Received: 10.07.2019 / Accepted: 25.07.2019 / Published online: 31.07.2019
Haematological indices are essential diagnostic tools used to evaluate the health status of fish. Many publications have been stated by different works that qualitative and quantitative variations in haematological parameters; for instance White Blood Cells (WBCs), Red Blood Cells (RBCs), Haematocrit (Hct), Haemoglobin (Hb) content, Mean Corpuscular Haemoglobin Concentration (MCHC), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Volume (MCV) in fish, offer an indication of the health status of the fish. The uses of probiotics as biological control agents in aquaculture have replaced the usage of chemotherapeutics, is an approach in the build-up in aquaculture environments. In the cultured fish, the use of probiotics in monospecies or multispecies forms has been reported to stimulate specific and non-specific immune parameters including lysozyme activities and phagocytic, expression of various cytokines as well as improvement of blood profiles of many fish increasing resistance diseases and to other environmental perturbations such as physiological stressors. Interestingly, many researchers have shown that haematological indices in fish continue to offer a valuable diagnostic tool; and progress is made in establishing a reasonable range of values for blood parameters of different fish species. Also, many interventions have shown that probiotics used in aquaculture have potential in improving blood profiles of fish; although there are not many summarised information regarding the effects of probiotics on haematological parameters in fish. The purpose of this review is to synthesise the influences of probiotics on haematological parameters in fisha.
Aquaculture; Blood cells; Haematology; Immune System; Probiotics; Physiological stressors
Bolliger and Everds (2010) define haematology as the study of the physiology and pathology of the cellular elements of blood. The biological and environmental factors including age, weight, sex, food, bacteria, viruses, fungi and water quality parameters can influence the importance of the haematological parameters of fish (Das and Das, 1993). Haematological indices affect physiological state of the fish and provide a significant hint on the well-being of the fish (Harikrishnan et al., 2010). Haematological indices are used as vital diagnostic tools to assess the health status of fish (Pradhan et al., 2012). Reports on haematological indices show a correlation of the physiology and evolution in organisms. However, information on fish physiology has become more essential due to diagnostic assessment and economic significance of fish (Pradhan et al., 2012). Many publications have reported on qualitative and quantitative variations in haematological/blood parameters such as White Blood Cells (WBC) count, Red Blood Cells (RBCs) count, Haematocrit (Hct) and Haemoglobin (Hb) in fish, as a sign of the wellbeing of the fish.
The status of the above haematological parameters in fish depend many factors, for instance the level of contaminants (concentration), temperature, nutrition, and fish species among others. Therefore, alteration in haematological parameters is considered as a prognostic tool as well as a primary warning signal of the disturbance in homeostatic defence abilities of fish (Sharma and Langer, 2014). Interestingly, the haematological study in fish offer valuable diagnostic tools and progress in establishing normal range values for blood parameters of diverse fish species (Pradhan et al., 2012).
In the culture of fish, the use of monospecies or multispecies have been reported to enhance fish health particularly enhancement of antibody levels, lysozymes and cytokine activities (Agrawal, 2005; Allameh et al., 2017) as well as haematological parameters in fish (Dawood et al., 2017). Extensive research and comprehensive reviews have been written on the contributions of probiotics for the enhancement of fish health regarding some of the above mentioned immune parameters. Although many interventions have been made by researchers regarding the use of probiotics in improving blood profiles of fish, (Dahiya et al., 2012; Kiron and Watanabe, 2010; da Paixão et al., 2017) there isn't any summarised information regarding the effects of probiotics on haematological parameters in fish. The purpose of the review is to reveal the influences of probiotics on haematological parameters in fish.
In the circulatory system, blood aids many transport functions. RBCs, WBCs, and plasma (extracellular space around these cells) are the three main modules with the blood cells (Farrell, 2011). The other body tissues within the blood differ in its extracellular volume, which is usually twice as large as the intracellular volume (that occupied by the cells) (Farrell, 2011). Principally, blood function as a transport medium for gases and solutes that include oxygen, carbon dioxide, nutrients, ammonia, and hormones (Farrell, 2011). Basically, the concentrations of the various components of blood are regulated while some are tightly regulated (homeostatic roles) include electrolyte (ion) concentrations, pH, arterial oxygen and carbon dioxide tensions, and osmolarity (Farrell, 2011). Relatively, arrangement of blood is represented by the relative heights of the packed layers of RBCs, WBCs, and plasma (Farrell, 2011). The term used for the percentage of the column of blood occupied by RBCs is Haematocrit (Hct), while percentage of the column of blood occupied by WBCs is leucocrit (Lct). Usually, fish blood comprises 60–80% plasma, 20–40% RBCs, and 0.5– 2.0% WBCs (Farrell, 2011). Others haematological parameters including MCH, MCHC, and MCV can be calculated or derived using information from the RBCs, Haematocrit and Haemoglobin measurements (Dahiya et al., 2012).
