1. Introduction
The pool barb, also called the spotfin swamp barb or stigma barb (
Puntius sophore), is a tropical freshwater fish species belonging to the Cyprinidae family. It is found in Asian nations and is indigenous to inland waters in Asia [
1]. The market’s need for both fresh and processed products, as well as their nutritional and decorative value, are driving up the demand for this widely distributed small indigenous fish species (<25 cm) in freshwater [
1,
2]. Though
P. sophore has been classified as lower risk to near threatened in recent research, the wild populations are declining quickly because of intense fishing pressure [
3]. There is fear that this fish may disappear unless proper steps are urgently taken to protect the fish from extinction. Nevertheless, there are few studies in the literature regarding their growth and physiological performances in captivity.
Stocking density is an essential aspect in fish culture operations since it directly affects the growth and survival of the fish, which in turn affects production. According to Ahmed et al. [
4], social interactions and poor water quality are where stocking densities have the greatest negative consequences on fish; thus, the appropriate stocking density varies depending on the species raising system, feed utilization, growth potential, management techniques, and best size at harvest [
5]. Fish metabolism, development, and stress are significantly impacted by the stocking density, and this impact is frequently species-specific [
6]. The severity, duration of the stressor, and physiological and behavioural changes that mobilize energy sources result in decreased performance and slower growth [
7,
8]. These effects may be brought on by physiological stress or a decline in water quality, such as a drop in dissolved oxygen levels and an increase in ammonia levels [
8]; therefore, it is crucial to establish the proper stocking density for each fish species at each stage of their lives in all varieties of production systems [
5].
Dietary supplements to enhance growth and disease resistance through nutrition have drawn a lot of interest. Probiotics are one of the more promising biological prevention and control methods among dietary supplements. Increased digestive enzyme activity, the production of inhibitory chemicals against various dangerous microbes, immune system regulation, and the prevention of gut pathogen colonization by competitive exclusion are some positive benefits of probiotic use [
9]. Probiotics’ effects in aquaculture deserve special attention in terms of economic evaluation, as they can help farmers make a profit by producing high-quality fish [
10]. Aquaculture routinely employs Bacillus spp. as a probiotic [
11,
12]. They have been shown to improve fish immunity and protect them from a number of diseases and convert carbohydrates into lactic acid [
13].
The alteration of the gut microbiota and establishment of beneficial microorganisms, as well as higher specific and total digestive enzyme activity in the brush border membrane, all of which promote nutritional digestibility and feed utilization, are all benefits of probiotics. In aquaculture, probiotics have been shown to have many benefits in aquaculture, including enhancing growth, immunity, disease resistance, and water quality [
14,
15]. Probiotics can be categorized as single-strain or multi-species probiotics based on the number of probiotic species or genera. A variety of single-strain probiotics are available. For instance,
Saccharomyces cerevisiae, a probiotic yeast, has been effectively assessed and is well known for its capacity to strengthen defence mechanisms and increase immunity in a range of finfish species [
16,
17]. Numerous fish species have been shown to benefit from the probiotic properties of
Bacillus species, particularly
B. subtilis [
18,
19]. In aquaculture, lactic acid bacteria (LAB) have also been effectively used as possible probiotics [
20]. Due to its growth-promoting and immunomodulatory properties,
Enterococcus faecium probiotic applications have become quite popular in aquaculture [
21,
22]. Conversely, multispecies probiotics are generally more beneficial to the host than single- or separate-strain probiotics since they are a combination, blend, or cocktail of two or more probiotic species or genera [
23,
24].
By creating microbial flocs from the addition of an external carbon source, such as molasses, rice bran, or wheat bran, among others, the biofloc system is a revolutionary culture technology that enables the growth of microalgae, protozoans, rotifers, and nematodes [
25]. Fish can eat the floc biomass that forms in bodies of water as a source of additional food and as a water purifier [
26]. Avnimelech [
27] notes that fish are also exposed to microbial compounds through bioflocs, such as β-1, 3-glucan, peptidoglycan, lipopolysaccharides, etc., which activate and maintain the non-specific immune system; however, opportunistic and pathogenic microbes may be present in microbial communities [
28]. What are the potential treatments for these potentially dangerous agents that could develop and multiply in the rearing water before eventually entering the host body through feeding and osmoregulation processes [
29]?
The haematological value predicts the survival condition or physiological stress of organisms that respond to endogenous or exogenous changes [
30], especially with environmental status [
31], simply acting as a biomarker in the different habitat of fish [
32] as well as for changes of chemical and environmental stress, ecological condition, temperature, food habit, different periods of the reproductive cycle, and chemical exposure [
33]. Haematological studies help to improve the aquaculture system because it is a vital tool to recognize the healthy individuals from diseased or stressed ones [
34]. The current study was created with the following objectives in mind: (1) to observe the effect of different stocking densities on the growth and haematological responses of
P. sophore and (2) to compare the growth performances and haematological profile of
P. sophore diets using biofloc and probiotics as diet supplementation. These efforts will help to determine the optimal choice of stocking density and food for ensuring fish growth and physiological performance.
4. Discussion
Water temperature (°C), pH, nitrate nitrogen (mg L
−1), and TH (mg L
−1) levels were all within the ideal range for the species during the 60-day experimentation period in all experimental groups [
41]. As a result, the outcomes for fish welfare measures like growth, survival, and physiological conditions were unaffected by differences in the water quality parameters between the fish tanks.
