1 Background

The aquaculture industry in Nigeria has undergone rapid expansion in recent years (FDF, 2007). With the demand for fish, farmers have intensified production resulting in higher stocking and feeding rates (Tiamiyu and Solomon, 2011). Concurrent with the increase in production, the industry has experienced an increase in fish health problems (Oladele et al., 2010; 2011) as experienced by other animal agriculture industries following the development of intensive production systems (Post, 1993; Roberts and Ellis, 2001).

Clarias gariepinus in Nigeria grows well under various culture systems including earth pond systems (Fagbenro et al., 2004; FAO, 2014).The Southern Guinea Savanna agro-ecological zone with a bimodal rainfall (averaging 1,000 mm to 1,300 mm per year) and maximum temperatures 26.0 to 38.0°C (Jidere et al., 2007) with several drainage and wet land systems is suitable for farming C. gariepinus in earthen ponds (Teugels, 1990; Ugwumba and Nnabuife, 2008; Ejiola and Yinka, 2012).

Serum biochemical parameters have been described as excellent physio-pathological indicators of fish health (Roberts, 2012). These parameters have been utilized as a diagnostic tool not only for physiological alterations occasioned by stress (Hrubec et al., 2001; Wagner and Congleton, 2004), but also for detecting structural alterations in organs/systems produced by diseases (Sandnes et al., 1988; Svodova and Vykusova, 1991); exposures to toxicants and or nutritional insufficiencies (Tacon, 1992; Artacho et al., 2000; Adeyemo et al., 2016).

The changes in some serum biochemical indices of farmed Clarias gariepinus caused by stress due to exposure to environmental pollutants, pathogen challenge, or nutritional toxicities have been reported (Onusiruke and Ufodike, 2000; Oladele et al., 2011; Adeyemo et al., 2016). According to Lio-po and Lim (2002) more data is needed for monitoring of managements and or therapeutic interventions in the culture of this fish species. Hence the present data would provide useful information for disease diagnosis C. gariepinus farmed in earthen ponds in the Guinea Savanna agro-ecological zone of Nigeria.

2 Materials and Methods

The Southern Guinea Savanna agro-ecological zone of Nigeria lies within latitudes 8°4’ and 11°3’N and longitudes 2°41’ and 13°33’E (Jidere et al., 2007). For the purpose of this study, the zone was delineated into three areas (area A consisting of Abuja and environs (9.08°N, 7.40°E); area B consisting of Lafia and environs (8.51°N, 8.52°E) and area C consisting of Makurdi and environs (7.73°N, 8.54°E). The sampling areas are at least between 200 km apart from one another. For each area sampled, longitude, latitude, and elevation data were recorded using a hand-held Global Positioning System device (eTrex GPS, Garmin Corporation, Olathe, KS).

2.1 Selection of samples

A total of two hundred and forty (240) apparently healthy C. gariepinus farmed in 24 earthen ponds (ten farms from each sampling area) were used for the study. Selection of farm was based upon good management/farming practice pond culture water sauced from a deep bore hole; non use of antibiotics (Langdon and Jones, 2002), and chemotherapeutic agents accompanied with regular and correct feeding using certified commercial diets; while the selection of sample fish was based upon an exclusion criteria of weight (250 g ≤ fish ≤ 450 g as juveniles and 451 g ≤ fish ≤ 1000 g as adults) with the absence of lesions and discernible ecto parasites using the method of Stoskopf (1993). Fish sampling was accomplished between the months of July 2016 and July 2017.

2.2 Collection of blood for serum biochemical analysis

Blood samples for clinical chemistry determination were collected from the caudal vein via caudal veni-puncture using a 5.0 ml syringe fitted with a 19 gauge needle. About 2 to 3 ml blood per fish were collected into a clean glass test tube. The blood sample was kept at room temperature for 30 minutes to clot. Afterwards, the test tube containing the clothed blood was centrifuged at 3000 revolutions per minute for 5 minutes using a table centrifuge, to enable a complete separation of the serum from the clotted blood. The clear serum supernatant was then carefully eluted with a clean syringe and needle and stored in an Ependorph tube at 0.0°C for the clinical chemistry determinations. Biochemical parameters were determined by a semi-automatic chemistry (RT-1904CV, China) analyser according to the manufacturer’s protocol. Reagents were supplied by Spectrum Diagnostic (Spectrum Lab, Egypt). The serum biochemistry reference interval was determined according to Friedrichs et al. (2012), following the guidelines of the National Committee for Clinical Laboratory Standards.

