The present study demonstrated the effects of dietary C. vulgaris supplementation and duration of feeding on growth performance, growth hormone levels, feed utilisation, and some biochemical parameters of red hybrid tilapia. Certain levels of interactions were observed between the supplementation levels and the duration of feeding. From the results (Table 2) there was significant interaction (p < 0.05) between supplementation levels and duration of feeding on weight gain (WG) and average growth rate (AGR) of the fish. The values were positively affected (increased weight gain) by increasing the supplementation levels during the medium and long durations of feeding. Similarly, the supplementation levels showed a significant effect (p < 0.05) on the fish’s specific growth rate (SGR) and survival rate (SR). The highest effect was seen at 5% supplementation levels (p < 0.05) for the specific growth rate. Although the values for the specific growth rate decreased with extension of the feeding duration, the findings were in concordance with Enyidi's (2017) report. According to Enyidi, African catfish (Clarias gariepinus) subjected to medium-term feeding with a C. vulgaris supplemented diet (5% kg − 1 of feed), showed a significantly higher weight gain, and specific growth rate. The findings were further supported by a study on olive flounder (Paralichthys olivaceus), which revealed the favourable effect of medium-term feeding with the Chlorella diet on weight gain and specific growth rate (Rahimnejad and Lee 2016). Similarly, Sergejevová and Masojídek (2012), observed that 2.5% Chlorella biomass dietary supplementation results in a significantly higher specific growth rate in starlet (Acipenser ruthenus) following a medium-term experimental feeding. In contrast, the short-term feeding of Nile tilapia (Oreochromis niloticus) with ≥ 30% C. vulgaris supplemented diet showed an unfavourable effect on daily weight gain and specific growth rate (Lupatsch and Blake 2013). The effect reported is not in line with the effects observed in the present study, as short-term feeding of 5% C. vulgaris presented an appreciable effect on weight gain and specific growth rate. The conflicting effect may be due to the dietary supplementation levels of C. vulgaris, as higher inclusion levels of alternative proteins could result in decreased growth performance (Lupatsch and Blake 2013; Esmaeili 2021), perhaps due to low digestibility of the alternative protein sources.
Organo-somatic indices are part of the indicators of fish well-being (Kiron et al. 2016). In spite of the significant effect of duration of feeding on condition factor (CF) and hepatosomatic index (HSI), there was no significant effect (p ˃ 0.05) of supplementation levels on CF and HSI of the fish. The finding did not correspond with that of a study by Enyidi (2017), who observed a decrease in HSI of African catfish fed diet supplemented with 5% C. vulgaris. However, the finding corroborates with Kim et al. (2013), who reported that medium-term feeding with diets whose fishmeal was partially replaced with 15% Spirulina pacifica did not significantly affesct the HSI values of parrot fish (Oplegnathus fasciatus). However, the supplementation levels showed a significant effect on the viscerosomatic index (VSI) during the short-term feeding, but the effect diminishes and blends with that of the control group at the end of medium and long-term feedings, respectively. The finding is in line with that of Kiron et al. (2016), who observed VSI values that were not significantly different from the control after feeding Atlantic salmon (Salmo salar) with 10% Desmodesmus sp. supplemented diet for medium-term feeding. Therefore, the experimental fish in the current study efficiently utilise the Chlorella supplemented diet with no accompanied untoward effect on organo-somatic indices.
The supplementation level had significantly improved the feed utilisation (p ≤ 0.05) of the red hybrid tilapia. The feed conversion ratio (FCR) of the Chlorella supplemented fish was not significantly different from the control during the short-term and long-term feeding trials, although, a lower FCR was seen at 5% supplementation level during the medium-term feeding. Nevertheless, the findings are in agreement with those of Enyidi (2017), and Lupatsch and Blake (2013), who reported significantly higher (p < 0.05) FCR in African catfish and Nile tilapia fed diets supplemented with C. vulgaris at 5 and ≥ 30% inclusion levels, respectively. Similarly, supplementation levels have showed a favourable effect on mean daily feed intake (MDFI). The MDFI increases with increased supplementation levels, with the highest value recorded at 5% supplementation level. The present finding seems to be consistent with the one reported by Lupatsch and Blake (2013), who reported that the daily feed intake of tilapia increases with the increased dietary inclusion of C. vulgaris. A study by Gouveia et al. (1996) reported that, a medium-term (67 days) dietary supplementation of C. vulgaris produces feed intake and weight gain values comparable to those produced by other feeds rich in pigments and vitamins. Therefore, the inclusion of C. vulgaris in fish diet could increase its acceptability in the diet.
