To our knowledge, no study had previously investigated the impacts of CaDF and KDF on growth parameters in this species. In our study, feeding of fingerling Huso huso with diets containing 0.2 g/Kg KDF and 0.15 g/Kg CADF for 60 days improved the growth parameters. Our results are in accordance with those of Hassaan et al. (2021) who observed the highest growth performance in Nile tilapia (Oreochromis niloticus) fed with 10 g kg− 1 KDF. Various studies show that the use of organic acids due to improving the metabolism and digestibility of proteins and minerals in the intestine, improves growth and nutrition beside enhanced appetite and changed the composition, diversity, and/or activity of the population of beneficial bacteria in the gut microbiota while inhibiting pathogenic bacteria in aquatic species (Sarker, 2005; Sarker et al., 2012; Hoseinifar et al., 2016; Ng and Koh, 2017; Wassef et al., 2019; Pelusio et al., 2020).
According to the results, fish fed with both of KDF and CaDF for 60 days had significantly higher activities of intestinal trypsin, α-amylase, lipase, and ALP. Accordingly, one of the primary causes of the KDF and CaDF's ability to stimulate development was the elevation of the activity of digestive enzymes. The breakdown of macromolecules and subsequent nutrient absorption in the gastrointestinal lumen are reportedly facilitated by increased digestive enzyme activity (Jang et al., 2019). Similar results regarding digestive enzymes were seen in hybrid tilapia fed potassium diformate (KDF) (Zhou et al., 2009), Oncorhynchus mykiss fed Dietary PrimaLac® and potassium diformate (KDF) (Farsani et al., 2019), Lateolabrax japonicus fed citric, lactic, and phosphoric acids (Huang et al., 2021), and Litopenaeus (Romano et al., 2015). Trypsin, a proteolysis enzyme, contributes to fish development and feed intake, but chymotrypsin is active when food availability or supply is constrained (Rungruangsak-Torrissen et al., 2006). Additionally, increased enterocyte nutritional absorption in fish is indicated by increased intestinal ALP activity, which is critical for both carbohydrate and lipid absorption (Calhau et al., 2000; Gawlicka et al., 2000).
Our findings showed that the 0.2 of KDF and CaDF have beneficial effects on the serum albumin level of Huso huso. Several studies demonstrated that different organic acids simultaneously increased serum albumin values in different fish species (Yesilbag and Colpan, 2006; Natsir et al., 2017; Hassaan et al., 2017; Hassaan et al., 2021). As an organism's response to internal and external circumstances, blood serum protein is a highly responsive biochemical system. An increase in serum albumin concentration in fish indicates an improved level of innate immunity that may also be linked to improved function of organs involved in protein production, including the liver (Tothova et al., 2016). Hence, in the current study, the increased serum albumin values might be attributed to the elevated values of protein synthesis in liver tissue of fish fed with KDF and CaDF incorporated diets.
Cortisol and glucose are serum stress indicators (Hajirezaee et al., 2019; Mohammadi et al., 2020). In response to stressful situations, internal tissue releases cortisol into the blood. To meet the energy demands of cells, glucose is produced through glycogenolysis (breakdown of glycogen into glucose) or gluconeogenesis (breakdown of proteins into glucose) in the liver (Sheridan, 1986; Hontela et al., 1993; Zhang et al., 2015). Therefore, the lower levels of these in H. huso fed with KDF and CaDF probably indicate higher resistance against common stresses in laboratory conditions. Contrary to our findings, Yılmaz and Ergün (2018) observed no difference between the serum cortisol and glucose levels in Oncorhynchus mykiss fed with diets containing Trans-cinnamic acid. The reason for this difference may be related to the difference in the type of organic acid, species and the dose used in different studies. Studies have shown that acidic anions facilitate the absorption of cations from minerals such as calcium (Edwards & Baker, 1999). In our study, serum calcium and sodium were affected significantly by adding CaDF and KDF which possibly was a result of better mineral absorption and usage by enteric epithelial cells (Roediger 1980). Similar to our results, Hassaan et al. (2021) observed that the level of serum calcium was increased in Nile tilapia (Oreochromis niloticus) fed a diet containing either 5 g or 10 g kg− 1 KDF. Overall, our findings showed that the application of KDF and CaDF improves the H. huso hematopoietic system, and they are safe as feed supplements.
