Probiotics for freshwater fish farming can be administered as single or multiple mixtures. The expected benefits of probiotics include disease prophylaxis, improved growth, and feed conversion parameters, such as the feed conversion rate (FCR) and specific growth rate (SGR).
According to the Food and Agriculture Organization/World Health Organization (FAO/WHO) definition, probiotics are ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’. In aquatic environments, the concept of probiotics also includes microorganisms that exert a beneficial effect not only by colonizing the host but also by being present in the water 
. Bacteria, yeast, and algae are extensively utilized as probiotics in aquaculture 
Probiotics for freshwater aquaculture may consist of a single strain or a range of microorganisms. Bacillus
spp., lactic acid bacteria (LAB), such as Bacillus
sp., and yeast (Saccharomyces cerevisiae
) diluted in the water or incorporated into fish feeds, and nitrifying/denitrifying bacteria, diluted in the water are often utilized as single or multiple probiotic mixtures in research and commercial formulations for freshwater fish farming 
The expected benefits include disease prophylaxis, prevention of disease spread, an improvement in food conversion efficiency (FCE), and an increase in the growth of farmed fish via the production of antioxidant enzymes, antioxidant activity, and promotion of healthy gut microbiota proliferation, all of which produce an enhancement in immunity and reduction in vulnerability to fish stress 
As a result, the potential benefits of probiotic treatment can be observed on the growth and efficiency of feeding and on the environmental parameters and aquaculture pollution. Probiotic management of diseases and prophylaxis, for example, can also reduce the need of antibiotics, one significant environmental issue of aquaculture. Likewise, environmental benefits of probiotics have been observed in experiments that monitored the water quality parameters of freshwater fishponds/tanks treated with probiotics diluted in the water or incorporated in the feed.
A lower feed conversion rate (FCR), for example, can be achieved when probiotics improve digestion by producing digestive enzymes, such as protease, amylase, and cellulase 
, and because of more efficient digestion, the organic load of fish farms may be reduced.
The potential benefits of probiotics on fish health also contribute to improvements in the growth, feed conversion efficiency, and water quality of freshwater fish species. Probiotics can modulate both local mucosal and systemic immune responses in farmed fish 
, thereby improving and maintaining fish health, preserving gut epithelial integrity, producing pathogen anti-virulence factors, and secreting antioxidant enzymes, resulting in a decrease in oxidative stress and cell damage, as well as a decrease in gut inflammation, in farmed fish 
. B. probiotica
has been used as a feed supplement and was demonstrated to result in an increase in the growth of a variety of fish species 
. Additionally, Bacillus
strains have been shown to benefit the environment by reducing ammonia levels and harmful algal blooms 
LABs are also frequently used in commercial preparations of aquaculture probiotics. Two enzymes in LAB cells, Mn-Kat (Mn-dependent pseudocatalase) and Heme-Kat, (Heme-dependent catalase), can degrade hydrogen peroxide (H2
). LAB can also chelate iron to reduce Fe2+
. Decreased O2–
levels in LAB cells can also inhibit Fe2+
. Other micronutrient factors and biochemical pathways, such as glutathione, thioredoxin, and vitamins C and E, enhance the antioxidant capacity of LAB cells, thus contributing to redox homeostasis 
The effects of probiotics can be classified into two groups according to the aim of the treatment:
- Fish growth and welfare parameters, including effects on fish growth and feed conversion parameters, gut microbiota and anatomy, immunity, and resistance to pathogens.
- Environmental parameters, including fishponds and/or tanks (water quality, diversity of aquatic microbiota).
2. Optimal Feeding Regimes and Improved Feed Conversion Are Prerequisites for Reducing the Environmental Impact Caused by Freshwater Fish Farms
Sustainable development of freshwater (FW) aquaculture requires minimal environmental impact and monitoring of the organic load released in the aquatic ecosystem, while safeguarding fish welfare and the productivity of the sector.
Aquaculture of intensively cultivated fish uses intensive fish farming methods and is based mainly on fishponds with recirculating or openly flowing water. Environmental conditions, including water quality, stocking density, and temperature, can affect fish growth and the feed conversion efficiency (FCE) of farmed fish 
. Furthermore, dietary regimes or pathological problems can compromise digestion and affect FCE 
. Feed quality and quantity are crucial for fish welfare and growth. Little or no growth occurs if the fish do not consume the feed or are not capable of utilizing the feed due to a nutrient deficiency 
The optimal feeding of farmed fish necessitates an understanding of the digestion process, the digestive system, and the parameters that affect fish metabolism and why these parameters can influence feed conversion and the organic load generated by fish farms. Feed conversion varies significantly depending on the feed composition and management practices used in a fish farm 
. The organic load and impact of freshwater fish farms in the aquatic ecosystem can be reduced by manipulation of the dietary regimes, for example, by adjusting the quantity of feed according to feed manufacturer feeding tables and by reducing the phosphorus content in the feed 
. The feed conversion ratio (FCR) is the common measure that quantifies the efficiency by which fish convert feed with respect to weight increase. FCE measures how efficiently farmed fish convert fish feed to weight gain, whereas FCR represents the amount of fish feed required to gain one unit of weight.
