Impacts of Crowding Stress on Aquatic Animals and Its Mitigation Through Feed Additives Supplementation – A Review

Stocking density is critical in determining a successful aquaculture practice (Bi et al., 2023). A high stocking density may be profitable and cost-effective in maximising space utilisation in an aquatic environment, but this practice forces the farmed species to compete for basic necessities such as food, space, and dissolved oxygen (Bi et al., 2023). Earlier studies claimed that a high stocking density could reduce fish growth performance and immunity, thus diminishing the total aquaculture production (Dediu et al., 2021). Therefore, stocking density has been recognised as a major source of chronic stress, which entails proper management for good aquaculture practices.

Stocking density may lead to biological or crowding stress, directly impacting fish gene expression, welfare, and production (Dediu et al., 2021; Jia et al., 2016). Furthermore, crowding stress disturbs the normal physiological response required for internal home-ostasis maintenance (Bayunova et al., 2002; Long et al., 2019). Crowding stress also causes endocrine changes related to stress hormones, namely corticosteroids and catecholamines (Herrera et al., 2019). Endocrine alterations can influence an organism’s physiological aspects, such as haematological indices, antioxidant capacity, and immune system (Herrera et al., 2019), manifested as adverse effects on growth performance, disease resistance, behaviour, and survival (Barton, 2002) (see Figure 1).

Figure 1.

Impacts of crowding stress on aquatic animals

Crowding stress harms the growth performance of aquatic animals (see Table 1). Current research on the impact of crowding stress is limited to several aquatic species, such as sturgeon, Acipenser queldenstaedtii, Nile tilapia, Oreochromis niloticus, Senegalese sole, Solea senegalensis, Chinese sturgeon, Acipenser sinensis, hybrid sturgeon and minor carp, Labeo bata. The study outcomes indicated that high stocking density causes crowding stress, increased stress hormones, and glucose imbalance in aquatic animals, negatively impacting their growth performance. Nonetheless, there are several cases where crowding stress had no adverse effects on the farmed aquatic animals. These discrepancies warrant further investigation to substantiate the impact of crowding stress on the growth performance of other aquatic animal species, particularly crustaceans, due to the lack of literature.

At low stocking density, sturgeons (A. queldenstaedtii) demonstrated superior growth performance compared to their counterpart at high stocking density (Çelikkale et al., 2005). Despite the insignificant results, the low-stocking density group recorded a better final weight and survival rate than the high-density group. Likewise, studies on juvenile of Senegalese sole, Solea senegalensis (Andrade et al., 2015; Costas et al., 2013, 2008; Salas-Leiton et al., 2008), larvae and fingerling of lake sturgeon, A. fulvescens (Fajfer et al., 1999), Atlantic sturgeon, A. oxyrinchus (Mohler et al., 2000) reported similar findings. The insignificant results in growth performance between the low and high stocking density groups may be attributed to the adaptation of the experimental fish to the high stocking density during the acclimatisation period. Another possible explanation is the fish farmed at high stocking density were fed with higher feed ration to fulfil their dietary requirements, thus sustaining themselves even under non-optimal conditions as observed in the Senegalese sole (Salas-Leiton et al., 2008). Similarly, (Costast al., 2013) reported that Senegalese sole farms in high and low stocking exhibited comparable growth performance with the condition that the high stocking density fish are supplemented with a higher protein diet.

In some cases, high stocking density can promote the growth performance of aquatic animals, as reported in juvenile Siberian sturgeon, A. baerii. Despite that, high stocking density remains a risk for causing significantly (P<0.05) lower growth performance as observed in hybrid sturgeon farming (Bi et al., 2023; Dediu et al., 2021), Chinese sturgeon (Long et al., 2019), Atlantic cod (Lambert and Dutil, 2001), Nile tilapia (Gibtan et al., 2008), European seabass (Saillant et al., 2003) and rainbow trout (Ellis et al., 2002). In severe cases, crowding stress caused mortality in hybrid sturgeon (Bi et al., 2023), Nile tilapia, Oreochromis niloticus (Garcia et al., 2013), and minor carp, Labeo bata (Karnatak et al., 2021). Thus, researchers proposed the use of the biofloc system to ease crowding stress, which was proven effective in juvenile common carp farmed at high stocking density (Adineh et al., 2022; Yousefi et al., 2023 a).