Growth hormone in fish is associated with diverse roles in different tissues and cell types. The hormone mediates homeostasis, immunological pathways, reproduction, behavioural activities and neuroendocrine functions (Jönsson and Bjornsson, 2002). The current work presented a significant effect of dietary supplementation of C. vulgaris on the growth hormone (GH) concentration of red hybrid tilapia. The levels of the GH increase with increased supplementation levels. Similarly, a study by Khan et al. (2015) revealed an elevated serum GH concentration in juvenile mahseer (Tor putitora) supplemented with a graded level of L-Ascorbyl-2-Polyphosphate over a longer duration of feeding. Miao et al. (2012) reported the positive impact of exogenous growth hormone on body length and weight gain of Nile tilapia (Oreochromis niloticus). Similarly, the duration of feeding showed a significant effect on GH levels. The serum GH concentration drops as the feeding period extends, with the long-term feeding presenting the lowest GH concentrations across both the control and the treatment groups. The trend is directly proportional to FCR and SGR reported earlier. The present finding is in line with the report by Won & Borski, (2013), who reported that there is an increased secretion of GH during hyper-anabolism, which stimulates protein assimilation and raises the FCR and SGR values as a result of an elevated feeding. The secreted GH binds its receptor (growth hormone receptor GHR) and triggers the production of hepatic insulin-like growth factor I (IGF I). This increases somatic growth and ultimately sends negative feedback on growth hormone secretion and hence a drop in the GH concentration (Won and Borski 2013).
Certain plant products have a history of enhancing total serum protein and globulin levels in fish, which signals immune system activation (Das et al. 2009). There was a significant interaction effect between the supplementation levels and duration of feeding on total serum protein (TP) and albumin globulin ratio (A:G) values. These effects were more glaring at 5% supplementation levels during the medium and long-term feeding. Similarly, Xu et al. (2014) observed an elevated level of total protein in gibel carp (Carassius auratus gibelio) fed diets supplement with lower inclusion levels of Chlorella, following a medium-term feeding (60 days). Likewise, Das et al. (2009) observed an increase in total protein levels in rohu (Labeo rohita) fed diets supplemented with Euglena viridis at ≤ 1g/ kg after a long-term feeding. There was significant interaction (p < 0.05) effect between the supplementation levels and feeding duration on serum globulin concentration. The trend of globulin values was the same at 1 and 3% supplementation levels at the medium and long-term feeding. The highest globulin value was observed at 5% supplementation level during the medium-term feeding. An elevated globulin concentration could depict the capability of the Chlorella supplemented diets to enhance the mononuclear phagocyte system, hence an improved immune response (Kumar et al. 2012). The result of the present study concerning higher globulin values in the Chlorella supplemented diet is similar to the findings of Xu et al. (2014); that medium-term feeding of gibel carp with Chlorella supplemented diets elevates the levels of globulin in the fish serum. The result also confirmed the observation of Das et al. (2009), who observed increased serum globulin levels following feeding rohu (Labeo rohita) diets supplemented with Euglena viridis over short, medium and long-terms feeding.
Nevertheless, the serum albumin concentration of the red tilapia supplemented with Chlorella differed from total protein and globulin concentrations by not presenting any interaction effect between the supplementation levels and feeding duration. The significant effect of supplementation on the albumin concentration increased with the increased supplementation level. The result was more prominent during the medium and long-term feeding. In addition, there was a significant effect of duration of feeding on albumin levels; the effect was conspicuous at long-term feeding. Similarly, short and medium-term supplementation of Chlorella spp. could not cause a marked change in albumin levels of gibel carp (Xu et al. 2014). In addition, Saberi et al. (2017) suggested that only long-term feeding with low-level C. vulgaris supplementation could cause a marked change in albumin concentration in tilapia. Interestingly, the result conforms with Haghighi et al. (2017) findings in rainbow trout (Oncorhynchus mykiss) fed diets supplemented with Aloe vera at lower inclusion levels over a long duration of feeding. Therefore, long-term feeding with C. vulgaris supplemented diet could result in higher total serum protein concentration in tilapia.