The positive impact of organic acids on the health status of liver cells in H. huso, as indicated by the significant reductions in AST and LDH levels in fish fed with supplemented KDF and CaDF foods. This suggests that acidified diets may have potential benefits for improving fish health, such as enhancing intestinal balance and digestibility, reducing toxins, and increasing their immune status (Kesbiç, 2018; Naderi Farsani et al., 2019).. These findings are consistent with previous studies conducted on Nile tilapia (Meshrf, 2014; Hassaan et al., 2016; Soltan et al. 2017) and rainbow trout (Naderi Farsani et al., 2019), and other fish species, where acidified diets were found to improve fish health and performance. It is important to note, however, that the effects of acidified diets on fish health may vary depending on the specific fish species, the composition and dose of the organic acids used, and other environmental factors. These findings highlight the potential benefits of acidified diets in aquaculture operations, particularly in terms of improving fish health and reducing the risk of disease outbreaks.
As biomarkers of oxidative stress, compounds and substances such as CAT, SOD, GSH, and MDA (As an indicator of lipid peroxidation) serve as indicators of damage (Birnie-Gauvin et al., 2017). Acidifiers improve the state of oxidative stress in organisms and reduce the risk of infection in organisms by increasing anti-oxidative and antimicrobial activity (Fridovich, 1995; Chelikani et al., 2004; Molayemraftar et al., 2022). With the increased probability of the presence of lactic acid bacteria in the digestive tract following the use of acidifiers, the stress indicators in the blood, decrease. This may be due to the anti-stress effects of the beneficial bacteria present in the digestive tract of fish fed with the diet. The food contains acidifier, which improves the antioxidant defense mechanism and reduces stress indicators in the blood (Lushchak, 2016). The present results showed that supplemented KDF and CaDF diets stimulated serum SOD and especially CAT activities which are two pioneer antioxidant enzymes. Eventually, these cumulatively suppress lipid peroxidation and increase the health index. Similar to our results, Hassaan et al. (2021) observed that the activities of SOD and CAT were highest in Nile tilapia (Oreochromis niloticus) fed diet contained either 5 g or 10 g kg− 1 KDF. Also, Nascimento et al. (2021) showed that Citric acid minimizes oxidative stress in Amazonian fish (Colossoma macropomum) when fed plant protein-based diets. Moreover, Huang et al. (2021) showed that an acidifier blend (citric, lactic, and phosphoric acids) reduces MDA levels in juvenile Japanese sea bass (Lateolabrax japonicus). Hence, the results of this study revealed up-regulation of SOD, CAT, and MDA in fish fed with KDF and CaDF, showing that KDF and CaDF reduced cell damage. Moreover, these results indicated that treatment with the acidifiers (KDF and CaDF) leads to a reduction in lipid peroxidation, which leads to the induction of the secretion of antioxidant enzymes and the elimination of free radicals and eventually improved the oxidative stability responses in Huso huso. However, these studies show that organic acids and their salts can protect the fish body from cellular oxidative damage. Finding out the various details of the effect of organic acids on antioxidant defense responses will be the subject of future studies.
It is interesting to note the potential benefits of organic acids in altering the intestinal environment to prevent the growth of harmful bacteria. The findings of Castillo et al. (2014) suggest that KDF and CaDF may induce low pH in the intestine, which can inhibit the proliferation of pathogenic microorganisms. Similarly, Reyshari et al. (2019) observed increased lactic acid bacterial counts in Asian sea bass fed diets supplemented with NaDF, further supporting the idea that organic acids can have a positive impact on gut microbiota. However, it is also important to consider the findings of Dai et al. (2018), indicating that citric acid supplementation did not affect the gut microbiota of Scophthalmus maximus L. This suggests that the effects of organic acids on gut microbiota may vary depending on the specific type of acid and the species of fish being studied. Overall, these studies provide valuable insights into the potential benefits of organic acids for improving gut health in fish. Further research is necessary to fully understand the mechanisms underlying their effects and to explore their applications in aquaculture.