Glucose is an essential nutrient for animals that provides energy in the form of adenosine triphosphate (ATP). Many studies utilised the blood glucose level to indicate chronic stress, particularly crowding stress (Iwama et al., 2015). High blood glucose levels have been linked to high stocking density (Long et al., 2019), but this is not the case for some studies as the species has acclimatised itself in a crowded environment (Dediu et al., 2021). Therefore, blood glucose levels may not be the most precise measurement of stress (Dediu et al., 2021).

Cortisol is an important hormone that regulates glucose production in aquatic animals via gluconeo-genesis and glycogenolysis pathways (Iwama et al., 2015). This stress hormone level can be used to reflect the stress experienced by an organism (Adineh et al., 2021 b; Fatima et al., 2017) and has been applied in earlier studies (Mommsen et al., 1999). An organism under stress will trigger the secretion of catecholamines hormone into the body (see Figure 2). Once present in blood, catecholamines stimulate the hypothalamic-pituitary-interrenal (HPI) axis to release cortisol into the circulatory system. Cortisol is responsible for mobilising energy reserves in the body by regulating and activating liver glycogenolysis and inhibiting glycolysis (Mazeaud et al., 1977). A high cortisol level will activate glycogenolysis and inhibit glycolysis, resulting in higher blood glucose production, which is common in aquatic animals under high stocking density (Dediu et al., 2021; Jia et al., 2016) and crowding stress (Ghafarifarsani et al., 2023; Hoseini et al., 2019 b; Hosseini and Hoseini, 2012). These adaptive responses provide extra energy for cells in their bodies to handle crowding (Barton, 2002), hence reducing the growth performance of aquatic animals in high stocking density environments.

Figure 2.

Mechanism of growth impairment of aquatic animals under crowding stress

Haematological indices are also good stress indicators (Jeney and Jeney, 2002) for aquatic animals under high stocking density, including total haemoglobin, total red blood cells, high mean corpuscular haemoglobin concentration, and low mean corpuscular volume (Dediu et al., 2021). The changes in haematological indices are adaptive responses for aquatic animals with high oxygen requirements and metabolic demands to maintain haemostasis (Dediu et al., 2021), particularly under crowding stress (Kleinert et al., 2019). Therefore, low-density aquatic animal farming remains ideal in aquaculture production, as Garcia et al. (2013) described in Nile tilapia, yielding superior production and uniform fish size. Nevertheless, several aquatic animals are adaptive to high-density farming, such as African catfish (Abdul Kari et al., 2021) and Senegalese sole (Andrade et al., 2015), with the condition that the farmed aquatic animals must be supplemented with high protein diet to mitigate crowding stress.

Aquatic animals farmed at high stocking density face the risk of immunosuppression and reduced antioxidant levels due to crowding stress (see Table 2), characterised by inhibiting metabolic and antioxidant enzyme activities. Despite evidence on the adverse effects of crowding stress on animal health and antioxidant status, several studies have reported no significant impacts on the farmed species. Therefore, further study is required to elucidate the effects of crowding stress on aquatic animals, particularly crustaceans.

Aquatic animals farmed in a high-density environment generally exhibit poorer immune function and a higher prevalence of disease infection (Aranguren et al., 2002; Mauri et al., 2011). For instance, Nile tilapia farmed in a high-density cage culture can exhibit higher disease infection and mortality (Garcia et al., 2013). High stocking density can also lead to immunosuppression, as observed in the common carp, Cyprinus carpio (Adineh et al., 2021 b; Hoseini et al., 2019 a), turbot, Scophthalmus maximus (Jia et al., 2016), grass carp, Ctenopharyngodon idella (Li et al., 2023; Lin et al., 2018), and gilthead seabream, Sparus aurata (Montero et al., 1999). In addition, crowding stress diminishes red and white blood cells, leading to immune system impairment in grass carp, Ctenopharyngodon idella (Lin et al., 2018). Nonetheless, immunosuppression did not occur in several studies on crowding stress, indicating the plasticity of the immune system that could be influenced by certain factors and metabolic changes (Al-Bakheit et al., 2016; Bi et al., 2023).

