sperm with intact DNA displayed halos, while fragmented DNA lacked this characteristic. Sperm morphometric measurements Each of the fresh milt samples, three replicates from each group, was diluted at a ratio of 1:49 (v/v) using a saline solution. A sperm smear was prepared by carefully spreading 5 µL of the diluted sample onto a clean microscope slide. The smears were subsequently fixed with methyl alcohol and stained using eosin and methylene blue. The sperm's morphometric measurements, including sperm head length, head width, midpiece width, and tail length, were done under magnification 1000x, according to (Musa 2010). Sperm morphometric analysis was done by counting 200 sperm cells from each milt sample. The sperm abnormalities classification, including head and middle piece diameters, as well as tail length, was based on the damage proposed by Paulino et al. (2016). Enzyme activity assays Catalase (CAT) activity was measured using the kit (Solarbio Life Sciences, Cat No. BC0200, China), according to the manufacturer's protocol. The assay was performed in 100 µL of milt sample. The reaction was initiated by combining the sample with a reaction mixture containing hydrogen peroxide (H₂O₂), and the breakdown of H₂O₂ was detected spectrophotometrically as a reduction in absorbance at 240 nm. The result was expressed as U/ml milt. Glutathione peroxidase (GPX) activity was measured for 100 µL milt by the GSH-PX assay kit (Elabscience, Cat No. E-BC-K096-S, USA), according to the kit instructions. The procedure is based on the oxidation of reduced glutathione in the presence of cumene hydroperoxide as a substrate, with the decrease in absorbance recorded at 340 nm, and the activity of the enzyme was determined as mU/ml milt. The activity of superoxide dismutase (SOD) was measured by the SOD assay kit (Solarbio Life Sciences, lot no. 809G113, China). The assay was performed according to the manufacturer’s protocol in the kit, using 100 µL of milt sample. The method depends on inhibiting the reduction of nitroblue tetrazolium by superoxide radicals and measuring the recorded absorbance at 560 nm. The enzyme activity was determined as U/ml milt. The assays were performed in triplicate to confirm reproducibility and accuracy. Negative controls (without enzyme) and positive controls (using standard enzyme solutions) were included in each batch of assays to validate the results. Gene expression analyses A sample of approximately 25 µl of fish milt, three replicates from each group, were used for RNA isolation using TRIzol (easy-RED, iNtRON Biotechnology, SKU:17063, Korea), according to Heidary and Pahlevan (2014). TOPscript™ RT-PCR DryMIX (Enzynomics, RT411, Korea) was used to synthesize cDNA from RNA, according to the method of Mohammady et al. (2021). Quantitative real-time PCR (qPCR) for amplifying the genes, HSP70 and CC chemokine , was performed using specific primers and SYBR™ Green Universal Master Mix (Applied Biosystems, cat. no. 4309155, USA). The primer sequences for amplification of HSP70 were forward (5'-CTCCACCCGAATCCCCAAAA-3'), and reverse (5'-TCGATACCCAGGGACAGAGG-3') (Hassan et al. 2017). The PCR primers used for the amplification of CC chemokine were forward (5'-ACAGAGCCGATCTTGGGTTACTTG-3') and reverse (5'-TGAAGGAGAGGCGGTGGATGTTAT-3') (Nakharuthai et al. 2016). Additionally, reference housekeeping gene β-actin primers had the sequences, forward (5'-TGGCAATGAGAGGTTCCG-3') and reverse (5'-TGCTGTTGTAGGTGGTTTCG-3') (Tanomman et al. 2013) and used for normalization (Livak and Schmittgen 2001). The reaction was performed using the Applied Biosystems 7500 instrument, with an initial denaturation step at 95 °C for 10 min, followed by 40 cycles in which the conditions were set at 95 °C for 10 sec, 60 °C, for CC chemokine , and 64 °C, for HSP70, for 30 sec, followed by 72 °C for a further 30 sec. A final extension was done at 72 °C for 10 min. Specific PCR products were confirmed by the melting curve analysis at the end of the amplification process. The numerical values represented the fold change concerning the control group (basic diet) when using the 2 −ΔΔCt method. Statistical analyses Data analyses, including normality, homogeneity tests, descriptive statistics, and one-way analysis of variance (ANOVA), were done by Statistical Analysis System, SAS, software, version 9.22. Duncan’s multiple range tests were used to compare differences among means. The values were presented as means ± standard deviation. Statistical differences were deemed significant when P < 0.05. Gene expression data analysis was conducted using the packages, ggplot2, dplyr, and tidyverse within the RStudio tool, version 2023.3.0.386 (Wickham and Grolemund 2023). The statistical analysis of gene expression data was performed using ANOVA, followed by Tukey’s post-hoc test to determine statistically significant differences. Gene expression values were reported as mean ± standard error (SE) to account for variability within samples. Results The physical characteristics of milt The milt volume and pH values across treatment groups are presented in Table 2. ZnO-K group recorded the highest significant milt volume, 1.40 ± 0.10 ml/male, while the PSO group had the lowest milt volume (0.80 ± 0.09 ml/male). Milt pH ranged from 6.700 ± 0.026 in the control group to 7.125 ± 0.025 in the ZnO-K group. The ZnO-K group also exhibited the highest sperm concentration (5.676 X 10 9 sperm/ml), with a minimal difference compared to the CEO group (5.540 X 10 9 sperm/ml). The PSO group displayed the lowest sperm concentration (3.844 X 10 9 sperm/ml), with no significant differences among the groups (Table 2). Sperm motility and velocity The ZnO-K group demonstrated the highest percentage of motile spermatozoa (66.21%), significantly higher than other groups, while the PSO group exhibited the lowest motility percentage (38.99%). Also, ZnO-K supplementation significantly enhanced sperm velocities, with curvilinear velocity (VCL) at 43.95 ± 0.89 µm/s and average path velocity (VAP) at 31.36 ± 1.04 µm/s (Table 2). Although the straight-line velocity (VSL) in the ZnO-K group (32.19 ± 1.90 µm/s) was higher than that of CEO group (23.35 ± 1.84 µm/s), the difference was not statistically significant. However, no significant differences in sperm velocities, VCL and VSL, were observed between the control, CEO, and PSO groups. Sperm vitality, DNA integrity, and morphological measurements The percentage of live spermatozoa, indicated by intact plasma membranes, was significantly higher in the ZnO-K (65.29% ± 0.55) and CEO (70.78% ± 4.42) groups compared to the other groups (Table 3). DNA integrity analysis (Table 3 and Fig. 1) revealed that spermatozoa from the ZnO-K and CEO groups exhibited the highest percentages of intact DNA, with values of 60.08% ± 1.741 and 54.23% ± 6.729, respectively. However, both the ZnO-k and CEO groups demonstrated significantly higher DNA integrity percentages than those of the control (28.08% ± 6.881) and PSO (34.88% ± 7.593) groups (Table 3). Morphological analysis revealed that the sperm head was spherical across all groups, with significant variations in head width, control (2.15 ± 0.15 µm), ZnO-K group (1.08 ± 0.13 µm), CEO group (1.84 ± 0.10 µm), and PSO group (2.56 ± 0.30 µm). The ZnO-K group exhibited the longest sperm tail (19.66 ± 0.57 µm) (Fig. 2). The PSO group displayed sperms with larger head widths and shorter tails, compared to other groups (Table 3, Fig. 2). Effect of dietary supplements on enzymatic activities in the sperms The enzymatic activity values of catalase (CAT), glutathione (GPX), and superoxidase dismutase (SOD) were presented in Table (4). The ZnO-K group exhibited CAT activity (47.33 ± 1.452 U/ml), significantly higher than those of the control (32.67 ± 2.603 U/ml) and the other treatment groups. A similar trend was observed, where the ZnO-K group showed a significant elevation in GPX activity (65.67 ± 5.547 mU/ml), compared with CEO group (41.67 ± 1.763 mU/ml) and PSO group (32.00 ± 2.081 mU/ml). Moreover, the ZnO-K group demonstrated a significant increase in SOD activity (60.67 ± 3.382 U/ml), in comparison with the other groups. Effect of dietary supplements on gene expressions in the sperms Quantitative PCR revealed a remarkable increase in the expression of HSP70 in the sperms of CEO and ZnO-K groups (Fig. 3a). However, the CEO group presented a level of HSP70 expression, which was significantly greater than that observed in the ZnO-K group, an observation in parallel with Tukey's post-hoc test. In contrast, treatment with PSO caused a significant reduction in the expression of HSP70 , relative to all groups (Fig. 3a). Similarly, the gene CC chemokine expression was significantly upregulated in the CEO and ZnO-K treatments compared to the control (P < 0.05) (Fig. 3b). However, there was no significant difference in the CC chemokine expression between CEO and ZnO-K treatments. Tukey's post-hoc test approved that PSO supplementation caused a significant downregulation in CC chemokine expression. Discussion The present study examined the impact of diet supplements, ZnO-K, CEO, and PSO, on the quality and quantity of sperms of O. niloticus . Integration of advanced tools, including CASA parameters, DNA integrity, enzyme assays, and gene expressions, validated sperm examination and introduced a robust sperm quality character profile under the effect of the studied supplements. The ZnO-K was found to have a significant elevation of milt volume and sperm concentration. Moreover, the milt volume, recorded for a male, in the current ZnO-K group was greater than those observed in O. niloticus males fed on a diet supplemented with Tribulus terrestis extract and 17α-methyl testosterone (Hassona et al. 2020 ) or a diet containing Spirulima platensis , as a feed additive (El-daim et al. 2021 ). Generally, Zinc is a substantial component for elevating spermatogenesis in fishes (Yamaguchi et al. 2009 ). Despite the volume of milt/male recorded in CEO group, CEO is comprised of up to 90% limonene, an agent with known antioxidant activity, which improves sperm quality (Eddin et al., 2021 ). However, this is the first assessment of CEO's impact on sperm health in Nile tilapia, and more research on its mode of action is needed. The stable milt pH across treatments (6.700-7.125) agrees with the findings by Ahamed et al. ( 2020 ), who found no dietary effect on milt pH in O. mossambicus . Sperm motility and velocity are critical examining parameters for semen quality (Gallego and Asturiano 2019 ). The observed sperm velocity values for ZnO-K group located within the typical permissible ranges of healthy sperm, observed by other studies on Nile tilapia (Gennotte et al. 2012 ; Dzyuba et al. 2019 ; Su et al. 2024 ). Sperm velocity values in CEO group were significantly lower than those in ZnO-K group, and that may be due to the presence of citrus-derived monoterpenoid aldehyde isomers, geranial, and neral, which may impair mitochondrial function, and consequently, sperm velocity parameters (Cavalleri et al. 2018 ). Comparable marginal differences in sperm motility were observed in mice that received citrus lemon oil (Rahayu and Hanizar 2021 ). PSO group exhibited the lowest sperm velocity values, among treated groups, suggesting PSO anti-nutritional components, such as phytates, which can bind to essential mineral nutrients required for sperm motility (Elinge et al. 2012 ; Nayyef et al. 2023). Generally, the current records of sperm velocity values differed from those documented in another study on tilapia (Sarmento et al. 2017 ), likely due to variations in dietary supplementation, feeding durations, and sperm motility measurement methodologies. A disparity was also noted when