Effect of probiotics in Haematocrit (hct)
Gallaugher (1994) defined Haematocrit (hct) as the measure of blood capacity to carry oxygen. In an organism, the higher the hct value, the higher the blood’s moving ability of oxygen. Hct values in teleost have been reported to range from 0% to 70% (Fletcher and Haedrich, 1987; Graham and Fletcher, 1983; Graham and Fletcher, 1985; Wells and Baldwin, 1990; Wells and Weber, 1991). Typically, hct values have been reported to range from 17% - 44% in rainbow trout (R M G Wells and Weber, 1991), 52.5% mackerel (Auxis rochei), 53% tuna (Thunnus thynnus), 43% blue marlin (Makaira nigricans) 8.5% hagfish (Eptatretus cirrhatus) (Satchell, 1991; Fánge, 1992). Higher hct values in organism indicate healthier conditions (Gallaugher, 1994) and can also indicate a response to increase stress thus serving as an antistress response to compensate for increased demand for oxygen for metabolic energy (Gallaugher, 1994).
For instance supplementation with primalac probiotics (Nikandishan Farjad Commerce Corporation, Tehran, Iran) containing LactoBacillus acidophilus, LactoBacillus casei, Enterococcus faecium and Bifidobacterium bifidium have been reported to increase the hct levels in Caspian roach fry subjected to salinity stress compared to those fed with the control diet (Imanpoor and Roohi, 2015). Also, infection with pathogenic bacteria Streptococcus iniae was shown an increase in hct and survival of Nile tilapia immersed in Bacillus sp.
In other research reports, the application of a mixed probiotic species of Lactococcus rhamnosus and Lactococcus lactis in red seabream (Dawood et al., 2017), Bacillus sp. in Nile tilapia (Feliatra et al., 2018), a combination of B. cereus and B. subtilis in Nile tilapia (Garcia-marengoni et al., 2015) and L. rhamnosus on rainbow trout (Kiron and Watanabe, 2010) have been reported to increase the Hct levels. These reports on fish fed probiotic-supplemented diets were indicated to have better health status compared to those fed control diets. Taken all the above into consideration, fish fed probiotic-supplemented diets might have higher hct levels and could respond better to stressors. This indicates that the anti-stress response in fish can be improved after feeding probiotic-supplemented diets as have been previously reported (Feliatra et al., 2018).
Effects of probiotics on Haemoglobin (Hb) concentration
According to Bolliger and Everds (2012), Haemoglobin (Hb) concentration is defined as the amount of total Hb per volume of whole blood and is determined by spectrophotometric method after lysis of red blood cells. Riggs (1976) reported that the purpose of Hb appears to conform to diverse metabolic needs of animals with constant environmental changes and also play a significant role in carrying oxygen from the gas-exchange organs to peripheral tissues (de Souza and Bonilla-Rodriguez, 2007). In fish, Hb form a line bet/ween the organism and the environment (Landini et al., 2002) but specifically through environmental changes and modifications in temporal and spatial variations, with oxygen availability as compared to terrestrial animals (Brix et al., 2004; de Souza and Bonilla-Rodriguez, 2007). Accounts on the utilisation of probiotic-supplemented diets hold a promise in increasing the Hb levels of fish.