Crowding stress reportedly elevated serum biochemical parameters, such as glucose, cortisol, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (Ghafarifarsani et al., 2023). Likewise, crowding stress elevated cortisol and glucose levels in common carp, Cyprinus carpio (Ghafarifarsani et al., 2023; Hoseini et al., 2019 b; Hosseini and Hoseini, 2012) as an adaptive response and provided additional energy (Barton, 2002). A high cortisol level in fish leads to an impaired immune system (Tort, 2011). Crowding stress did not adversely impact the immune and antioxidant status of several aquatic animals, such as Senegalese sole, Solea senegalensis (Andrade et al., 2015). There were no significant differences (P>0.05) between the high and low stocking density groups, and the haematocrit results demonstrated blood parameters such as cortisol and glucose levels remained constant in both groups (Andrade et al., 2015). Similar cases were also recorded in other aquaculture species, including European seabass, Dicentrarchus labrax (Pascoli et al., 2011), gilthead seabream, Sparus aurata (Tort et al., 1998), and rainbow trout, Oncorhynchus mykiss (Morgan et al., 2008). These discrepancies suggest that aquatic animals are adaptive to a high stocking-density environment (Andrade et al., 2015).

Low lysozyme activity has been reported in aquatic animals under stressful environments with high stocking density or infected by disease, including Siberian sturgeon, Acipenser baerii (Chesneau, 2018) and Chinese sturgeon, Acipenser sinensis (Long et al., 2019) at high stocking density. Thus, lysozyme activity is a suitable indicator of the health status of aquatic animals. Moreover, a high stocking density could inhibit and block metabolic and antioxidant enzyme activities, inducing oxidative stress (Gao et al., 2017; Junqueira et al., 2004) and physiological imbalances in aquatic animals (Aksakal et al., 2011). Despite that, several studies discovered no significant (P>0.05) difference in the antioxidant capacity of tongue sole, Cynoglossus semilaevis (Liu et al., 2016), largemouth bass, Micropterus salmonides, and Senegalese sole, Solea senegalensis (Andrade et al., 2015) at different stocking densities, suggesting the adaptability of farmed fish under high stocking density.

High stocking density also reportedly triggers oxidative stress and induces lipid peroxidation activity. For instance, antioxidant capacity induced lipid peroxidation and reduced superoxide dismutase (SOD) and glutathione (GSH-PX) activities in the serum of hybrid sturgeon farmed under high stocking density (Bi et al., 2023; Liu et al., 2014; Long et al., 2019; Yousefi et al., 2020). Lipid peroxidation produces malondialdehyde (MDA), a metabolite that can be measured in blood serum to indicate oxidative stress level. In addition, downregulation of skin immune-related genes, such as interleukin-1β (IL-1β), insulin-like growth factor – (IGF-), and lysozyme (LZM), were observed in juvenile turbot, Scophthalmus maximus under high stocking density (Jia et al., 2016). The study also found that high stocking density inhibits mucus enzyme activities, induces oxidative stress in the skin, and decreases lysozyme activity in juvenile turbot (Jia et al., 2016).

Heat shock protein (Hsp) expression parallels stress exposure; hence, it is a good stress indicator for aquatic animals (Sanders, 1993). The Hsps are classified based on molecular weight: small Hsps (< 40kDa), Hsp60, Hsp70, Hsp90, and Hsp100. These proteins are produced as a protective mechanism against protein denaturation in the events of hyperthermia, cytotoxicity from pesticides, heavy metals, ethanol, and hypoxia (Dorts et al., 2009; Lee and Corry, 1998; Lewis et al., 1999; Lindquist and Craig, 1988), and crowding stress (Aksakal et al., 2011).