comparing the current results with those of Bombardelli et al. ( 2023 ), who reported higher sperm velocities in genetically improved farmed tilapia. This difference may be attributed to the use of wild tilapia broodstock in the present study. Nevertheless, the differences in the measurement of fish sperm velocity reported in different studies underline the need for a standardized framework for measuring sperm velocity. Standardization would increase the reliability of studies, allow for comparability between results, and promote the development of validated methodologies for the measurement of fish sperm velocities (Blackburn et al. 2022 ). Moreover, other examining tools, such as DNA integrity test, antioxidant enzyme analyses, and gene expression analysis, applied in this study, supported the evaluation of sperm quality. The plasma membrane integrity of the ZnO-K group can be explained by the stimulation of antioxidant enzymes (CAT, GPX, SOD), which play an important role in sperm quality maintenance (Soaudy et al. 2021 ). In addition, the bactericidal activity of CEO (Li et al. 2018 ) would also contribute to sperm membrane integrity. The current ZnO-K group ’ s longer sperm tails, which were positively connected with greater sperm velocities, were consistent with those of Barbus (Alavi et al. 2008 ). The correlation between sperm midpiece size, and mitochondrial sheath length, is critical for promoting sperm motility (Morita et al. 2004 ). However, zinc plays a role in optimizing sperm midpiece size and mitochondrial sheath length, and consequently, improving sperm motility (Arruda et al. 2021). DNA integrity, a key sperm quality indicator, was highest in the ZnO-K group, likely due to Zinc’s role in stabilizing sperm chromatin and mitigating oxidative stress (Björndahl and Kvist 2011 ; Huang et al. 2021 ). The occurrence of fragmented DNA in the control group may refer to unidentified direct or indirect oxidative stresses on the fish spermatozoa. However, fragmented DNA has been recorded previously in sperms of a control group of O. niloticus (Bombardelli et al. 2023 ). However, the low antioxidant reserves in fish sperm, due to limited cytoplasmic content (Cosson 2019 ), make them particularly susceptible to oxidative damage (Cabrita et al. 2014 ). Sperms of ZnO-K group recorded the highest levels of CAT, GPX, and SOD activities, demonstrating Zinc's capacity to enhance the sperm antioxidant enzymes. Zinc is a cofactor for numerous antioxidant enzymes, reducing oxidative damage and improving overall reproductive health (Fallah et al. 2018 ). This study supported previous findings on the relationship between the activities of the enzymes, SOD, and GPX, and the prevention of oxidative stress-induced sperm damage in various fish species (Shaliutina-Kolešová et al. 2018 ). However, zinc supplementation has been found enhancing the synthesis of zinc-binding enzymes with antioxidant properties, thereby improving sperm quality (Cheng and Chen 2021 ). In the current investigation, elevated enzyme activities were consistent with the enhanced sperm motility and DNA integrity observed in ZnO-K group, thus, validating other sperm quality parameters. This study added a knowledge about the relationship between the quality of tilapia sperm and the expression levels of the genes, heat shock HSP70 , and CC chemokine , supporting the crucial role of ZnO-K and CEO, as antioxidant key players, in enhancing the expression of immune response genes in crayfish, Procambarus clarkii (Kong et al. 2024 ) and zebrafish, Danio rerio , (Mahjoubian et al. 2023 ). Also, current results expanded our knowledge about the positive impact of citrus essential oil on the expression of other genes, such as the cytokine gene TNF-α , the antioxidant gene SOD , and the somatotropic axis growth-mediation gene IGF-1 , in several organs of adult Nile tilapia (Mohamed et al. 2021 ). However, limited knowledge is known about the mechanisms of the effect of PSO on the expression of HSP70 and CC chemokine in fish sperm and further studies should be done to clarify the suppression of these gene expressions by current PSO supplementation. In conclusion, the present study highlighted the potential role of ZnO-K, as a dietary supplement, in enhancing O. niloticus sperm quality, with different levels. This improvement is attributed to the multifaceted role of this antioxidant in promoting sperm motility, velocity, preserving DNA integrity, enhancing antioxidant enzymatic defenses, and upregulating immunity-related gene expressions. Additionally, CEO demonstrated a positive impact on sperm parameters, including concentration, velocity, viability, DNA integrity, and the higher expression of HSP70 and CC chemokine . In contrast, PSO, exhibited the least efficacy, suggesting the need for further optimization or its combination with other supplements to maximize its potential benefits in improving sperm quality. Declarations Funding Declaration No funding Author Contribution Marwa M. Ali Contribution: Conceptualization, Investigation, methodology, Data analyses, writingKamal Fathy ElborayContribution: sampling, Investigation, methodology, writing, editingEngy T. MegahedContribution: methodology, data analyses, writing Hany T. Abu-TalebContribution: sampling, Project administration, VisualizationAlshimaa E. ElsayedContribution: methods, data analysesEman Y. MohammadyContribution: fish rearing, feedingMona S. AmerContribution: methods, data analysesSoliman A. MorsiContribution: sampling, methodsEman M. AbbasContribution: editing, reviewMohamed S. HassaanEditing reviewHosam Elsaiedgroup leader corresponding author Acknowledgment All members of the Blue Gene Bank of the National Institute of Oceanography and Fisheries, NIOF, introduced their deep gratitude to the Academy of Scientific Research and Technology, ASRT, Egypt, for supporting the current research under the call “blue economy”. 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Aquaculture, 433: 173-182.‏ Greiling AM, Schwarz C, Gierus M, Rodehutscord M (2018) Pumpkin seed cake as a fishmeal substitute in fish nutrition: effects on growth performance, morphological traits and fillet color of two freshwater salmonids and two catfish species. Archives of Animal Nutrition, 72: 239–259. Hassan AM, El Nahas AF, Mahmoud S, et al (2017) Thermal stress of ambient temperature modulate expression of stress and immune-related genes and DNA fragmentation in Nile tilapia ( Oreochromis niloticus (Linnaeus, 1758). Appl Ecol Environ Res 15: Hassona NN, Zayed MM, Eltras WF, Mohamed RA (2020) Dietary supplementation of Tribulus terrestris extract improves growth and reproductive performances of the male Nile tilapia ( Oreochromis niloticus ). Aquac Res 51:4245–4254 Heidary M, Pahlevan M (2014) TRIzol-based RNA Extraction: A Reliable Method for Gene Expression Studies. 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Czech Journal of Animal Science 63: Soaudy MR, Mohammady EY, Ali MM, et al (2021) Potential effects of dietary ZnO supported on kaolinite (ZnO-K) to improve biological parameters, reproduction indices, lipid profile and antioxidant enzymes activities for broodstock of Nile tilapia ( Oreochromis niloticus ). Anim Feed Sci Technol 281:115117 Solanki GB, Singh VK, Kavani FS, et al (2023) Seasonal influence on expression of heat shock proteins (HSP70 and HSP90) vis-à-vis functional competence of Gir bull semen. Anim Biotechnol 34:3739–3748 Su Y, Wu Y, Ye M, et al (2024) Star1 gene mutation reveals the essentiality of 11-ketotestosterone and glucocorticoids for male fertility in Nile Tilapia ( Oreochromis niloticus ). Comp Biochem Physiol B Biochem Mol Biol 273:110985 Tanomman S, Ketudat-Cairns M, Jangprai A, Boonanuntanasarn S (2013) Characterization of fatty acid delta-6 desaturase gene in Nile tilapia and heterogenous expression in Saccharomyces cerevisiae. Comp Biochem Physiol B Biochem Mol Biol 166:148–156 Vilela DAR, Silva SGB, Peixoto MTD, et al (2003) Spermatogenesis in teleost: insights from the Nile tilapia ( Oreochromis niloticus ) model. Fish Physiol Biochem 28:187–190 Wickham H, Grolemund G (2023) R for Data Science, 2nd edn. O’Reilly Media, Sebastopol, CA, USA Yamaguchi S, Miura C, Kikuchi K, et al (2009) Zinc is an essential trace element for spermatogenesis. Proceedings of the National Academy of Sciences 106:10859–10864 Zhang C-N, Li X-F, Tian H-Y, et al (2015) Effects of fructooligosaccharide on immune response, antioxidant capability and HSP70 and HSP90 expressions of blunt snout bream ( Megalobrama amblycephala ) under high ammonia stress. Fish Physiol Biochem 41:203–217 Tables Table 1: Ingredients and proximate composition of the basal diet (g/kg diet). Ingredients Basal diet Fish meal 150 Soybean meal 540 Yellow corn 120 Wheat bran 120 Soybean oil 40 Zinc-free premix a 30 Proximate analysis (%) Dry matter 89.55 Crude protein 350 Crude lipid 72.0 Ash 49.2 Fiber content 533 NFE b 529.4 GE c (kJ/g dry matter) 20.19 a Vitamin and mineral mix (mg or g / Kg diet): MnSO4, 40 mg; MgO, 10 mg; K2SO4, 40 mg; KI, 0.4 mg; CuSO4, 12 mg; Ferric citrate, 250 mg; Na2SeO3, 0.24 mg; Co, 0.2 mg; retinol, 40000 IU; cholecalciferol, 4000 IU; α-tocopherolacetate, 400 mg; menadione, 12 mg; thiamine, 30 mg; riboflavin, 40 mg; pyridoxine, 30 mg; cyanocobalamin, 80 mcg;;nicotinic acid, 300 mg; folic acid, 10 mg; biotin, 3 mg; pantothenic acid, 100 mg; inositol, 500 mg; ascorbic acid, 500 mg. 2 B. acidophullus was prepared to obtain (1.47 × 107 CFU kg- 1 approximately. b NFE (Nitrogen free extract) = 100- (crude protein + lipid + ash +fibre content). c Gross energy, calculated using gross calorific values of 23.63, 39.52 and 17.15 Kj g - 1 for protein, fat and carbohydrate. Table (2) Milt volume, pH, sperm concentration, motility, and velocity parameters Items Control group + SD ZnO-K group + SD CEO group + SD PSO group + SD P -value Volume (ml/male) 1.20 b ± 0.052 1.40 a ± 0.10 0.90 c ± 0.054 0.80 c ± 0.026 0.0001 pH 6.700 a ± 0.026 7.125 a ± 0.025 7.000 a ± 0.022 7.00 a ± 0.018 0.0850 Sperm concentration (sperms ml -1 milt) 5.285x10 9 a ± 1.133 5.676x10 9 a ± 0.879 5.540x10 9 a ± 1.325 3.844x10 9 a ± 0.742 0.587 % Motility 48.0 3 b ±7.26 66.2 1 a ± 6.09 42.36 b ±4.68 38.99 b ±3.07 0.0077 Velocity (µm sec -1 ) VCL 24.49 b ± 0.677 43.95 a ± 0.89 28.07 b ± 0.751 25.03 b ± 0.327 0.0150 VSL 21.72 b ± 1.313 32.19 a ± 1.90 23.35 ab ± 1.841 20.88 b ± 0.838 0.0490 VAP 20.94 b ± 0.899 31.36 a ± 1.036 20.19 b ± 0.498 18.48 c ± 0.318 0.0217 Values in the same row with different superscripts are significantly different (P<0.05). Table (3) Sperm vitality, DNA integrity, and morphometric measurements Items Control group + SD ZnO-K group + SD CEO group + SD PSO group + SD P -value % Live sperms 49.97 b ± 0.74 65.29 a ± 0.55 70.78 a ± 4.42 41.14 c ± 1.32 0.0001 % DNA integrity 28.08 b ± 6.881 60.08 a ± 1.714 54.23 a ± 6.729 34.88 b ± 7.593 0.0040 Morphological measurements (µm) Head length 2.82 a ± 0.09 2.56 a ± 0.11 2.41 a ± 0.13 3.08 a ± 0.31 0.0795 Head width 2.15 ab ±0.15 1.80 b ±0.13 1.84 b ±0.10 2.56 a ± 0.30 0.0300 Midpiece width 0.57 a ±0.040 0.66 a ±0.05 0.59 a ±0.05 0.51 a ±0.06 0.2520 Tail length 18.83 a ± 0.46 19.66 a ±0.57 17.87 b ±0.65 14.92 c ±0.52 0.0001 Values in the same row with different superscripts are significantly different (P<0.05). Table 4 Enzymatic activities in sperms of O. niloticus males Items Control group + SD ZnO-K group + SD CEO group + SD PSO group + SD P -value CAT (U/ml) 32.67 b ± 2.603 47.33 a ± 1.452 28.00 bc ± 1.527 24.00 c ± 2.886 0.0004 GPX (mU/ml) 48.00 b ± 4.582 65.67 a ± 5.547 41.67 bc ± 1.763 32.00 c ± 2.081 0.0017 SOD (U/ml) 40.00 b ± 4.041 60.67 a ± 3.382 43.00 b ± 3.511 36.67 b ± 3.179 0.0058 Values in the same row with different superscripts are significantly different (P<0.05). CAT: Catalase (U/ml) as unit per milliliter; GPX: Glutathione peroxidase mU/ml) as milliunit per milliliter; SOD: Superoxide dismutase (U/ml) as unit per milliliter. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 23 Jun, 2025 Read the published version in Fish Physiology and Biochemistry → Version 1 posted Reviewers agreed at journal 22 Apr, 2025 Reviewers agreed at journal 21 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers invited by journal 03 Apr, 2025 Submission checks completed at journal 03 Apr, 2025 First submitted to journal 25 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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A and B, enlargements of sperm heads. N, the head of sperm shows non-fragmented DNA,with a halo. D, head of sperm shows fragmented DNA, without halo. Scale bar = 5 µm; bar in A or B = 1.5 µm\u003c/p\u003e","description":"","filename":"figuresfishphysiologybioechmistry1.png","url":"https://assets-eu.researchsquare.com/files/rs-6146222/v1/a382c8daf3116a8704af49ac.png"},{"id":81630083,"identity":"384345be-647d-49cb-8835-00b64463508d","added_by":"auto","created_at":"2025-04-29 11:18:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1007642,"visible":true,"origin":"","legend":"\u003cp\u003eSperm morphological structures for males\u003cem\u003e \u003c/em\u003efed on diets supplemented with different antioxidants, 1. Head sperm, 2. Midpiece and 3. Tail. Scale bar = 10 µm.\u003c/p\u003e","description":"","filename":"figuresfishphysiologybioechmistry2.png","url":"https://assets-eu.researchsquare.com/files/rs-6146222/v1/19f4a8dacc76a0fdb616179e.png"},{"id":81630075,"identity":"167d0842-62f1-4dcd-8de2-dc137191eaa4","added_by":"auto","created_at":"2025-04-29 11:18:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80113,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of the supplements ZnO-K, citrus essential oil, and pumpkin seed oil on the gene HSP70 expression in the sperms of O. niloticus. Bars with different superscript letters (a, b, c, d) illustrate significant differences (p \u0026lt; 0.05). Error bars +/- 1 SD.\u003c/p\u003e\n\u003cp\u003e(b) Effect of the supplements ZnO-K, citrus essential oil, and pumpkin seed oil on the gene CC chemokine expression in the sperms of O. niloticus. Bars with different superscript letters (a, b, c) illustrate significant differences (p \u0026lt; 0.05). Error bars +/- 1 SD.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"figuresfishphysiologybioechmistry3.png","url":"https://assets-eu.researchsquare.com/files/rs-6146222/v1/2e0df3ae2ac5d1ca8f3901c6.png"},{"id":85686596,"identity":"54631eb7-daa2-4525-ada3-754bec48d784","added_by":"auto","created_at":"2025-06-30 16:08:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3893135,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6146222/v1/a302aefe-d8cc-425d-9394-88ba2ea8644c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ameliorative potential of dietary supplements, ZnO-K, citrus essential oil, and pumpkin seed oil, on sperm quality in Nile tilapia: Insights from CASA, DNA integrity, antioxidant enzymes, and gene expressions ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe sustainability of \u003cem\u003eOreochromis niloticus\u003c/em\u003e breeding programs relies heavily on sperm quality, a primary determinant of the success of fertilization and overall reproductive success. Oxidative stress, being a result of an imbalance between reactive oxygen species (ROS) and antioxidant mechanisms, detrimentally impacts sperm motility, viability, and DNA integrity (Cabrita et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). As such, improving sperm quality through dietary supplementation with antioxidant enrichment is essential for sustainable \u003cem\u003eO. niloticus\u003c/em\u003e production (Sarmento et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bombardelli et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNutritional antioxidants exhibited a great potential in enhancing sperm motility, DNA stabilization, antioxidant enzyme activity, and stress resistance-related gene expression in aquatic organisms (Ciereszko and Dabrowski \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Gammanpila et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mahanty et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). ZnO-K, having antioxidant activity, has been effective at 90 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet in improving biological parameters, reproduction indices, lipid profile, and antioxidant enzyme activities for \u003cem\u003eO. niloticus\u003c/em\u003e (Soaudy et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, previous manual measures of the semen quality of Nile tilapia under the effect of ZnO-K had several limitations, including avoiding sperm velocity measurements, and sperm DNA integrity, the efficient sperm quality parameters (Soaudy et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although research on CEO in \u003cem\u003eO. niloticus\u003c/em\u003e is scarce, essential oils from lemongrass have positively influenced tilapia growth and antioxidant status (Al-Sagheer et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while citrus lemon extracts have improved sperm quality in mice (Rahayu and Hanizar \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and health in \u003cem\u003eO. niloticus\u003c/em\u003e (Mohamed et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). PSO, with its high content of polyunsaturated fatty acids, flavonoids, and antioxidants, has shown efficacy in enhancing rat sperm quality by mitigating oxidative stress (Benalia et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Aghaie et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Although the impact of pumpkin seed oil has been studied on human sperm, there is a lack of knowledge of the effect of this oil on fish sperm quality. However, previous studies heightened the impact of pumpkin seeds on only fish growth performance (Dada and Ejete-Iroh, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Greiling et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEvaluating fish sperm quality involved several advanced methodologies. CASA provides precise measurements of motility parameters, such as curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP), across various fish species (Rurangwa et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Caldeira et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although limited in \u003cem\u003eO. niloticus\u003c/em\u003e, DNA integrity assays have found application in other fish species, like zebrafish and Arctic charr (Gos\u0026aacute;lvez et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jeuthe et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCAT, GPX, and SOD are antioxidant enzymes that neutralize oxidative damage, ensuring sperm structural integrity and function (Drevet \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Agarwal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Perumal \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Gene expression analysis also offers an understanding of the molecular mechanisms that regulate sperm function and its reaction to stressors. Various limitations of sperm quality evaluation can be avoided by examining the molecular pathways of stress responses mediated through the genes \u003cem\u003eHSP70\u003c/em\u003e and \u003cem\u003eCC chemokine\u003c/em\u003e (Caballero-Campo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eHSP70\u003c/em\u003e was identified as a significant protein kinase activity inhibitor accountable for protecting cells from apoptosis in the absence of similar stressors, indicating its crucial role in cellular resistance and adaptation response to stress (Ferraz et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eHSP70\u003c/em\u003e expression level is significantly correlated with sperm motility and reproductive success (Solanki et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The gene \u003cem\u003eCC chemokine\u003c/em\u003e plays a role in the regulation of immune responses, implying its function in reproductive biology (Palomino and Marti \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The current research is a significant advancement in the molecular biology of fish reproduction as it is the first investigation to examine \u003cem\u003eHSP70\u003c/em\u003e and \u003cem\u003eCC chemokine\u003c/em\u003e gene expressions in tilapia sperm.\u003c/p\u003e \u003cp\u003eRecent studies have highlighted the positive impacts of antioxidant additives on sperm quality in \u003cem\u003eO. niloticus\u003c/em\u003e (Sarmento et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hassona et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Soaudy et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bombardelli et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nonetheless, these findings have not incorporated integrated advanced tools to comprehensively assess the multifaceted traits linked to enhancing sperm quality. This study aims to evaluate the potential of ZnO-K, CEO, and PSO as dietary supplements to enhance sperm quality in \u003cem\u003eO. niloticus\u003c/em\u003e, utilizing a holistic approach that integrates advanced sperm quality assessment tools, including CASA parameters, DNA integrity, antioxidant enzyme assays, and gene expressions. These integrated examining tools could provide a robust profile for sperm quality characters for currently studied food additives. In addition, this study addressed the knowledge gap about the impact of dietary supplements on the expression of the genes, \u003cem\u003eHSP70\u003c/em\u003e and \u003cem\u003eCC chemokines\u003c/em\u003e, in \u003cem\u003eO. niloticus\u003c/em\u003e sperm.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eFish management and experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild brood \u003cem\u003eO\u003c/em\u003e.\u003cem\u003e\u0026nbsp;niloticus\u003c/em\u003e males were collected from Lake Nasser, Egypt, one of the largest habitats for this species (Elsaied et al. 2019). A total of 192 adult males, with a mean total length of 27.15 ± 0.43 cm and a mean weight of \u0026nbsp; 421.31 ± 6.26 g, were selected for the study. Males were assigned to four groups, with three replicates for each group, to enhance the results' robustness (Soaudy et al. 2021). \u0026nbsp;The fish groups were evenly distributed among indoor concrete ponds, each measuring 4.50 m in length, 2.25 m in width, and 0.75 m in height. The water temperature was maintained between 25 °C and 27 °C throughout the experimental period, with dissolved oxygen at 6.0 ± 0.03 mg l\u003csup\u003e-1\u003c/sup\u003e, total ammonia at 0.15 ± 0.02 mg l\u003csup\u003e-1\u003c/sup\u003e, nitrite (NO\u003csub\u003e2\u003c/sub\u003e) at 0.02 ± 0.004 mg l\u003csup\u003e-1\u003c/sup\u003e and nitrate (NO\u003csub\u003e3\u003c/sub\u003e) at 0.74 ± 0.06 mg l\u003csup\u003e-1\u003c/sup\u003e. All physicochemical water parameters were monitored according to standard methods APHA (1989).