2.3 Statistical analysis

Data were assessed for normality using the Kolmogorov-Smirnov test and confirmed to be normally distributed (P<0.05). Using the methods of Horn and Pesce (2003), outliers were screened and eliminated when found. The lowest and highest values measured for each analyst and the 25^th and 75^th percentiles were mathematically determined. The difference in serum biochemical parameters between male and females and between juveniles and adults were statistically analyzed using appropriate tests with the accepted level of significance set at P≤0.05.

3 Results

The mean values water quality parameters obtained from the sampling areas are presented in Table 1. Farms in Abuja environs had the highest water hardness (168.00 ± 10.19 mg/l CaCO₃), while farms in Lafia area had the least water hardness (142.39 ± 17.04 mg/l CaCO₃). The dissolved oxygen concentration and water temperature of ponds in Abuja area are 5.62 ±1.07 (mg/l) and 21.3 ± 2.01 (°C) while those for Lafia and environs are 4.97 ± 1.91 (mg/l) and 29.60 ± 1.65 (°C). The PH of pond water in sampled farms in Abuja, Lafia and Makurdi areas are 7.28 ± 0.02; 8.11 ± 1.05 and 7.78 ± 1.04 respectively. The electrical conductivity of the pond water ranged from 296.62 ± 5.18 (in Abuja areas) to 415.09 ± 17.24 (in Makurdi sampling area).

3.1 Selected serum biochemistry values of Clarias gariepinus sampled earthen ponds in the Southern Guinea Savanna Agro-ecological zone of Nigeria

The serum biochemistry results for juvenile males and juvenile females collected from the three sampling areas were not different form one another, and are as presented in Table 2a and Table 2b. The Serum total protein ranged from 31.22 g/dl to 42.11 g/dl with the 25^th percentile (Q₁) having a serum total protein value of 33.55 g/dl while the 75^th percentile (Q₃) had a value of 38.05 g/dl (Table 2a). Serum glucose ranged between 17.18 mg/dl and 26.33 mg/dl while the AST values for male juvenile C. gariepinus ranged between 90.78 IU/L and 101.91 IU/L with a 25^th percentile and 75^th percentile values of 60.105 IU/L and 100.24 IU/L respectively. In this group of fish, serum cholesterol concentration ranged from 91.00 to 99.09 mg/dl, and serum triglycerides concentration ranged between 77.21 and 83.67 mg/dl with a median value of 78.99 mg/dl (Table 2a). In juvenile female C. gariepinus, the serum globulin concentration ranged from 8.39 g/dl to 50.83 g/dl with a 25^th percentile and 75^th percentile values of 26.85 g/dl and 41.87 g/dl as shown in Table 2b. These values are significantly (P < 0.05) higher than values obtained for serum albumin (Table 2b).

The result for adult male C. gariepinus sampled from the Southern Guinea Savanna agro-ecological zone are as presented in Table 3a. It shows that mean total protein is 41.26 g/dl, the 25^th and 75^th percentiles are 38.96 g/dl and 43.57 g/dl respectively. In this group, the serum globulin concentration was also considerably higher than those of albumin among adult female C. gariepinus (32.08 g/dl for Globulin, 9.18 g/dl for Albumin). In adult female C gariepinus sampled from this area, the serum total protein ranged from 33.74 g/dl to 41.44 g/dl with a 25^th and 75^th percentiles values of 37.70 g/dl and 33.74 g/dl respectively. The serum globulin ratio ranged between 24.11 g/dl and 33.22 g/dl with a 25^th and 75^th percentile values of 29.056 g/dl and 31.944 g/dl respectively and serum alkaline phosphatase (ALP) activity ranged between 19.43 and 39.01 IU/L (Table 3b).

A statistical evaluation of serum biochemical parameters of fishes sampled from Abuja shows that the mean values obtained for Albumin, Globulin, Albumin-Globulin ratio, AST and ALT were not significant different (P>0.05) for all the groups whereas, the values obtained for serum total protein varied significantly (P<0.05) between sexes and developmental stages (Table not shown).