Various plant extracts can be used as phytobiotics to mitigate crowding stress in aquaculture (see Table 3). Phytobiotics can be utilised as a feed additive between 40 and 112 days to enhance the immune system and antioxidant capacity of the aquatic species. Furthermore, phytobiotics could improve blood parameters, immune system, antioxidant capacity, and disease resistance and mitigate crowding stress in aquaculture. Nevertheless, studies in this area are still in their infancy and require further investigation to explore the potential of phytobiotics in aquaculture.

Earlier studies have reported that oak, Quercus castaneifolia leaf extract has high antioxidant and antibacterial properties (Almeida et al., 2008; Bahador and Baserisalehi, 2011) that could reduce free radical formation and inhibit the growth of pathogenic bacteria. Besides improving growth performance, the plant extract enhanced the antioxidant enzyme activities such as SOD, CAT, and GPx to mitigate oxidative stress in the common carp (Paray et al., 2020). Oak leaf extract may also stimulate immune components, such as lysozyme elevation, to increase disease resistance in the fish (Hoseinifar et al., 2019; Paray et al., 2020). Conversely, oak leaf extract could not regulate the cortisol level of fish under stress for faster recovery compared to the untreated control group (Paray et al., 2020). Meanwhile, dietary Rheum officinale extract significantly regulated cortisol and glucose levels to mitigate crowding stress in common carp (Xie et al., 2008). Green tea extract also mitigated anaesthesia-induced stress in black rockfish, Sebates schlegeli (Hwang et al., 2013), while Urtica dioica extract significantly reduced crowding stress in Victoria labeo, Labeo victorianus by lowering cortisol and glucose levels (Ngugi et al., 2015).

Rheum officinale bail is a Chinese herbal medicine for constipation, jaundice, ascites, and asthma (Akbar, 2020). Previous studies have claimed this herbal remedy enhances the immune system and relieves stress (Jian and Wu, 2004; Yin et al., 2006). Anthraquinone extract from rhubarb bail potentially improves blood parameters, disease resistance against Aeromonas hydrophila, and crowding stress in aquaculture (Xie et al., 2008). Nevertheless, overusing Chinese herbs in farmed aquatic species causes gut microbiota imbalance (Liu et al., 2004) and affects the palatability of medicated feed, reducing their growth performance (Xie et al., 2008).

Lemon (Citrus limon) peel reportedly possesses medicinal value such as antimicrobial, anticancer, and antioxidant properties (Espina et al., 2011). This high-fibre agricultural waste has been widely explored as a feed additive in aquaculture. Previous studies revealed that dried lemon peel (DLP) positively impacted the growth performance, haematological parameters, and immune system in various aquatic animals, such as common carp, Cyprinus carpio (Sadeghi et al., 2021), Nile tilapia, Oreochromis niloticus, African catfish, Clarias gariepinus (Abdel Rahman et al., 2019), gilthead seabream, Sparus aurata (Beltrán et al., 2017) and rohu, Labeo rohita (Harikrishnan et al., 2020).

DLP potentially mitigated crowding stress in rainbow trout, Oncorhynchus mykiss, by enhancing their immune system and antioxidant capacity, besides lowering the blood cortisol level (Chekani et al., 2021). The cortisol level is significantly lower in the group of fish that received DLP than the fish from the control group, which was also observed in juvenile Labeo victorianus (Ngugi et al., 2017). On the other hand, there was no significant (P>0.05) difference in cortisol levels between the DLP-treated and untreated Nile tilapia and African catfish (Abdel Rahman et al., 2019). Dietary DLP also significantly (P<0.05) increased the total immunoglobulin, alternative complement (ACH50), and lysozyme activity in rainbow trout compared to the control group. Currently, the mechanism of DLP in boosting the immune system remains clear, but studies have explained that these enhancements are caused by the bioactive compounds found in DLP (Baba et al., 2016; Beltrán et al., 2017; Xi et al., 2017). The DLP also enhances the antioxidant capacity of aquaculture species by significantly increasing the SOD and CAT activities (P<0.05) compared to the control group. The SOD and CAT are the frontliners of the anti-oxidant defence against pathogenic stress (Zhang et al., 2013).