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental diets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour experimental diets were formulated (Table 1). Diet 1 served as the basal control without supplementation. Diet 2 was supplemented with 0.06 g kg\u003csup\u003e-1\u003c/sup\u003e ZnO-K (Soaudy et al. 2021), which was synthesized using a hydrothermal method according to Mohammady et al. (2021) and Hrenovic et al. (2012), with slight modifications. Citrus oil and pumpkin seed oil (100% pure) were purchased from local markets in Egypt. Diet 3 contained 10 g kg\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ecitrus essential oil, CEO (Kesbiç et al. 2020). Diet 4 was supplemented with 15 g kg\u003csup\u003e-1\u003c/sup\u003e pumpkin seed oil, PSO (Dada and Ejete-Iroh 2015). Each of ZnO-K, CEO, and PSO was mixed thoroughly with other ingredients in the basal diet (350 g kg\u003csup\u003e-1\u003c/sup\u003e crude protein) (Table 1), using a feed mixer (Hobart Corporation, Troy, OH, USA) and blended with soybean oil. Distilled water was added to the premixed ingredients to form a dough, which was then extruded through a hand-operated noodle maker. The pellets were dried at room temperature and stored and sealed in cellophane bags at −4°C until use. Gross energy was calculated based on Brett (1973) and proximate composition was analyzed according to AOAC (2012). Fish males were fed at 3% of their body weight, twice daily, for 60 days (Vilela et al. 2003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of fish milt\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMilt samples were collected from all males in each replicate by gently applying pressure to the abdomen, avoiding contamination with water, feces, or urine, according to Abascal et al., 2007. Milt was collected in sterile, pre-chilled Eppendorf tubes on ice, and the milt analysis parameters were measured immediately after sampling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComputer Assisted Semen Analysis (CASA) parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSperm motility and velocity were assessed using a CASA system (Spermolyzer, Mira Lab, https://mira-lab.com/product/spermolyzer/) equipped with a high-speed digital camera and software tailored for fish sperm analysis. The CASA system was adjusted for capturing 30 frames per second for individual sperm cells (400–700 per sample), and parameters were set according to the method of Alcántar‐Vázquez et al. (2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm motility and velocity measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA sample of 20 µl milt, three replicates from each group, was diluted (1:20 v/v) in Hank’s balanced salt solution. For the analysis, 5 µL of diluted semen solution was dropped on a CASA slide, which was placed on the microscope stage at 25 \u003csup\u003eO\u003c/sup\u003eC. Sperm motility was activated by adding pond-filtered water and the sperm movements were captured, under 200× magnification, within a minute of activation. In total, six tracks were captured, four tracks from the corners and 2 tracks from the center of the slide, to quantitatively measure spermatozoa motility (Alcántar‐Vázquez et al 2022). \u0026nbsp;The velocity parameters assessed with CASA included curvilinear velocity (VCL, µm s \u003csup\u003e−1\u003c/sup\u003e), measuring the average velocity of sperm along their actual curvilinear path; straight line velocity \u0026nbsp;(VSL, µm s\u003csup\u003e−1\u003c/sup\u003e), the time-average velocity of a sperm head along the straight line between its first and last detected positions; and average path velocity (VAP, µm s\u003csup\u003e−1\u003c/sup\u003e), the velocity along the average path of the spermatozoon\u0026nbsp;(Boyers et al. 1989; Kime et al. 2001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm vitality assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSperm vitality was assessed via plasma membrane integrity using eosin-nigrosin staining (Kledmanee et al. 2013). Each of the fresh milt samples, three replicates from each group, was diluted at a ratio of 1:49 (v/v) with 0.9% NaCl saline solution, and 5 µl of diluted milt were mixed quickly with 5 µl of 0.5% Eosin Y stain, for 30 sec, followed by 10% Nigrosin stain. The integrity of the spermatozoa plasma membrane was determined for 200 sperm cells in each microscopic field,\u0026nbsp;under magnification 400x.\u0026nbsp;The spermatozoa with intact plasma membranes appeared colorless and differentiated from the stained dead sperm cells, which lacked membrane structural integrity. The number and percentage of live and dead sperms were calculated by CASA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpermatozoa DNA integrity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSperm DNA integrity was evaluated using three milt replicates from each group, using the sperm chromatin dispersion (SCD) method, the Halo Test, according to the kit (BASO Biotech Co. Ltd., Lot M22606, Taiwan) manual instruction with modifications. Fresh milt was diluted in phosphate-buffered saline buffer (pH 7.4), according to Bombardelli et al. (2023). About 7 μL of diluted milt aliquot was mixed with 120 μL of low melting agarose (0.5%) at 37 \u003csup\u003e◦\u003c/sup\u003eC and evenly spread on glass slides pre-coated with 1.5% agarose. \u0026nbsp;The embedded sperms were then treated with a lysis solution containing high salt and detergents to remove nuclear proteins, allowing DNA relaxation (Bombardelli et al. 2023). The slides were then exposed to a denaturation solution, NaOH (300 mM)/EDTA (200 mM), to induce DNA denaturation in fragmented sperm. Finally, the slides were washed, dehydrated, and stained with DNA-specific dyes in the kit. DNA integrity in sperm cells was visualized under 400x magnification and analyzed using CASA system. Sperm with intact DNA displayed halos, while fragmented DNA lacked this characteristic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm morphometric measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach of the fresh milt samples, three replicates from each group, was diluted at a ratio of 1:49 (v/v) using a saline solution. A sperm smear was prepared by carefully spreading 5 µL of the diluted sample onto a clean microscope slide. The smears were subsequently fixed with methyl alcohol and stained using eosin and methylene blue. The sperm's morphometric measurements, including sperm head length, head width, midpiece width, and tail length, were done under magnification 1000x, according to (Musa 2010). Sperm morphometric analysis was done by counting 200 sperm cells from each milt sample. The sperm abnormalities classification, including head and middle piece diameters, as well as tail length, was based on the damage proposed by Paulino et al. (2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme activity assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCatalase (CAT) activity was measured using the kit (Solarbio Life Sciences, Cat No. BC0200, China), according to the manufacturer's protocol. The assay was performed in 100 µL of milt sample. The reaction was initiated by combining the sample with a reaction mixture containing hydrogen peroxide (H₂O₂), and the breakdown of H₂O₂ was detected spectrophotometrically as a reduction in absorbance at 240 nm. The result was expressed as U/ml \u0026nbsp;milt. Glutathione peroxidase (GPX) activity was measured for 100 µL milt by the GSH-PX assay kit (Elabscience, Cat No. E-BC-K096-S, USA), according to the kit instructions. The procedure is based on the oxidation of reduced glutathione in the presence of cumene hydroperoxide as a substrate, with the decrease in absorbance recorded at 340 nm, and the activity of the enzyme was determined as mU/ml milt. The activity of superoxide dismutase (SOD) was measured by the SOD assay kit (Solarbio Life Sciences, lot no. 809G113, China). The assay was performed according to the manufacturer’s protocol in the kit, using 100 µL of milt sample. The method depends on inhibiting the reduction of nitroblue tetrazolium by superoxide radicals and measuring the recorded absorbance at 560 nm. The enzyme activity was determined as U/ml milt. The assays were performed in triplicate to confirm reproducibility and accuracy. Negative controls (without enzyme) and positive controls (using standard enzyme solutions) were included in each batch of assays to validate the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA sample of approximately 25 µl of fish milt, three replicates from each group, were used for RNA isolation using TRIzol (easy-RED, iNtRON Biotechnology, SKU:17063, Korea), according to Heidary and Pahlevan (2014). \u0026nbsp;TOPscript™ RT-PCR DryMIX (Enzynomics, RT411, Korea) was used to synthesize cDNA from RNA, according to the method of Mohammady et al. (2021). Quantitative real-time PCR (qPCR) for amplifying the genes,\u003cem\u003e\u0026nbsp;HSP70\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCC chemokine\u003c/em\u003e, was performed using specific primers and SYBR™ Green Universal Master Mix (Applied Biosystems, cat. no. 4309155, USA). The primer sequences for amplification of \u003cem\u003eHSP70\u003c/em\u003e were forward (5'-CTCCACCCGAATCCCCAAAA-3'), and reverse (5'-TCGATACCCAGGGACAGAGG-3') (Hassan et al. 2017). The PCR primers used for the amplification of \u003cem\u003eCC chemokine\u003c/em\u003e were forward (5'-ACAGAGCCGATCTTGGGTTACTTG-3') and reverse (5'-TGAAGGAGAGGCGGTGGATGTTAT-3') (Nakharuthai et al. 2016). Additionally, reference housekeeping gene \u003cem\u003eβ-actin\u003c/em\u003e primers had the sequences, forward (5'-TGGCAATGAGAGGTTCCG-3') and reverse (5'-TGCTGTTGTAGGTGGTTTCG-3') (Tanomman et al. 2013) and used for normalization (Livak and Schmittgen 2001). The reaction was performed using the Applied Biosystems 7500 instrument, with an initial denaturation step at 95 °C for 10 min, followed by 40 cycles in which the conditions were set at 95 °C for 10 sec, 60 °C, for \u003cem\u003eCC chemokine\u003c/em\u003e, and 64 °C, for \u003cem\u003eHSP70,\u003c/em\u003e\u0026nbsp; for 30 sec, followed by 72 °C for a further 30 sec. A final extension was done at 72 °C for 10 min. Specific PCR products were confirmed by the melting curve analysis at the end of the amplification process. The numerical values represented the fold change concerning the control group (basic diet) when using the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Data analyses, including normality, homogeneity tests, descriptive statistics, and one-way analysis of variance (ANOVA), were done by Statistical Analysis System, SAS, software, version 9.22. Duncan’s multiple range tests were used to compare differences among means. The values were presented as means ± standard deviation. Statistical differences were deemed significant when \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Gene expression data analysis was conducted using the packages, ggplot2, dplyr, and tidyverse within the RStudio tool, version 2023.3.0.386 (Wickham and Grolemund 2023). The statistical analysis of gene expression data was performed using ANOVA, followed by Tukey’s post-hoc test to determine statistically significant differences. Gene expression values were reported as mean ± standard error (SE) to account for variability within samples.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe physical characteristics of milt\u003c/h2\u003e \u003cp\u003eThe milt volume and pH values across treatment groups are presented in Table\u0026nbsp;2. ZnO-K group recorded the highest significant milt volume, 1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 ml/male, while the PSO group had the lowest milt volume (0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 ml/male). Milt pH ranged from 6.700\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026 in the control group to 7.125\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025 in the ZnO-K group. The ZnO-K group also exhibited the highest sperm concentration (5.676 X 10\u003csup\u003e9\u003c/sup\u003e sperm/ml), with a minimal difference compared to the CEO group (5.540 X 10\u003csup\u003e9\u003c/sup\u003e sperm/ml). The PSO group displayed the lowest sperm concentration (3.844 X 10\u003csup\u003e9\u003c/sup\u003e sperm/ml), with no significant differences among the groups (Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSperm motility and velocity\u003c/h2\u003e \u003cp\u003eThe ZnO-K group demonstrated the highest percentage of motile spermatozoa (66.21%), significantly higher than other groups, while the PSO group exhibited the lowest motility percentage (38.99%). Also, ZnO-K supplementation significantly enhanced sperm velocities, with curvilinear velocity (VCL) at 43.