In practice, fish farmers may fail to follow the recommended feeding rations due to inadequate monitoring of size dispersion and total fish biomass, conditions that create uncertainty in the estimation of fish weight 
. Furthermore, fish metabolism and feed consumption vary according to thermal conditions and several other parameters. Suboptimal temperatures and overfeeding can result in wasted feed and/or uneaten or poorly digested feed, all of which can result in an increase in FCR. For example, at low winter temperatures, farmed rainbow trout can exhibit a decrease in FCE, digestibility of dry matter, nitrogen levels, and energy derived from the diet, thus resulting in an increase in solid nitrogen waste output per kg of produced fish 
illustrates an example in which changes in FCR affect the release of phosphorus by fish farms. In this specific example, temperature affected the FCR of farmed rainbow trout fish farms, and increasing seasonal temperatures caused an increase in the appetite and feeding requirements of the fish. This process caused an increase in feed consumption but reduced the feed conversion efficiency (FCE), resulting in higher FCR that in turn affected phosphate levels. In fact, with a seasonal rise in temperature, a small increase (4.4%) of FCR occurred whereas the levels of phosphate loading increased by 21.1% (calculated from Azevedo et al. 
). It is reasonable to assume that a reduction in the organic load generated by trout farms could be achieved via more accurate estimation of fish biomass in each pond. Aquatic pollution is a significant environmental issue for freshwater ecosystems, affecting the ecological status of the benthos and the welfare fish in inland waters 
An example of how changes in the feed conversion ratio (FCR) due to temperature effects on the fish feeding rate and metabolism can affect the phosphorus load released by farmed rainbow trout. This change is due to an increase in FCR (such as when more feed is required to produce one kg of weight gain) that will result in a proportionally higher amount of phosphate released into the aquatic environment (data calculated from Azevedo et al. 
3. How Probiotics Can Improve FCR, Fish Health, and Fish Growth and Help Reduce the Environmental Impact Caused by Freshwater Fish Farms
The expected benefits of probiotics on freshwater farmed fish are graphically illustrated in Figure 2. Probiotics can alter fish and water microbiota. Expected beneficial effects at the level of the aquatic ecosystem include improvement of the water quality in fish farm waters via control of the nitrification–denitrification process, resulting in reduced levels of ammonia and algal bloom. Expected beneficial effects of probiotics at the organism level are mainly related to the gut and include improvements in gut function, intestinal cellular integrity, inflammation control, pathogen inhibition, release of anti-virulence factors, protection from free radicals, and enhanced immunity. In other words, probiotics can improve fish health and growth feed conversion, and lead to a reduction in the organic load and antibiotics used in freshwater fish farms, thus reducing the environmental impact of aquaculture pollution.
Figure 2. A flow diagram with the action of probiotics at the level of the aquatic ecosystem and at the organism level, resulting in improved fish health, feed conversion, and growth, and reduction of freshwater aquaculture pollution.
3.1. Probiotics Can Improve the Digestion of Fish Diets and Support the Replacement of Fish Oils and Fish Proteins as Ingredients of Fish Feeds
A historical trend in this industry for accurately determining the protein requirements for each species, improve FCR, and lessen the environmental impact of aquaculture on aquatic ecosystems has been evolving. Several changes driven by research and global market forces in the composition of aquaculture feeds have been found. An obvious trend toward reducing the dependence of fish feed on fisheries and replacing fish protein and fish oils with alternative sources of proteins and fat has been found 
. Probiotics can aid in the development of new fish feeds and lead to an improvement in gut health and feed conversion when used as supplements in conventional fish diets. Furthermore, probiotics can cause a reduction in the pathological issues occurring in the gut when alternative sources are used for the manufacture of fish feeds 
. Likewise, gut function and immunity of beluga sturgeons fed a soya-bean-containing diet showed an improvement when experimental fish feed was supplemented with probiotics 
, illustrating the improvements that probiotics produce on fish health and growth, paving the way for the development of alternative fish feed, and therefore, reducing the reliance of the aquaculture fish feed industry on fisheries and fish meal 
3.2. Probiotics Can Reduce Subacute Intestinal Pathological Problems, Improve Feed Conversion, and Reduce Disease Outbreaks, Mortality, and Antibiotic Usage
Optimal fish feeding, fish welfare, and reduction of the environmental impact of fish farms are crucial parameters for the economic viability and sustainability of the sector 
. The health status and the efficiency of the FCE of farmed fish may vary according to management and production methods 
. Successful aquaculture requires safeguarding of the health of growing fish and optimization of the feed conversion, thereby achieving better FCR, reducing the amount of feed required to produce farmed fish, reducing the environmental impact generated by fish feed production, and reducing aquaculture wastes generated by wasted or poorly digested feed 
Poor digestion can result from intestinal pathological problems 
. Intestinal pathological problems resulting from experimental fish diets reflecting an obstacle in the replacement of fish proteins with alternative sources have frequently been reported 
. Intestinal health is crucial for animal growth and the efficiency of animal feeds 
. Intestinal pathological problems in farmed fish can be associated with the disruption of intestinal function and reduction in the efficiency of feed conversion 
. Subacute intestinal pathological problems, such as sub-acute intestinal inflammation 
, can affect feed digestibility 
. Intestinal pathological issues may affect normal intestinal functions and produce a higher intestinal pathological index score; the subsequent inflammation can therefore result in higher excretion of nutrients resulting from impaired digestion 
3.3. Probiotics Can Improve Water Quality of Freshwater Fishponds and Help Reduce the Environmental Impact of Freshwater Fish Farms
Probiotics can result in improved fish growth and water quality of freshwater fishponds/tanks 
, resulting in a reduction of some environmental issues related to fish farm effluents. Probiotics are members of the healthy intestinal microbiota, and evidence to suggest a positive effect of probiotics in improving the digestion, health, and growth of farmed fish can be found. Microbial modulation by probiotics may also help improve host nutritional status, and the growth of farmed fish fed with probiotic supplements exhibited improved feed conversion, a significant parameter for the sustainability of aquaculture