Rosemary, Rosmarinus officinalis, leaf is another phytobiotic that can be used as a feed additive to reduce crowding stress in common carp due to the high stocking density farming system. This medicinal herb contains bioactive compounds with various health benefits, including flavonoids and tannins, which act as free radical scavengers and inhibit lipid peroxidation (Almeida et al., 2008; Benedí et al., 2004; Zheleva-Dimitrova et al., 2010). For instance, dietary rosemary leaf demonstrated the ability to control cortisol and glucose levels in fish under crowding stress (Yousefi et al., 2019). Furthermore, combining Saint John’s wort, Hypericum perforatum, lemon balm, Melissa officinalis, and rosemary promoted the antioxidant capacity and mitigated crowding stress in juvenile Atlantic salmon, Salmo salar. Phenolic compounds, particularly in rosemary and lemon balm, contributed to antioxidant capacity enhancement in fish (Barros et al., 2013; Peng et al., 2007).

Pomegranate peel is another promising feed additive to ease crowding stress. This plant possesses various bioactive compounds, such as polyphenols, which are known for their pharmacological effects, including anti-diabetic, antioxidant and immunostimulatory (Akhtar et al., 2015; Malviya et al., 2014; Singh et al., 2019). Furthermore, studies have revealed that dietary pomegranate peel positively impacted the growth and health of Nile tilapia and rainbow trout (Avazeh et al., 2021; Badawi and Gomaa, 2016; Monir et al., 2020). This agriculture by-product also effectively lowered the plasma cortisol glucose in the blood of common carp subjected to crowding stress (Yousefi et al., 2023 b).

Shewanella putrefaciens Pdp11 significantly (P<0.05) mitigated crowding stress by upregulating immune gene transcription, modulating gut microbiota, and stimulating disease resistance against Vibrio harveyi and V. parahaemolyticus in Solea senegalensis (Tapia-Paniagua et al., 2014) (see Table 4). Dietary S. putrefaciens Pdp11 also upregulated immune-related genes, such as C7, HP, NARS, HAMP1, TNFAIP9, and NCCRP1, responsible for producing antimicrobial peptides against pathogenic invasion. Likewise, these genes were upregulated in S. senegalensis lipopolysaccharide supplementation (Osuna-Jiménez et al., 2009; Prieto-Álamo et al., 2009). Meanwhile, S. putrefaciens Pdp11 potentially reduced the harmful bacteria family Actinobacteria, Photobacterium, and Vibrio spp. in the intestines of S. senegalensis (de La Banda et al., 2010; Tapia-Paniagua et al., 2010).

Dietary gallic acid significantly reduced glucose and cortisol levels in common carp, Cyprinus carpio, to mitigate crowding stress (Ghafarifarsani et al., 2023) (see Table 5). A similar finding has been reported in broilers (Biswas et al., 2023). However, the mechanism of gallic acid in relieving crowding stress remains unclear. Dietary yeast, Xanthophyllomyces dendrorhous (sexual stage Phaffia rhodozyma) extract at a dose of 0.5 g/kg of feed for 40 days enhanced the antioxidant capacity and mitigated crowding stress in juvenile Atlantic salmon, Salmo salar.

Yeast extract contains bioactive compounds, primarily carotenoids. Carotenoids are vitamin A precursors acting as free radical scavengers to eliminate excess energy, preventing cellular oxidative damage (Amengual et al., 2011; Loto et al., 2012; Martinez-Moya et al., 2015). Astaxanthin is the main bioactive compound in yeast extract, a carotenoid that can inhibit lipid peroxidation activity (Contreras et al., 2015). Other carotenoids in yeast extract include lycopene, β-carotene, and phoenicoxanthin (Contreras et al., 2015; Córdova et al., 2016). The β-glucan is a primary bioactive compound in the yeast cell wall, known for the antioxidant properties and MDA inhibition (Lee et al., 2011). Therefore, astaxanthin and β-glucan in the yeast extract may be responsible for boosting the antioxidant capacity of the experimental fish by inhibiting lipid peroxidation, lowering MDA level, protecting cells from oxidative damage, and mitigating crowding stress.