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89 \u0026micro;m/s and average path velocity (VAP) at 31.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 \u0026micro;m/s (Table\u0026nbsp;2). Although the straight-line velocity (VSL) in the ZnO-K group (32.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90 \u0026micro;m/s) was higher than that of CEO group (23.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84 \u0026micro;m/s), the difference was not statistically significant. However, no significant differences in sperm velocities, VCL and VSL, were observed between the control, CEO, and PSO groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSperm vitality, DNA integrity, and morphological measurements\u003c/h2\u003e \u003cp\u003eThe percentage of live spermatozoa, indicated by intact plasma membranes, was significantly higher in the ZnO-K (65.29% \u0026plusmn; 0.55) and CEO (70.78% \u0026plusmn; 4.42) groups compared to the other groups (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eDNA integrity analysis (Table\u0026nbsp;3 and Fig.\u0026nbsp;1) revealed that spermatozoa from the ZnO-K and CEO groups exhibited the highest percentages of intact DNA, with values of 60.08% \u0026plusmn; 1.741 and 54.23% \u0026plusmn; 6.729, respectively. However, both the ZnO-k and CEO groups demonstrated significantly higher DNA integrity percentages than those of the control (28.08% \u0026plusmn; 6.881) and PSO (34.88% \u0026plusmn; 7.593) groups (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eMorphological analysis revealed that the sperm head was spherical across all groups, with significant variations in head width, control (2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u0026micro;m), ZnO-K group (1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u0026micro;m), CEO group (1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 \u0026micro;m), and PSO group (2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 \u0026micro;m). The ZnO-K group exhibited the longest sperm tail (19.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 \u0026micro;m) (Fig.\u0026nbsp;2). The PSO group displayed sperms with larger head widths and shorter tails, compared to other groups (Table\u0026nbsp;3, Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of dietary supplements on enzymatic activities in the sperms\u003c/h2\u003e \u003cp\u003eThe enzymatic activity values of catalase (CAT), glutathione (GPX), and superoxidase dismutase (SOD) were presented in Table\u0026nbsp;(4). The ZnO-K group exhibited CAT activity (47.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.452 U/ml), significantly higher than those of the control (32.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.603 U/ml) and the other treatment groups. A similar trend was observed, where the ZnO-K group showed a significant elevation in GPX activity (65.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.547 mU/ml), compared with CEO group (41.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.763 mU/ml) and PSO group (32.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.081 mU/ml). Moreover, the ZnO-K group demonstrated a significant increase in SOD activity (60.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.382 U/ml), in comparison with the other groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of dietary supplements on gene expressions in the sperms\u003c/h2\u003e \u003cp\u003eQuantitative PCR revealed a remarkable increase in the expression of \u003cem\u003eHSP70\u003c/em\u003e in the sperms of CEO and ZnO-K groups (Fig.\u0026nbsp;3a). However, the CEO group presented a level of \u003cem\u003eHSP70\u003c/em\u003e expression, which was significantly greater than that observed in the ZnO-K group, an observation in parallel with Tukey's post-hoc test. In contrast, treatment with PSO caused a significant reduction in the expression of \u003cem\u003eHSP70\u003c/em\u003e, relative to all groups (Fig.\u0026nbsp;3a).\u003c/p\u003e \u003cp\u003eSimilarly, the gene \u003cem\u003eCC chemokine\u003c/em\u003e expression was significantly upregulated in the CEO and ZnO-K treatments compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;3b). However, there was no significant difference in the \u003cem\u003eCC chemokine\u003c/em\u003e expression between CEO and ZnO-K treatments. Tukey's post-hoc test approved that PSO supplementation caused a significant downregulation in \u003cem\u003eCC chemokine\u003c/em\u003e expression.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study examined the impact of diet supplements, ZnO-K, CEO, and PSO, on the quality and quantity of sperms of \u003cem\u003eO. niloticus\u003c/em\u003e. Integration of advanced tools, including CASA parameters, DNA integrity, enzyme assays, and gene expressions, validated sperm examination and introduced a robust sperm quality character profile under the effect of the studied supplements.\u003c/p\u003e \u003cp\u003eThe ZnO-K was found to have a significant elevation of milt volume and sperm concentration. Moreover, the milt volume, recorded for a male, in the current ZnO-K group was greater than those observed in \u003cem\u003eO. niloticus\u003c/em\u003e males fed on a diet supplemented with \u003cem\u003eTribulus terrestis\u003c/em\u003e extract and 17α-methyl testosterone (Hassona et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) or a diet containing \u003cem\u003eSpirulima platensis\u003c/em\u003e, as a feed additive (El-daim et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Generally, Zinc is a substantial component for elevating spermatogenesis in fishes (Yamaguchi et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Despite the volume of milt/male recorded in CEO group, CEO is comprised of up to 90% limonene, an agent with known antioxidant activity, which improves sperm quality (Eddin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, this is the first assessment of CEO's impact on sperm health in Nile tilapia, and more research on its mode of action is needed. The stable milt pH across treatments (6.700-7.125) agrees with the findings by Ahamed et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who found no dietary effect on milt pH in \u003cem\u003eO. mossambicus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSperm motility and velocity are critical examining parameters for semen quality (Gallego and Asturiano \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The observed sperm velocity values for ZnO-K group located within the typical permissible ranges of healthy sperm, observed by other studies on Nile tilapia (Gennotte et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dzyuba et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Sperm velocity values in CEO group were significantly lower than those in ZnO-K group, and that may be due to the presence of citrus-derived monoterpenoid aldehyde isomers, geranial, and neral, which may impair mitochondrial function, and consequently, sperm velocity parameters (Cavalleri et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Comparable marginal differences in sperm motility were observed in mice that received citrus lemon oil (Rahayu and Hanizar \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). PSO group exhibited the lowest sperm velocity values, among treated groups, suggesting PSO anti-nutritional components, such as phytates, which can bind to essential mineral nutrients required for sperm motility (Elinge et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nayyef et al. 2023). Generally, the current records of sperm velocity values differed from those documented in another study on tilapia (Sarmento et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), likely due to variations in dietary supplementation, feeding durations, and sperm motility measurement methodologies. A disparity was also noted when comparing the current results with those of Bombardelli et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who reported higher sperm velocities in genetically improved farmed tilapia. This difference may be attributed to the use of wild tilapia broodstock in the present study. Nevertheless, the differences in the measurement of fish sperm velocity reported in different studies underline the need for a standardized framework for measuring sperm velocity. Standardization would increase the reliability of studies, allow for comparability between results, and promote the development of validated methodologies for the measurement of fish sperm velocities (Blackburn et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, other examining tools, such as DNA integrity test, antioxidant enzyme analyses, and gene expression analysis, applied in this study, supported the evaluation of sperm quality.\u003c/p\u003e \u003cp\u003eThe plasma membrane integrity of the ZnO-K group can be explained by the stimulation of antioxidant enzymes (CAT, GPX, SOD), which play an important role in sperm quality maintenance (Soaudy et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, the bactericidal activity of CEO (Li et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) would also contribute to sperm membrane integrity. The current ZnO-K group\u003csup\u003e\u0026rsquo;\u003c/sup\u003es longer sperm tails, which were positively connected with greater sperm velocities, were consistent with those of \u003cem\u003eBarbus\u003c/em\u003e (Alavi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The correlation between sperm midpiece size, and mitochondrial sheath length, is critical for promoting sperm motility (Morita et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, zinc plays a role in optimizing sperm midpiece size and mitochondrial sheath length, and consequently, improving sperm motility (Arruda et al. 2021).\u003c/p\u003e \u003cp\u003eDNA integrity, a key sperm quality indicator, was highest in the ZnO-K group, likely due to Zinc\u0026rsquo;s role in stabilizing sperm chromatin and mitigating oxidative stress (Bj\u0026ouml;rndahl and Kvist \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The occurrence of fragmented DNA in the control group may refer to unidentified direct or indirect oxidative stresses on the fish spermatozoa. However, fragmented DNA has been recorded previously in sperms of a control group of \u003cem\u003eO. niloticus\u003c/em\u003e (Bombardelli et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the low antioxidant reserves in fish sperm, due to limited cytoplasmic content (Cosson \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), make them particularly susceptible to oxidative damage (Cabrita et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSperms of ZnO-K group recorded the highest levels of CAT, GPX, and SOD activities, demonstrating Zinc's capacity to enhance the sperm antioxidant enzymes. Zinc is a cofactor for numerous antioxidant enzymes, reducing oxidative damage and improving overall reproductive health (Fallah et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This study supported previous findings on the relationship between the activities of the enzymes, SOD, and GPX, and the prevention of oxidative stress-induced sperm damage in various fish species (Shaliutina-Kolešov\u0026aacute; et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, zinc supplementation has been found enhancing the synthesis of zinc-binding enzymes with antioxidant properties, thereby improving sperm quality (Cheng and Chen \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the current investigation, elevated enzyme activities were consistent with the enhanced sperm motility and DNA integrity observed in ZnO-K group, thus, validating other sperm quality parameters.