The welfare and productivity of fish are dependent on the level of stressors in fish, and a rise in the level of blood glucose is one of the bio-indicators of heightened stress in fish (Jiang et al. 2017). The present study indicated the significant effect of supplementation levels of C. vulgaris on the serum glucose level of the experimental fish. After a long feeding duration, the dietary C. vulgaris supplementation significantly lowered the serum glucose level, relative to the control group’s values. The feeding duration has also shown an effect on glucose concentration, with the lowest glucose concentration values observed at the medium-term feeding irrespective of the supplementation levels. The result is in concordance with that of Rahimnejad and Lee (2016), who reported that medium-term feeding on diets supplemented with C. vulgaris at 5–10% does not significantly affect the plasma glucose concentration of olive flounder (Paralichthys olivaceus). In addition, the findings of the current study are in line with that of Koo et al. (2001). They observed a significantly lower serum glucose level in Juvenile Olive Flounder (Paralichthys olivaceus) fed diets supplemented with C. vulgaris at 2–4% for long-term feeding. The result also agrees with the findings of Henrique et al. (1998). The authors noticed that long-term feeding of seabream (Sparus aurata) with vitamin C supplemented diet significantly lowers the plasma glucose level. Therefore, long-term feeding of fish with diets supplemented with C. vulgaris could reduce the stress in fish.
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are the main liver enzymes used as bio-indicators of the functionality of a liver. An increase in the serum concentration of these enzymes is signalling damage to the liver, muscles, kidney and/or gills (Jyotirmayee and Das 2015). The supplementation levels did not affect the serum AST and ALT levels in the present study. The finding is similar to that of Rahimnejad and Lee (2016), who observed that medium-term feeding of Olive Flounder (Paralichthys olivaceus) with C. vulgaris diet at 5–15% supplementation levels showed no significant effect on AST and ALT serum levels. Although, there was a significant effect of duration of feeding on liver enzymes levels, with the AST serum levels appearing significantly higher during the short-term feeding, as the period of feeding extended, the concentration decreased. On the other hand, the ALT serum concentration was lower during the short-term feeding, and the value increased as the duration of feeding extend. The trend is similar to the pattern observed in the control group of rohu (Labeo rohita) fed C. vulgaris diet for 90 days (long-term feeding) (Jyotirmayee and Das 2015). AST is found in many organs such as; the liver, pancreas, kidneys, cardiac muscle, brain, lungs, white blood cells and red blood cells, while, ALT is present in abundance in the liver (Küpeli et al. 2006). Therefore, the dietary supplementation of C. vulgaris in the current study appeared safer by not causing an observable damaging effect on the vital organs of red hybrid tilapia.
Respiratory burst activity is mainly mediated by neutrophils and macrophages (Uribe et al. 2011). Neutrophils are the first cells deployed when there is an inflammatory reaction and are integral components of innate immunity in teleost. In addition, they also limit the abundance of several pathogens (Sowmya and Sachindra 2013). These cells eliminate bacteria during the respiratory burst via the production of reactive oxygen species that are toxic to the pathogenic agents (Uribe et al. 2011). The result of the present study confirmed the significant effect of the supplementation levels of C. vulgaris on the respiratory burst activity of tilapia. The activity steadily increased with the extension of the feeding period; the long-term feeding presented the highest respiratory burst activity values. Medium-term feeding with C. vulgaris supplemented diets enhances the free radicals scavenging activity of Olive Flounder (Paralichthys olivaceus) (Rahimnejad and Lee, 2016). Similarly, medium-term feeding of a diet supplemented with garlic enhances the respiratory burst activity of Nile tilapia (Oreochromis niloticus) (Aly et al. 2008).
Lysozyme is a bactericidal enzyme identified in various fishes. The enzyme is present in mucus secretion/membranes, lymphoid tissue, and serum of most aquatic vertebrates. Lysozymes are mainly found in monocytes/macrophages and neutrophils. The enzyme act by hydrolysing the peptidoglycans of bacterial cell walls, eventually resulting in cell lysis. In addition, it also acts via the opsonisation pathway (Uribe et al. 2011; Hayball and Puccetti 2012). Moreover, its activity is not related to other immune substances (Hayball and Puccetti 2012). The current study revealed the significant effect of supplementation levels on serum lysozyme activity, with a 5% supplementation level being consistently higher during short, medium and long-term feeding. Similarly, the duration of feeding has indicated an effect on lysozyme activity. Although, the lysozyme activities were not significantly different in the short and medium-term feeding, but significantly higher in long-term feeding. A similar result of elevated lysozyme activity was seen in rohu (Labeo rohita) fed diets supplemented with a microalgae (E. viridis), the effect persisted throughout the short, medium and long durations of feeding (Das et al. 2009). correspondingly, long-term feeding with diets supplemented with Dunaliella salina, significantly increased the lysozyme concentration of Oncorhynchus mykiss (Amar et al. 2004). Long-term feeding of diets supplemented with carotenoid elevates the serum lysozyme activity of common carp, Cyprinus carpio (Sowmya and Sachindra 2013). The same trend was observed following medium-term feeding of koi carp (Cyprinus carpio) with diets supplemented with C. vulgaris at 2–10% (Khani et al. 2017).