The supplementation of dietary pennyroyal, Mentha pulegium, essential oil improved the growth performance, enhanced the immune system, increased antioxidant capacity, and mitigated crowding stress in common carp (Yousefi et al., 2023 a). Pennyroyal essential oil was found to activate digestive enzyme activity in the fish and promote gut health, enhancing the fish’s growth performance (Kaur et al., 2018; Ringø et al., 2016). In addition, pulegone, an organic compound that is abundant in essential oil, was reported to enhance antioxidative response and ease stress due to crowding in the fish (Yousefi et al., 2023 a).

Chrysin is a bioactive compound present in various natural sources such as honey, propolis, blue passion, and mushrooms (Mani and Natesan, 2018). The medicinal properties of this compound have been widely reported, including immunostimulatory, anti-inflammatory, antimicrobial, and antioxidant activities (Stompor-Gorący et al., 2021; Zeinali et al., 2017). Thus, chrysin could be included as a feed additive to boost fish production and mitigate crowding stress in an aquaculture system. In a previous study, rainbow trout that received a diet containing chrysin for 56 days demonstrated improved antioxidative response and immune system (Yousefi et al., 2023 c).

A probiotic combination of Lactobacillus acidophilus (LAB) and resveratrol (RE) was found to mitigate crowding stress in common carp, Cyprinus carpio (Gabr et al., 2023) (Table 6). The functional roles of LAB include enhancing digestion and nutrient absorption, digestive enzyme activity, growth-related metabolite production, and eliminating harmful gut bacteria (Assan et al., 2022; Balcázar et al., 2006). Meanwhile, RE can ameliorate intestinal damage, reduce protein degradation, enhance antioxidant capacity, and improve lipid and glucose metabolism (Zhang et al., 2013; Salomão et al., 2019; Tan et al., 2019; Wilson et al., 2015). Furthermore, the LAB and RE combination enhanced the antioxidant capacity by increasing antioxidant enzymes to protect the antioxidant defence system, thus relieving crowding stress in common carp (Gabr et al., 2023). Previous studies also revealed that probiotics can protect the antioxidant mechanism against free radicals and oxidative stress in fish (Hoseinifar et al., 2020) and other animals (Heshmati et al., 2018).

Combining selenium and garlic extract as feed additives positively impacted grass carp, Ctenopharyngodon Idella, exhibiting improved growth performance, antioxidative response, and mitigated crowding stress (Adineh et al., 2021 a). Selenium was reported as an essential component of glutathione peroxide enzyme, which protects the cellular membrane from oxidative impairment (Rotruck et al., 1973). Meanwhile, garlic is a widely used phytobiotic that is utilised as a feed additive to enhance the growth and health of various aquatic species (Adineh et al., 2021 a). Flavonoids and sulphur are two primary components of garlic that are rich in antioxidants and can eliminate free radicals in fish (Chung, 2006; Sharma et al., 2010). Therefore, the selenium and garlic combination is ideal in fish farming.

High stocking density in aquaculture causes crowding stress in farmed species, impairing their growth performance and health status. Researchers have proposed using feed additives, namely probiotics, phytobiotics, and synthetic immune stimulants, to mitigate this issue by modulating the immune system, antioxidant capacity, and intestinal condition of aquatic animals. Furthermore, feed additives have exhibited protective effects against crowding stress, but the mechanism of these components has yet to be fully elucidated. Moreover, limited studies have explored the use of probiotics as feed additives to mitigate crowding stress in fish farming.

Currently, studies have revealed that feed additives can potentially control cortisol and glucose levels in fish after being exposed to crowding stress. Most studies incorporated phytobiotic as feed additives between 0.5% and 5% of the diet to manage the effects of crowding stress in fish farming. Alternatively, synthetic immune stimulants have demonstrated the potential as a feed additive and reducing stress due to crowding in fish farming at an even lower inclusion level (≤0.5% of diet). A combination of synthetic immune stimulants and probiotics or phytobiotics also demonstrated positive outcomes in mitigating stress in fish farming. Future studies should investigate the appropriate dosage and optimise feed additives administration to identify the best practices for sustainable aquaculture production. Furthermore, future studies should continue to explore the application of the biofloc technology in fish farming, which was found to reduce crowding stress due to high stocking density.