\u003c/p\u003e \u003cp\u003eThis study added a knowledge about the relationship between the quality of tilapia sperm and the expression levels of the genes, heat shock \u003cem\u003eHSP70\u003c/em\u003e, and \u003cem\u003eCC chemokine\u003c/em\u003e, supporting the crucial role of ZnO-K and CEO, as antioxidant key players, in enhancing the expression of immune response genes in crayfish, \u003cem\u003eProcambarus clarkii\u003c/em\u003e (Kong et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and zebrafish, \u003cem\u003eDanio rerio\u003c/em\u003e, (Mahjoubian et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Also, current results expanded our knowledge about the positive impact of citrus essential oil on the expression of other genes, such as the cytokine gene \u003cem\u003eTNF-α\u003c/em\u003e, the antioxidant gene \u003cem\u003eSOD\u003c/em\u003e, and the somatotropic axis growth-mediation gene \u003cem\u003eIGF-1\u003c/em\u003e, in several organs of adult Nile tilapia (Mohamed et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, limited knowledge is known about the mechanisms of the effect of PSO on the expression of \u003cem\u003eHSP70\u003c/em\u003e and \u003cem\u003eCC chemokine\u003c/em\u003e in fish sperm and further studies should be done to clarify the suppression of these gene expressions by current PSO supplementation.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study highlighted the potential role of ZnO-K, as a dietary supplement, in enhancing \u003cem\u003eO. niloticus\u003c/em\u003e sperm quality, with different levels. This improvement is attributed to the multifaceted role of this antioxidant in promoting sperm motility, velocity, preserving DNA integrity, enhancing antioxidant enzymatic defenses, and upregulating immunity-related gene expressions. Additionally, CEO demonstrated a positive impact on sperm parameters, including concentration, velocity, viability, DNA integrity, and the higher expression of \u003cem\u003eHSP70\u003c/em\u003e and \u003cem\u003eCC chemokine\u003c/em\u003e. In contrast, PSO, exhibited the least efficacy, suggesting the need for further optimization or its combination with other supplements to maximize its potential benefits in improving sperm quality.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Declaration\u003c/h2\u003e \u003cp\u003e No funding\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMarwa M. Ali Contribution: Conceptualization, Investigation, methodology, Data analyses, writingKamal Fathy ElborayContribution: sampling, Investigation, methodology, writing, editingEngy T. MegahedContribution: methodology, data analyses, writing Hany T. Abu-TalebContribution: sampling, Project administration, VisualizationAlshimaa E. ElsayedContribution: methods, data analysesEman Y. MohammadyContribution: fish rearing, feedingMona S. AmerContribution: methods, data analysesSoliman A. MorsiContribution: sampling, methodsEman M. AbbasContribution: editing, reviewMohamed S. HassaanEditing reviewHosam Elsaiedgroup leader corresponding author\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eAll members of the Blue Gene Bank of the National Institute of Oceanography and Fisheries, NIOF, introduced their deep gratitude to the Academy of Scientific Research and Technology, ASRT, Egypt, for supporting the current research under the call \u0026ldquo;blue economy\u0026rdquo;. We thank all staff at the Inland Water and Aquaculture Research Center, NIOF, Al Qanatir AL Khayriyyah, Egypt, for technical help and support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbascal FJ, Cosson J, Fauvel C (2007) Characterization of sperm motility in sea bass: The effect of heavy metals and physicochemical variables on sperm motility. Journal of Fish Biology, 70: 509\u0026ndash;522. \u003c/li\u003e\n\u003cli\u003eAgarwal A, Durairajanayagam D, Halabi J, et al (2014) Proteomics, oxidative stress and male infertility. Reprod Biomed Online 29:32\u0026ndash;58\u003c/li\u003e\n\u003cli\u003eAghaie S, Nikzad H, Mahabadi JA, et al (2016) Protective effect of combined pumpkin seed and ginger extracts on sperm characteristics, biochemical parameters and epididymal histology in adult male rats treated with cyclophosphamide. 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Iran J Fish Sci 20:846\u0026ndash;862\u003c/li\u003e\n\u003cli\u003eElinge CM, Muhammad A, Atiku FA, et al (2012) Proximate, mineral and anti-nutrient composition of pumpkin (\u003cem\u003eCucurbita pepo\u003c/em\u003e L) seeds extract. International Journal of plant research 2:146\u0026ndash;150\u003c/li\u003e\n\u003cli\u003eElsaied HE, Soliman T, Abu-Taleb HT, et al (2019) Phylogenetic characterization of eukaryotic and prokaryotic gut flora of Nile tilapia, \u003cem\u003eOreochromis niloticus\u003c/em\u003e, along niches of Lake Nasser, Egypt, based on rRNA gene high-throughput sequences. Ecol Genet Genom 11:100037\u003c/li\u003e\n\u003cli\u003eFallah A, Mohammad-Hasani A, Colagar AH (2018) Zinc is an essential element for male fertility: a review of Zn roles in men\u0026rsquo;s health, germination, sperm quality, and fertilization. J Reprod Infertil 19:69\u003c/li\u003e\n\u003cli\u003eFerraz M de AMM, Carothers A, Dahal R, et al (2019) Oviductal extracellular vesicles interact with the spermatozoon\u0026rsquo;s head and mid-piece and improves its motility and fertilizing ability in the domestic cat. Sci Rep 9:9484. https://doi.org/10.1038/s41598-019-45857-x\u003c/li\u003e\n\u003cli\u003eGallego V, Asturiano JF (2019) Fish sperm motility assessment as a tool for aquaculture research: a historical approach. Rev Aquac 11:697\u0026ndash;724\u003c/li\u003e\n\u003cli\u003eGammanpila M, Yakupitiyage A, Bart AN (2007) Evaluation of the effects of dietary vitamin C, E and Zinc supplementation on reproductive performance of Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e). Sri Lanka Journal of Aquatic Sciences 12:1\u0026ndash;14\u003c/li\u003e\n\u003cli\u003eGennotte V, Franĉois E, Rougeot C, et al (2012) Sperm quality analysis in XX, XY and YY males of the Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e). Theriogenology 78:210\u0026ndash;217\u003c/li\u003e\n\u003cli\u003eGos\u0026aacute;lvez J, L\u0026oacute;pez-Fern\u0026aacute;ndez C, Hermoso A, Fern\u0026aacute;ndez JL, Kjelland M E (2014) Sperm DNA fragmentation in zebrafish (Danio rerio) and its impact on fertility and embryo viability: Implications for fisheries and aquaculture. Aquaculture, 433: 173-182.\u0026rlm;\u003c/li\u003e\n\u003cli\u003eGreiling AM, Schwarz C, Gierus M, Rodehutscord M (2018) Pumpkin seed cake as a fishmeal substitute in fish nutrition: effects on growth performance, morphological traits and fillet color of two freshwater salmonids and two catfish species. Archives of Animal Nutrition, 72: 239\u0026ndash;259. \u003c/li\u003e\n\u003cli\u003eHassan AM, El Nahas AF, Mahmoud S, et al (2017) Thermal stress of ambient temperature modulate expression of stress and immune-related genes and DNA fragmentation in Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e (Linnaeus, 1758). Appl Ecol Environ Res 15:\u003c/li\u003e\n\u003cli\u003eHassona NN, Zayed MM, Eltras WF, Mohamed RA (2020) Dietary supplementation of Tribulus terrestris extract improves growth and reproductive performances of the male Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e). Aquac Res 51:4245\u0026ndash;4254\u003c/li\u003e\n\u003cli\u003eHeidary M, Pahlevan M (2014) TRIzol-based RNA Extraction: A Reliable Method for Gene Expression Studies. Journal of Sciences, Islamic Republic of Iran 25:13\u0026ndash;17\u003c/li\u003e\n\u003cli\u003eHrenovic J, Milenkovic J, Daneu N, et al (2012) Antimicrobial activity of metal oxide nanoparticles supported onto natural clinoptilolite. Chemosphere 88:1103\u0026ndash;1107\u003c/li\u003e\n\u003cli\u003eHuang L, Yao G, Huang G, et al (2021) Association of Zinc deficiency, oxidative stress and increased double-stranded DNA breaks in globozoospermic infertile patients and its implication for the assisted reproductive technique. Transl Androl Urol 10:1088\u003c/li\u003e\n\u003cli\u003eJeuthe H, Palaiokostas C, Johannisson A (2022) DNA fragmentation and membrane integrity in sperm of farmed Arctic charr (Salvelinus alpinus). Aquaculture 547:737537\u003c/li\u003e\n\u003cli\u003eKesbi\u0026ccedil; OS, Acar \u0026Uuml;, Yilmaz S, Aydin \u0026Ouml;D (2020) Effects of bergamot (\u003cem\u003eCitrus bergamia\u003c/em\u003e) peel oil-supplemented diets on growth performance, hematology and serum biochemical parameters of Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e). Fish Physiol Biochem 46:103\u0026ndash;110\u003c/li\u003e\n\u003cli\u003eKime DE, Van Look KJW, McAllister BG, et al (2001) Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish. Comparative Biochemistry and Physiology Part C: Toxicology \u0026amp; Pharmacology 130:425\u0026ndash;433\u003c/li\u003e\n\u003cli\u003eKledmanee K, Taweedet S, Thaijongruk P, et al (2013) Effect of L-cysteine on chilled carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e) semen qualities. The Thai Journal of Veterinary Medicine 43:91\u0026ndash;97\u003c/li\u003e\n\u003cli\u003eKong M, Zhao W, Wang C, et al (2024) A Well-Established Gut Microbiota Enhances the Efficiency of Nutrient Metabolism and Improves the Growth Performance of \u003cem\u003eTrachinotus ovatus\u003c/em\u003e. Int J Mol Sci 25:5525\u003c/li\u003e\n\u003cli\u003eLi Z, Cai M, Liu Y, Sun P (2018) Development of finger citron (\u003cem\u003eCitrus medica\u003c/em\u003e L. var. sarcodactylis) essential oil loaded nanoemulsion and its antimicrobial activity. Food Control 94:317\u0026ndash;323\u003c/li\u003e\n\u003cli\u003eLiu K, Hao X, Wang Q, et al (2019) Genome-wide identification and characterization of heat shock protein family 70 provides insight into its divergent functions on immune response and development of \u003cem\u003eParalichthys olivaceus\u003c/em\u003e. PeerJ 7:e7781\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-\u0026Delta;\u0026Delta;CT method. Methods 25:402\u0026ndash;408. https://doi.org/10.1006/meth.2001.1262\u003c/li\u003e\n\u003cli\u003eMahanty A, Mohanty S, Mohanty BP (2017) Dietary supplementation of curcumin augments heat stress tolerance through upregulation of nrf-2-mediated antioxidative enzymes and hsps in \u003cem\u003ePuntius sophore\u003c/em\u003e. Fish Physiol Biochem 43:1131\u0026ndash;1141\u003c/li\u003e\n\u003cli\u003eMahjoubian M, Naeemi AS, Moradi-Shoeili Z, et al (2023) Oxidative stress, genotoxic effects, and other damages caused by chronic exposure to silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs), and their mixtures in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). Toxicol Appl Pharmacol 472:116569\u003c/li\u003e\n\u003cli\u003eMohamed RA, Yousef YM, El‐Tras WF, Khalafallaa MM (2021) Dietary essential oil extract from sweet orange (\u003cem\u003eCitrus sinensis\u003c/em\u003e) and bitter lemon (\u003cem\u003eCitrus limon\u003c/em\u003e) peels improved Nile tilapia performance and health status. Aquac Res 52:1463\u0026ndash;1479\u003c/li\u003e\n\u003cli\u003eMohammady EY, Soaudy MR, Abdel-Rahman A, et al (2021) Comparative effects of dietary zinc forms on performance, immunity, and oxidative stress-related gene expression in Nile tilapia, \u003cem\u003eOreochromis niloticus\u003c/em\u003e. Aquaculture 532, https://doi.org/10.1016/j.aquaculture.2020.736006\u003c/li\u003e\n\u003cli\u003eMorita M, Takemura A, Okuno M (2004) Acclimation of sperm motility apparatus in seawater-acclimated euryhaline tilapia \u003cem\u003eOreochromis mossambicus\u003c/em\u003e. Journal of Experimental Biology 207:337\u0026ndash;345. https://doi.org/10.1242/jeb.00748\u003c/li\u003e\n\u003cli\u003eMusa N (2010) Sperm activation in Nile tilapia \u003cem\u003eOreochromis niloticus\u003c/em\u003e and the effects of environmentally relevant pollutants on sperm fitness. Aquaculture eTheses http://hdl.handle.net/1893/2310\u003c/li\u003e\n\u003cli\u003eNakharuthai C, Areechon N, Srisapoome P (2016) Molecular characterization, functional analysis, and defense mechanisms of two CC chemokines in Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) in response to severely pathogenic bacteria. Dev Comp Immunol 59:207\u0026ndash;228\u003c/li\u003e\n\u003cli\u003eNayyef han, a. S. Aliama (2023) A study of the changes in phytate concentration and some parameters in men with oligospermia and azoospermia. History of Medicine 9:1009-1016.