4 Discussion

Serum biochemical parameters can vary within and between species of fish (Hrubec et al., 2001; Knowles et al., 2006), These values have also been reported to be dependent on the physico-chemical parameters of the culture water, health status and nutrition of the cultured organism (Farrell, 1997). The objective of the determination of serum biochemical reference intervals in apparently healthy subjects is to establish a basis for comparisons of suspected organ or system dysfunctions before the institution and or monitoring of interventions in farm or clinical settings (Langdon and Jones, 2002).

The serum total protein concentration range from 17.01 to 58.31 mg/dl were significantly higher than the values reported for Senegalese sole (Solea senegalensis) (Peres et al., 2014), however, the values are closer (30.8 to 61.6 g/dl) in feral Clarias gariepinus (Myburgh et al., 2008) from Zambezi River in Southern Africa. The serum albumin levels obtained were higher than values reported by Myburgh et al. (2008) but found similar to Atlantic salmon (Salmon salar) (Sandnes et al., 1988) and Red pacu (Piaractus brchyponus) (Sakamoto et al., 2001). Tacon (1992) observed the existence of a wide variability in the serum proteins amongst farmed fishes. The serum globulin levels was significantly less than 21.8 g/dl to 44.4 g/dl reported by Myburgh et al. (2008), this variability may however be attributable to genetic variability, health status of the fish or even to difference in water quality parameters (Hrubec et al., 2001). The serum globulin levels as observed in the present study are similar to the normal serum globulin values reported by Yilmaz et al (2006). The serum albumin-globulin ratio ranged from 1.0 to 13.3 as against 0.21 to 0.54 reported by Myburgh et al. (2008). The reason for this wide variability may have its root in genetic differences between both groups of fish; however, globulin concentration levels in healthy Clarias gariepinus are less than those of albumin.

4.1 Serum glucose concentration

The serum glucose concentrations obtained in this study are lower than the values reported for C. gariepinus treated with sub lethal concentrations of potassium permanganate (Kori-Siakpere et al., 2011)and apparently healthy feral samples from the Nile River (Sabiri et al., 2009); The results of the present study are however, similar to those reported for C. gariepinus by Yilmaz et al. (2006) and in the Silver catfish (Rhamdia quelen) (Lermen et al., 2004), as well as in Sea Bass (Dicentrarchus labrax) (Coz-Rakovac et al., 2005). An increase in serum glucose concentration in teleosts has been reported to be caused by a wide variety of environmental stressors including hypoxic conditions, starvation and capture stress (Hunn and Greer, 1991; Percin and Konyalioglu, 2008; Kori-Siakpere et al., 2011).

4.2 Serum alanine amino transaminase (ALT) and aspartate transaminase (AST) activity

The ALT values in the present study ranged from 6.13 IU/L to 27.14 IU/L and the AST values ranged from 37.05 IU/L to 121.64 IU/L. These values markedly differs from those obtained in feral Clarias gariepinus by Myburgh et al. (2008), who had reported values ranging from 0.0 IU/L to 27.0 IU/L for alanine amino transaminase (ALT) and 43.6 IU/L to 157.8 IU/L for aspartate amino transaminase (AST). ALT and AST belong to the non-plasma specific enzymes which are localized within tissue cells of the liver, heart, gills, kidneys, and muscles (Hunn and Greer, 1991; Percin and Konyalioglu, 2008). Heavy metal compounds, pesticides, sublethal concentration of phenols and organic pollution may induce changes in blood ALT and AST activities in the serum of fish (Asztalos et al., 1990). In this study, AST activities were lower than values reported in Esox luscius (252.00 to 583.8 IU/L), Lenhardt (1992); Salmo salar (278.0 ± 73.16 IU/L), Sandnes et al. (1988). But were higher than values reported in Piaractus branchyponus (49.10 IU/L), Sakamoto et al. (2001). AST activities in the sampled C. gariepinus catfish were similar to those reported in Oreochromis nilocticus (230.5 ± 219.6 IU/L), Chen et al. (2002) while ALT activities were higher than values reported for Salmo salar (6.00 IU/L), Sandnes et al. (1988); but were much lower than values reported in Oreochromis niloticus (Chen et al., 2002) and in Channel catfish Ictalurus punctatus (Tavares-Dias and Moraes, 2007).