For example, in contrast to the fish fed control diets, the application of a probiotic species of Lactococcus sporogenes in Clarius batrachus (indian magur) (Dahiya et al., 2012) and combined dosage of these probiotics L. sporogenes, L. acidophilus, B. subtilis, B. licheniformis, Saccharomyces cervirial in Cirrihinus mrigal (Sharma et al., 2013) Bacillus np5 in Oreochromis niloticus (Tanbiyaskura et al., 2015), LactoBacillus and Bifidobacterium in Clarias gariepinus (catfish) (Kiron and Watanabe, 2010), Bacillus subtilis, Saccharomyces cerevisiae in mori, L. acidophilus and β-glucan, in snakehead (Talpur and Ikhwanuddin, 2013), Bacillus cereus in juvenile nile tilapia (Garcia-marengoni et al., 2015) , B. Pamillus in Labeo rohta (Rajikkannu et al., 2015), B. Licheniforms and B. subtilis in Rutilis frisii kutum (Azarin et al., 2015) have been reported to increase the Haemoglobin levels. In these reports fishes fed probiotic-supplemented diets were indicated to have better health status compared to those fed control diets.
Effects of probiotics on RBCs
The amount of RBCs also known as Erythrocytes (ERI) in a given volume of blood is determined using an automated counter (Bolliger and Everds, 2012). In fish, RBCs are elliptical or ovoid, but species have changes in RBC dimensions and length from 10– 20 mm and 6–10 mm in width (Farrell, 2011). The primary role of RBCs is the transport of oxygen by the high concentrations of the respiratory pigment Haemoglobin and pH regulation (Farrell, 2011). Fish fed with probiotic-supplemented diets can intensify the RBCs levels of fish compared to that of those fed control/ unsupplemented diets (Azarin et al., 2015).
For example, the application of a mixed probiotic species of L. sporogenes in C. batrachus (Dahiya et al., 2012), a combined dosage of L. sporogenes, L. acidophilus, B. subtilis, B. licheniformis and S. cervirial in C. mrigal in (Sharma et al., 2013) and B. licheniformis and B. subtilis in Rutilis frisii kutum (Azarin et al., 2015), LAB in rainbow trout, L. plantarum in Nile tilapia (Faramaz et al., 2011) were reported to have significantly higher RBCs compared to fish feed supplemented diets. Also evidence of various scientific works depicts, the use of probioticsupplemented improved the RBCs level in the fish species.
Effects of probiotics on WBCs count
Medicine net defines WBCs count (leukocyte count) as the number of WBCs in the blood and can be measured as part of the Complete Blood Count (CBC). Fish WBCs function importantly in cellular defense and immunity. However, the functional effects of WBCs found in tissues (thymus, spleen and kidney) outside of the blood are also production sites (Farrell, 2011). Fish regulate the number of circulating WBCs, despite their minuscule representation in the fish blood (Leucocrit (Lct) = 0.3–1.0%) but toxicants and stress can depress leucocrit (Farrell, 2011).
The practices of probiotic-supplemented diets have been testified to hold capacity in increasing the WBC levels of fish compared to that of those fed control/unsupplemented diets. In contrast to the fish fed control diets, L. sporogenes as probiotic in C. batrachus (Dahiya et al., 2012), the application of a mixed probiotic species L. sporogenes, L. acidophilus, B subtilis, B. licheniformis and S. cervirial in C. mrigal (Sharma et al., 2013), B. subtilis in rainbow trout (Kamgar and Ghane, 2014), mixed probiotic species of B. subtilis and S. cerevisiae in C. mrigala (Ullah et al., 2018), L. acidophilus and B. subtilis in Nile tilapia (Aly et al., 2008) have been reported to increase the WBC levels. Fish fed with probiotic supplemented diets were indicated to have better immune responses compared to those fed control diets. Also fish fed with probiotic-supplemented diets might have higher WBC levels and could respond better to stressors. Feeding fish with probiotic-supplemented diets enhanced immune defense (Munir et al., 2018).
Effects of probiotics on other blood parameters
The first blood derivatives is the Mean Corpuscular Haemoglobin Concentration (MCHC), which represents the average Haemoglobin concentration within erythrocytes and calculated by dividing the whole blood Haemoglobin value (in grams per decilitre) by the hct (as a percentage) and multiplying by 100 expressed as grams per decilitre of erythrocytes (John and Harvey, 2012). The concentration of Haemoglobin within a RBC is usually expressed as Mean Cell Haemoglobin Concentration (MCHC) and over a wide range of fish species and conditions (physiological and environmental) (Farrell, 2011). The administration of probiotics as feed in an infected H. fossils (Mohideen and Haniffa, 2015) elicited alteration in MCHC, a combination of B. subtilis and S. cerevisiae in a C. mrigala (Ullah et al., 2018), B. subtilis in rainbow trout (Kamgar and Ghane, 2014) and B. pumilus in Labeo rohita (Rajikkannu et al., 2015).