\u0026rlm;\u003c/li\u003e\n\u003cli\u003ePalomino DCT, Marti LC (2015) Chemokines and immunity. Einstein (s\u0026atilde;o paulo) 13:469\u0026ndash;473\u003c/li\u003e\n\u003cli\u003ePaulino MS, Veras GC, Felizardo VO, et al (2016) Assessment of gametes in tilapia \u003cem\u003eOreochromis niloticus\u003c/em\u003e: Effects of body weight in a New Lineage. Journal of Fisheries Sciences com 10:63\u003c/li\u003e\n\u003cli\u003ePerumal P (2014) Effect of superoxide dismutase on semen parameters and antioxidant enzyme activities of liquid stored (5 C) Mithun (Bos frontalis) semen. Journal of Animals 2014:821954\u003c/li\u003e\n\u003cli\u003eRahayu VG, Hanizar E (2021) The effect of lemon (\u003cem\u003eCitrus limon\u003c/em\u003e) extracts on the quantity and quality of mice (\u003cem\u003eMus musculus\u003c/em\u003e) sperm. J Islam Sci and Tech 7:300\u0026ndash;316\u003c/li\u003e\n\u003cli\u003eRurangwa E, Volckaert FAM, Huyskens G, et al (2001) Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e). Theriogenology 55:751\u0026ndash;769\u003c/li\u003e\n\u003cli\u003eSarmento N, Martins EFF, Costa DC, et al (2017) Effects of supplemental dietary vitamin C on quality of semen from Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) breeders. Reproduction in Domestic Animals 52:144\u0026ndash;152\u003c/li\u003e\n\u003cli\u003eShaliutina-Kole\u0026scaron;ov\u0026aacute; A, Rui N, Ashtiani S, et al (2018) Oxidative stress and antioxidant enzyme defense system in seminal plasma of common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e) and rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) during spawning season. Czech Journal of Animal Science 63:\u003c/li\u003e\n\u003cli\u003eSoaudy MR, Mohammady EY, Ali MM, et al (2021) Potential effects of dietary ZnO supported on kaolinite (ZnO-K) to improve biological parameters, reproduction indices, lipid profile and antioxidant enzymes activities for broodstock of Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e). Anim Feed Sci Technol 281:115117\u003c/li\u003e\n\u003cli\u003eSolanki GB, Singh VK, Kavani FS, et al (2023) Seasonal influence on expression of heat shock proteins (HSP70 and HSP90) vis-\u0026agrave;-vis functional competence of Gir bull semen. Anim Biotechnol 34:3739\u0026ndash;3748\u003c/li\u003e\n\u003cli\u003eSu Y, Wu Y, Ye M, et al (2024) Star1 gene mutation reveals the essentiality of 11-ketotestosterone and glucocorticoids for male fertility in Nile Tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e). Comp Biochem Physiol B Biochem Mol Biol 273:110985\u003c/li\u003e\n\u003cli\u003eTanomman S, Ketudat-Cairns M, Jangprai A, Boonanuntanasarn S (2013) Characterization of fatty acid delta-6 desaturase gene in Nile tilapia and heterogenous expression in Saccharomyces cerevisiae. Comp Biochem Physiol B Biochem Mol Biol 166:148\u0026ndash;156\u003c/li\u003e\n\u003cli\u003eVilela DAR, Silva SGB, Peixoto MTD, et al (2003) Spermatogenesis in teleost: insights from the Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) model. Fish Physiol Biochem 28:187\u0026ndash;190\u003c/li\u003e\n\u003cli\u003eWickham H, Grolemund G (2023) R for Data Science, 2nd edn. O\u0026rsquo;Reilly Media, Sebastopol, CA, USA\u003c/li\u003e\n\u003cli\u003eYamaguchi S, Miura C, Kikuchi K, et al (2009) Zinc is an essential trace element for spermatogenesis. Proceedings of the National Academy of Sciences 106:10859\u0026ndash;10864\u003c/li\u003e\n\u003cli\u003eZhang C-N, Li X-F, Tian H-Y, et al (2015) Effects of fructooligosaccharide on immune response, antioxidant capability and HSP70 and HSP90 expressions of blunt snout bream (\u003cem\u003eMegalobrama amblycephala\u003c/em\u003e) under high ammonia stress. Fish Physiol Biochem 41:203\u0026ndash;217\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Ingredients and proximate composition of the basal diet (g/kg diet).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"257\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eIngredients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003eBasal diet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eFish meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eSoybean meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e540\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eYellow corn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eWheat bran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eSoybean oil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eZinc-free premix\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003e\u003cem\u003eProximate analysis (%)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eDry matter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e89.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eCrude protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e350\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eCrude lipid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e72.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eAsh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e49.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eFiber content\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e533\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eNFE\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e529.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.9535%;\"\u003e\n \u003cp\u003eGE\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(kJ/g dry matter)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0465%;\"\u003e\n \u003cp\u003e20.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Vitamin and mineral mix (mg or g / Kg diet): MnSO4, 40 mg; MgO, 10 mg; K2SO4, 40 mg; KI, 0.4 mg; CuSO4, 12 mg; Ferric citrate, 250 mg; Na2SeO3, 0.24 mg; Co, 0.2 mg; retinol, 40000 IU; cholecalciferol, 4000 IU; \u0026alpha;-tocopherolacetate, 400 mg; menadione, 12 mg; thiamine, 30 mg; riboflavin, 40 mg; pyridoxine, 30 mg; cyanocobalamin, 80 mcg;;nicotinic acid, 300 mg; folic acid, 10 mg; biotin, 3 mg; pantothenic acid, 100 mg; inositol, 500 mg; ascorbic acid, 500 mg. 2\u003cem\u003eB. acidophullus\u0026nbsp;\u003c/em\u003ewas prepared to obtain (1.47 \u0026times; 107 CFU kg- 1 approximately. \u003csup\u003eb\u003c/sup\u003e NFE (Nitrogen free extract) = 100- (crude protein + lipid + ash +fibre content). \u003csup\u003ec\u003c/sup\u003e Gross energy, calculated using gross calorific values of 23.63, 39.52 and 17.15 Kj g\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e for protein, fat and carbohydrate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (2)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMilt volume, pH,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esperm concentration, motility, and velocity parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eItems\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eControl group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eZnO-K group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCEO group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePSO group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVolume (ml/male)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.20\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 0.052\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.40\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 0.10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.90\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 0.054\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.80\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 0.026\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e6.700\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.026\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e7.125\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.025\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e7.000\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.022\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e7.00\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0850\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSperm concentration (sperms ml\u003csup\u003e-1\u003c/sup\u003e milt)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5.285x10\u003csup\u003e9\u003c/sup\u003e \u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 1.133\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5.676x10\u003csup\u003e9 a\u003c/sup\u003e \u0026plusmn; 0.879\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5.540x10\u003csup\u003e9 a\u003c/sup\u003e \u0026plusmn; 1.325\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e3.844x10\u003csup\u003e9 a\u003c/sup\u003e \u0026plusmn; 0.742\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.587\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% Motility\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e48.0\u003c/strong\u003e\u003cstrong\u003e3\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;7.26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e66.2\u003c/strong\u003e\u003cstrong\u003e1\u003csup\u003ea\u003c/sup\u003e\u0026plusmn; 6.09\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e42.36\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;4.68\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e38.99\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;3.07\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0077\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eVelocity (\u0026micro;m sec\u003csup\u003e-1\u003c/sup\u003e)\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVCL\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e24.49\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 0.677\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e43.95\u003csup\u003ea\u003c/sup\u003e\u0026plusmn; 0.