Fish growth was examined, and the findings showed that the growth rate varied depending on the stocking density. Even though the same meal was given to all of the treatments equally, the growth of
P. sophore was higher in the LD group than it was in the other treatments in terms of mean final length, final weight, body weight increase, FCR, FCE, SGR, RGR, and DGR. A slow growth rate appeared to be associated with HD and increased competition for food and space, with an inverse connection with stocking density, assuming that space had population-limiting effects [
42]. The current findings are also consistent with those of Narejo et al. [
43], who found that
Heteropneustes fossilis farming performed best at lower stocking densities, as well as those of Chakraborty [
44], who noted that
Bengala elonga had a higher size and higher survival rate when there was less density. They also claimed that fish in a concrete cistern fed on formulated feed had a larger size and a higher survival rate because of the decreased density. Pouey et al. [
45] also noted a negative correlation between the growth and high densities of young silver catfish reared in a closed water recirculation system and treated to three different SDs (3.75, 7.5, and 11.25 gL
−1, approximately). In the present study, the authors found that the lower SD produced the highest growth rate, yet mortality rates did not increase in high densities. An improper stocking density can lead to increased competition among individuals for food and housing space, thereby increasing individual growth variability, and potentially establishing a size hierarchy or social hierarchy [
46].
Probiotic diet supplementation in the current study improved
P. sophore’s growth and feed consumption. The data for the current study showed that the probiotic-containing diet had considerably higher TLG, BWG, FCE, SGR, RGR, and DGR values, and survival rates than the other diets. It revealed that one of the explanations could be probiotics, which have been demonstrated to enhance feed digestion by releasing digestive enzymes or changing the environment in the stomach, leading to greater development [
11]. They may adhere for a short period, colonize the gastrointestinal tract, and raise antibody levels [
47]. As a result, probiotics have been shown to have positive impacts on bacterial populations in the environment and immune system responses in aquatic organisms [
48]. The growth of multiple fish species, including Nile tilapia [
49], common carp (
Cyprinus carpio) [
50], and rainbow trout (
Oncorhynchus mykiss), has been shown to be impacted by
Bacillus spp. [
51]. Giri et al. [
52] found that the addition of probiotics using
Lactobacillus reuteri P16 enhanced the total bacteria in intestine of
Cyprinus carpio exposed to lead. Improving the bacterial population in fish guts can help improve nutrient absorption and metabolism which increase the immune system in the body of fish, leading to the disease resistance of the fish [
11]. Fish species such as
Oreochromis mossambicus [
53],
Macrobrachium resenbergii [
54],
Litopenaeus vannamei, and
P. conchonius [
55] have shown growth and better health when fed biofloc instead of conventional fish food [
25]. However, in this study, for
P. sophore reared on biofloc, their growth performances were significantly lower compared with other treatments. A 28% mortality was observed in the biofloc-containing treatment, whereas there was no mortality in the control or probiotic-containing diets. It revealed that there might be a reason why fish did not like to consume external carbon sources and microbial flocs as an additional protein source.
The physiological stress the fish are under and any factors that could have a negative impact on their health should be taken into account before making a choice to improve biomass production in aquaculture by increasing fish density. The haematological values recorded in this study were unfold the changes in health status in different stocking densities and dietary treatments for the better management of
P. sophore when cultured in captivity, thus assuring sustainable aquaculture and the longevity of the species. All of the treatments were in the normal range, and there was no significant difference between them (
p > 0.05), except between WBC and stocking densities [
56,
57,
58]. These findings imply that
P. sophore may be somewhat sensitive to high-density stress. The general impact of fish crowding that appeared as an increase in haemoglobin values was described by many authors as haemoconcentration [
59,
60]. Under conditions of high energy demand, such as prolonged stress, this reaction may be the reason why the blood’s ability to carry oxygen increases. Additionally, as a result of increased energy needs for feed acquisition and the development of dominant–subordinate interactions, fish that are crowded have higher metabolic rates.
Likewise, the blood parameter data revealed no significant differences between the various experimental diets except RBC, MCV, MCH and PLT levels, but it was recorded that fish fed probiotic diets had slightly improved physiological functions and health status when compared to fish from the control and biofloc groups. Fish with higher levels of haemoglobin in their blood probably transported oxygen to their tissues more effectively, which led to better growth [
61]. According to Jahan et al. [
36], adding more yeast probiotics to fish diets may result in higher levels of Hb and Glu. Probiotic use considerably raised the RBC and Hb levels in
P. sophore in the current investigation, possibly as a result of better dietary protein absorption. A positive effect was also found by Marzouk et al. [
62], as shown by a notable increase in RBC count and Hb conc. in both fish groups fed probiotics-supplemented diets. Jäger et al. [
63] recommended the supplementation of probiotics for facilitating the absorption of essential amino acids. When diets containing S. cerevisiae and probiotics were employed, Abdel-Tawwab et al. [
16] saw similar outcomes in
O. niloticus, Sharma et al. [
64] in
Cirrhinus cirrhosis, and Talpur and Ikhwanuddin [
65] in Lates calcarifer. The current research implies that administering probiotic supplements to feed diets will improve fish health which supports the findings of Khattab et al. [
66], who found that fish fed diets containing commercial probiotics composed of
Bacillius licheliformes and
Bacillius subtilis had blood haematological parameters that were significantly higher than those of the control group.
The treatments with biofloc had the highest LYM and MON levels despite no significant differences (
p > 0.05) in any of the blood parameters. Other blood values showed a similar trend. Compared to the clear water condition represented by the control, it appears that the biofloc condition had no detrimental effects on fish health. Similar findings were made by Long et al. [
67], who hypothesized that biofloc had no appreciable impact on blood haematological measures, including RBC, WBC, Hb, and HTC. The same range of haematocrit values for tilapia under the biofloc treatment (BFT) or recalculating aquaculture system (RAS) was also suggested by Azim and Little [
68]; however, in the current study, though the haematological parameters were not affected by an external carbon source, the growth and survival rate was lower compared to the other treatments.