The second blood derivative is the Mean Corpuscular Haemoglobin (MCH). MCH is defined as the average amount of Haemoglobin found in each RBC and is determined by dividing the Haemoglobin by the RBCs (Bolliger and Everds, 2012). In general, MCH is the least useful haematology parameter, because of it’s insensitive to change and provides little additional information than other RBC parameters (Bolliger and Everds, 2012).
The administration of probiotics as feed-in experimentally infected H. fossils elicited alteration of MCH (Mohideen and Haniffa, 2015). A combination of B. subtilis and S. cerevisiae in C. mrigala (Ullah et al., 2018), B. subtilis in rainbow trout (Kamgar and Ghane, 2014) and B. pumilus in Labeo rohita (Rajikkannu et al., 2015)
The final blood derivative is the Mean Corpuscular Volume (MCV). The average size of Red Blood Cells is known as MCV (Bolliger and Everds, 2012). When a haemocytometer determines cell counts, MCV is calculated. For instrumentgenerated cell counts, red blood cell volume is measured during the cell count described above, and the MCV is determined by the histogram (Bolliger and Everds, 2012). If the cells are counted using a haemocytometer, the MCV is determined by dividing the Haematocrit by the RBC (Bannerman, 1983; Bolliger and Everds, 2010; Hall, 2007). The administration of probiotics as feed-in infected H. fossils (Mohideen and Haniffa, 2015), Bacillus licheniformis and Bacillus subtilis fed in Kutum (Rutilus frisii kutum) fry (Azarin et al., 2015), Bacillus licheniformis and Bacillus subtilis in matrinxa˜ (Brycon amazonicus) breeders fed (Dias et al., 2012) and B. pumilus in Labeo rohita (Rajikkannu et al., 2015).
Ultimately, the levels of MCH, MCV and MCHC in the blood serve as the determinants of Haemoglobin in the RBC.
Table 1 describes the influence of monospecies and multispecies probiotics and how it affects haematological indices in some fish species.
Table 1: Probiotic effects on Haematological parameters in fish.
|Haematological effect||Species of fish||Reference|
|Lactococcus rhamnosus and Lactococcus lactis||Increase hct||Red seabream||Dawood et al., (2017)|
|Bacillus spp||Increase hct||Nile tilapia||Feliatra et al., 2018)|
|Bacillus cereus and Bacillus subtilis||Increase hct||Nile tilapia||Garcia-marengoni et al., (2015)|
|Lactococcus rhamnosus||Increase hct||Rainbow trout||Kiron and Watanabe, (2010)|
|Lactococcus sporogenes||Increase Hb||Indian magur (Clarius batrachus)||Dahiya et al., (2012)|
|L. sporogenes, L. acidophilus, B. subtilis, B. licheniformis, Saccharomyces cervirial||Increase Hb||Cirrihinus mrigal||Sharma, Sihag and Gahlawat, (2013)|
|Bacillus NP5||Increase Hb||Nile tilapia||Tanbiyaskura et al., (2015)|
|Lactobacillus and Bifidobacterium||Increase Hb||Catfish||Kiron and Watanabe, (2010)|
|L. acidophilus and β-glucan||Increase Hb||Snakehead||Talpur and Ikhwanuddin, (2013)|
|Bacillus cereus||Increase Hb||Juvenile Nile tilapia||Garcia-marengoni et al., (2015)|
|Bacillus pamillus||Increase Hb||Labeo rohta||Rajikkannu et al., (2015)|
|B. licheniforms and B. subtilis||Increase Hb||Rutilis frisii kutum||Azarin et al., (2015)|
|Lactococcus sporogenes||Increase RBCs||Clarius batrachus||Dahiya et al., (2012)|
|L. sporogenes, L. acidophilus, B. subtilis, B. licheniformis, Saccharomyces cervirial||Increase RBCs||Cirrihinus mrigal||Sharma, Sihag, and Gahlawat, (2013)|
|L. plantarum||Increase RBCs||Nile tilapia||Faramaz et al., (2011)|
|B. licheniforms and B. subtilis||Increase RBCs||Rutilis frisii kutum||Azarin et al., (2015)|
|Lactococcus sporogenes||Increase WBCs||Clarius batrachus||Dahiya et al., (2012)|
|L. sporogenes, L. acidophilus, B. subtilis, B. licheniformis, Saccharomyces cervirial||Increase WBCs||Cirrihinus mrigala||Sharma, Sihag and Gahlawat, (2013)|
|B. subtilis||Increase WBCs||Rainbow trout||Kamgar and Ghane, (2014)|
|B. subtilis and S. cerevisiae||Increase WBCs (Ullah et al., 2018)||C. mrigala||Ullah et al., (2018)|
|L. acidophilus and B. subtilis||Increase WBCs||Nile tilapia||Aly et al., (2008)|
Stress responses on probiotics in fish
Many research studies have shown that, stress is a major factor that hinders fish culture. However, the use of probiotics shows a promise in improving stress tolerance (Balcázar et al., 2006; Gatesoupe, 1999; Verschuere et al., 2003; Wang and Lin, 2008).