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e28.07\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 0.751\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e25.03\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 0.327\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0150\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVSL\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e21.72\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 1.313\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e32.19\u003csup\u003ea\u003c/sup\u003e\u0026plusmn; 1.90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e23.35\u003csup\u003eab\u003c/sup\u003e \u0026plusmn; 1.841\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e20.88\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 0.838\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0490\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVAP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e20.94\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 0.899\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e31.36\u003csup\u003ea\u003c/sup\u003e\u0026plusmn; 1.036\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e20.19\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 0.498\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e18.48\u003csup\u003ec\u003c/sup\u003e\u0026plusmn; 0.318\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0217\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues in the same row with different superscripts are significantly different (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (3) Sperm vitality, DNA integrity, and morphometric measurements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eItems\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eControl group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eZnO-K group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCEO group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePSO group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% Live sperms\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e49.97\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; 0.74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;65.29\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.55\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e70.78\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 4.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e41.14\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 1.32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% DNA integrity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; 28.08\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 6.881\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; 60.08\u003csup\u003ea\u003c/sup\u003e\u0026plusmn; 1.714\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e54.23\u003csup\u003ea\u003c/sup\u003e\u0026plusmn; 6.729\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e34.88\u003csup\u003eb\u003c/sup\u003e\u0026plusmn; 7.593\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0040\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eMorphological measurements\u0026nbsp;\u003c/u\u003e\u003c/strong\u003e\u003cstrong\u003e(\u0026micro;m)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 555px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHead length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; 2.82\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.09\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;2.56\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2.41\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e3.08\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0795\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHead width\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2.15\u003csup\u003eab\u003c/sup\u003e \u0026plusmn;0.15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;1.80\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.84\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2.56\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0300\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMidpiece width\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.57\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.040\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;0.66\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.59\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.51\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.06\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.2520\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTail length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;18.83\u003csup\u003ea\u003c/sup\u003e\u0026plusmn; 0.46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e19.66\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.57\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e17.87\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.65\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e14.92\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.52\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues in the same row with different superscripts are significantly different (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4 \u0026nbsp;Enzymatic activities in sperms of \u003cem\u003eO. niloticus\u003c/em\u003e males\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"688\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eItems\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZnO-K group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCEO group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePSO group \u003cu\u003e+\u003c/u\u003e SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAT (U/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.67\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 2.603\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e47.33\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.452\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.00\u003csup\u003ebc\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.527\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e24.00\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 2.886\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0004\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGPX (mU/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e48.00\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 4.582\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e65.67\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 5.547\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e41.67\u003csup\u003ebc\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.763\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.00\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 2.081\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0017\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSOD (U/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e40.00\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 4.041\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e60.67\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 3.382\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e43.00\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 3.511\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e36.67\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 3.179\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0058\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues in the same row with different superscripts are significantly different (P\u0026lt;0.05). CAT: Catalase (U/ml) as unit per milliliter;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGPX: Glutathione peroxidase mU/ml) as milliunit per milliliter; SOD: Superoxide dismutase (U/ml) as unit per milliliter.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sperm quality, O. niloticus, antioxidant supplements, CASA parameters, DNA integrity, enzyme bioassay, gene expression","lastPublishedDoi":"10.21203/rs.3.rs-6146222/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6146222/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSperm quality improvement is crucial to achieving the reproductive efficiency of \u003cem\u003eOreochromis niloticus\u003c/em\u003e. This study examined the effect of three dietary antioxidant supplements, kaolinite-doped zinc oxide (ZnO-K), citrus essential oil (CEO), and pumpkin seed oil (PSO), on sperm quality. Integrated sperm examination tools, including Computer Assisted Semen Analysis (CASA) parameters, spermatozoa DNA integrity, antioxidant enzyme bioassays, and gene expressions, were applied to validate sperm quality. One hundred and ninety-two adult males (mean weight 421.31\u0026thinsp;\u0026plusmn;\u0026thinsp;6.26 g) were divided into four groups, each with three replicates. The first control group was fed on a diet without supplements. The second group was fed on ZnO- K-containing diet (0.06 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); the third group was fed on a CEO-containing diet (10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); and the fourth group was fed on a PSO-containing diet (15 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). ZnO-K supplementation significantly elevated milt volume (1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 ml) and sperm concentration (5.676 x 10\u003csup\u003e9\u003c/sup\u003e sperm ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), as well as enhancing CASA parameters, including sperm motility, velocities, and DNA integrity. An increase in antioxidant activities of the enzymes, catalase, CAT, glutathione peroxidase, GPX, and superoxide dismutase, SOD, were observed in the ZnO-K-feeding group, recording 47.333\u0026thinsp;\u0026plusmn;\u0026thinsp;1.452 U ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e milt, 65.667\u0026thinsp;\u0026plusmn;\u0026thinsp;5.547 mU ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e milt and 60.667\u0026thinsp;\u0026plusmn;\u0026thinsp;3.382 U ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e milt, respectively. Notably, upregulation of the expressed genes, \u003cem\u003eHSP70\u003c/em\u003e, and \u003cem\u003eCC chemokines\u003c/em\u003e was recorded in sperms from ZnO-K- and CEO-feeding groups, compared with gene expression suppression in the PSO-feeding group. All these findings suggest that ZnO-K and CEO are efficient in enhancing the quality of \u003cem\u003eO. niloticus\u003c/em\u003e sperm, with the most pronounced effects shown by ZnO-K.\u003c/p\u003e","manuscriptTitle":"Ameliorative potential of dietary supplements, ZnO-K, citrus essential oil, and pumpkin seed oil, on sperm quality in Nile tilapia: Insights from CASA, DNA integrity, antioxidant enzymes, and gene expressions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 11:18:09","doi":"10.21203/rs.3.rs-6146222/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"327115131327773616439657578135055257219","date":"2025-04-22T04:22:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310213032725110427820046788878068783992","date":"2025-04-22T03:50:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T05:12:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49911826996397526166713290021359427774","date":"2025-04-04T03:47:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-04T03:34:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-03T13:02:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2025-03-25T07:28:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"593ed263-848c-4f6c-bac1-1e5e2f70ed2c","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:08:03+00:00","versionOfRecord":{"articleIdentity":"rs-6146222","link":"https://doi.org/10.1007/s10695-025-01529-4","journal":{"identity":"fish-physiology-and-biochemistry","isVorOnly":false,"title":"Fish Physiology and Biochemistry"},"publishedOn":"2025-06-23 16:05:45","publishedOnDateReadable":"June 23rd, 2025"},"versionCreatedAt":"2025-04-29 11:18:09","video":"","vorDoi":"10.1007/s10695-025-01529-4","vorDoiUrl":"https://doi.org/10.1007/s10695-025-01529-4","workflowStages":[]},"version":"v1","identity":"rs-6146222","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6146222","identity":"rs-6146222","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}