4.3 Serum alkaline phosphatase activity

Alkaline phosphatise (ALP) occurs in every tissue but its highest activities are found in the liver, bone, intestine and kidney (Campbell, 2012) and also of diagnostic aid in degenerative hepato-biliary diseases (Peres et al., 2014). Increases in serum ALP activity is most likely a result of hepatocellular injury and or biliary obstruction (Post, 1993). The serum alkaline phosphates activity of the sampled C. gariepinus catfish ranged from 18.53 to 42.11 IU/L with a median value of 22.40 IU/L. These values although similar to the values reported for C. gariepinus by Myburgh et al. (2008), they were significantly higher than the values reported by Knowles et al. (2006) for cultured Short Nose Sturgeon (Acipenser brevinostrum) and by Peres et al. in Senegalese sole (Solae senegalensis).

4.4 Serum creatinine, urea and uric acid concentrations

Creatinine is a metabolite of muscle creatine and urea is a product of the deamination of glucogenic amino acids via the alanine cycle. Physiologically, the kidney excretes urea, uric acid and creatinine; hence the utility of the serum concentration of these analytes as biomarkers for kidney function (Rock et al., 1987), and elevation of the serum concentration of these analytes has been reported to be indicative of impairments of kidney function (Loeb, 1991). The serum creatinine concentration ranged between 0.17 and 0.48 mg/dl while the serum urea concentration ranged from 1.98 to 4.12 mg/dl; this is similar to the findings of Yilmaz et al. (2006) and Myburgh et al. (2008). The serum uric acid concentration ranged from 1.00 to 1.67 mg/dl, with the minimum value being lower than values reported by Yilmaz et al. (2006) in the same fish species.

4.5 Serum lactase dehydrogenase concentration

Lactose dehydrogenase (LDH) is the enzyme responsible for the formation of lactic acid during glycolysis and its oxidation to pyruvate during respiration (Campbell, 2012). An increased amount of serum lactose dehydrogenase is brought about by cellular damage or necrosis (Knowles et al., 2006). Normal levels of LDH found in the serum are indicative of the body’s routine destruction of senescent cells and their replacements. The serum LDH concentration in this study is lower than values reported by Yilmaz et al. (2006) and it is in conformity with that of Myburgh et al. (2008) for C. gariepinus.

4.6 Serum lipids

The serum lipids consists of the serum total cholesterol, high density lipid, low density lipid and serum triglycerides. Cholesterols are natural fats present in the body as part of cell membranes, lipoproteins, bile acids and steroid hormones (Blake and Ridker, 2001). Serum high density lipids (HDL) is one of the five major lipoproteins which are complex particles composed of multiple proteins which transport all fat molecules around the body. HDL is the smallest of the lipoproteins particles that primarily transports cholesterol to the liver or other steroidogenic organs such as the adrenals, ovary and the testes by both direct and indirect pathways; the serum HDL concentration in the present study ranged between 9.00 and 29.01 mg/dl.

Serum triglycerides are lipid compounds composed of glycerols esterified to three fatty acid chains. Increased serum concentrations of triglycerides termed hypertriglyceridemia when present with low HDL and elevated LDL concentrations is indicative of insulin resistance (Yuan et al., 2007). According to Brusell (2001) and Farrell (1997; 2002), elevated serum triglyceride concentration is a clinical risk factor especially when accompanied by decreased concentration in serum LDL. Very high levels of serum triglycerides have been linked to the development of pancreatitis in man (Abdel-Maksoud and Hokanso, 2002). The serum triglycerides concentration obtained in this study ranged from 45.39 to 92.04 mg/dl.   

5 Conclusion

This study revealed the normal serum biochemistry of Clarias gariepinus farmed in earthen ponds in the Guinea savanna agro-ecological zone of Nigeria, serves as a reference for future researchers to assess the health as well as to predict the onset of disease, allowing appropriate interventions and prevent occurrence of losses. However, additional investigations should be carried out on the effects of capturing stress on the serum enzyme activities of this important fish species.

Authors’ contributions

Adeyemo B.T (ABT): Study design; Serum biochemistry analysis and write up of the article; Shola S.G (SGS): Study design and statistical analysis; Apochi J.O (AJO): Fish sample collection and analysis; Obande R.A (ORA): Fish sample collection and write up; Dagah Helen (DH): Serum biochemistry analysis; Odo Joel (OJ): Fish sampling and Statistical analysis. All authors read and approved the final manuscript.

Acknowledgements

Authors wishes to acknowledge Dr Alani Abdulahi and Dr Egbe Emmanuel for invaluable contributions in the serum biochemical analysis and for reviewing the initial manuscript.

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