For instance, Taoka et al. (2006) reported commercial probiotic Alchem Poseidon (comprising a mix of Bacillus subtilis, LactoBacillus acidophilus, Clostridium butyricum and Saccharomyces cerevisiae) increased stress tolerance in P. olivaceus, under closed recirculating system. Hernandez et al. (2010) reported that juvenile Porthole livebea er fed with Artemia nauplii enriched with L. casei withstand stress tolerance (after 1 hour of air exposure). D. labrax fed with LactoBacillus delbrueckii decrease the cortisol levels (Carnevali et al., 2006) as well as Sparus auratus fed with probiotics, LactoBacillus fructivorans and LactoBacillus plantarum decrease the cortisol level under acute stress situations (Rollo et al., 2006). Pagrus major fed with probiotics, LactoBacillus plantarum (LP20) indicate high tolerance against low salinity stress (Dawood et al., 2017). Also Yokoyama et al. (2005) reported that the Japanese flounder fed with probiotics Lactoferrin withstand high stress tolerance when subjected to high temperature. Also, transportation of the commercial Amazonian ornamental fishes such as marbled hatchet fish Carnegiella strigata (Gomes et al., 2008); cardinal tetra (Paracheirodon axelrodin) (Gomes et al., 2009) indicate decrease in cortisol levels with the fish fed with commercial probiotic Efinol®. Fish fed with probiotic Pdp11 improve stress tolerance to HSD than control diet in S. auratus (Varela et al., 2010). In S. auratus, HSD a chronic stressor, that triggers the stress axis, creating an elevation of plasma cortisol (Mancera et al., 2008; Polakof et al., 2006; Sangiao-Alvarellos et al., 2005).
The presence of B. subtilis in the diet of Nile tilapia decreased the stress associated with high stock density (Telli et al., 2014). Thy et al. (2017) reported that the striped catfish (P. hypophthalmus) subjected to ammonia stress after B. amyloliquefaciens 54A, and B. pumilus 47B diet supplementation indicate low mortality. The mixture of Bacillus species (B. subtilis, B. pumilus, B. amyloliqueficiens and B. licheniformis) enhanced yellow perch's stress tolerance to hypoxia and air-exposure (Eissa et al., 2018).
Haematological indices including WBCs, RBCs, Hb, Hct, MCHC, MCV and MCH are used as essential diagnostic tools to assess the health status of fish. The level of these indices which depends on species, age, and environmental conditions can be used to access the physiological state of fish. Although many potential probiotic bacteria have been identified for use in aquaculture, Lactic acid and Bacillus species are common probiotics that have been reported to modulate most of these haematological indices. Besides, research data available indicate probiotic investigation on haematology is mostly based on optimal culture conditions as well as responses following pathogen infections. However many environmental perturbations such as changes in temperature, the concentration of pollutants, nutrition among others are known to cause physiological stress in fish.
Therefore, we suggest that more research will be necessary to determine the mitigation effects of probiotics on haematological indices of fish under such conditions and also how probiotics can influence the haematological parameters in the fish because haematological parameters assessed the physiological state of the fish.
The study was supported by National Natural Science Foundation of China as the funder and the award number is 31572651.