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75 75 Provisional clinical chemistry parameters in the African Sharptooth ( gariepinus) 25 25

5 5 J G Myburgha*, C J Bothaa, D G Booyseb and F Reyersc

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reported in fish in other parts of the ABSTRACT world2,18. Controlled laboratory exposure Pollution affects aquatic systems worldwide and there is an urgent need for efficient of Sharptooth catfish to copper concen- monitoring. Fish are generally sensitive to their environment and are thus considered to trations that were measured in the be valuable bioindicator species. The African Sharptooth catfish (Clarias gariepinus)is Olifants River, Kruger National Park, sig- particularly important in this respect because of its very wide distribution. In order to use nificantly altered their blood chemistry C. gariepinus as a bioindicator species its baseline clinical chemistry must be defined. parameters19. Existing data are scarce, and the objective of this work was therefore to establish clinical Given its wide distribution, the African chemistry parameters for Blood was collected from male and female catfish C. gariepinus. Sharptooth catfish could be a suitable and a number of clinical chemistry parameters were determined. Plasma protein values, bioindicator of pollution in a variety of but particularly those of plasma albumin, were found to be very low,approximately half the value for dogs, but similar to the values in Channel catfish (Ictalurus punctatus). Plasma urea freshwater aquatic ecosystems. However, values in Sharptooth catfish were found to be much lower than in dogs, but only marginally to be able to detect molecular and cellular lower than in Channel catfish. Plasma creatinine in Sharptooth catfish, however,was only a level changes in response to pollutants, quarter of that of dogs and one third of that found in Channel catfish. These findings may prior knowledge of physiological baseline 5 have implications for using urea and/or creatinine as an index of renal glomerular filtration, parameters is essential . There is, unfortu- as is done in mammals. Plasma enzyme activity ranges were much lower in Sharptooth nately, a dearth of information on the catfish than in dogs, particularly for alkaline phosphatase (ALP) and alanine amino- clinical chemistry of the African Sharptooth transferase (ALT). By comparison, Channel catfish have an even lower ALT activity range catfish. The objective of this paper is, but an ALP range that is very similar to dogs. The implications for using these enzymes as therefore, to define baseline clinical markers for liver disease are not clear from these data, as factors such as plasma half-life and chemistry parameters of C. gariepinus that tissue distribution remain to be determined. The very low plasma thyroxine (T4) levels have could be used as indicators of organ func- important implications for laboratory personnel, who will have to set up calibration and tion or damage, to assess the impact of standardisation adaptations for the methods that are generally designed for human pollution in aquatic ecosystems. samples. Although the sample size was too small for reliable comparisons, it appeared that there was little difference in the parameters measured between male and female fish. The MATERIALS AND METHODS values obtained are a useful starting point for using C. gariepinus as a bioindicator species. Key words: bioindicator, Clarias gariepinus, clinical chemistry, plasma enzymes, proteins, Experimental and facilities renal function, Sharptooth catfish, thyroxine, T . 4 Male (n = 13) and female (n = 15) African Myburgh J G, Botha C J, Booyse D G, Reyers F Provisional clinical chemistry parameters in Sharptooth catfish (Clarias gariepinus) the African Sharptooth catfish (Clarias gariepinus). Journal of the South African Veterinary with body mass ranging from 600–1000 g Association (2008) 79(4): 156–160 (En.). Department of Paraclinical Sciences,Faculty of Veteri- were obtained from an earth dam at the nary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110 South . University of Pretoria experimental farm. The fish were kept indoors in a 1500 tank containing municipal water. An external INTRODUCTION is one of the most important individual filter system was used to constantly circu- Aquatic ecosystems are subject to pollu- species in traditional freshwater fisheries late the water. The filter system was tion that may have dire effects3,18. Deleteri- in Africa15. It is widely distributed in Africa, cleaned and at least a third of the water in ous effects of pollution are often preceded where it occurs in almost any freshwater the tank was replaced every week. by subtle molecular and cellular changes habitat, but favours floodplains, large Commercial trout pellets were fed that can be detected in some inhabitants sluggish rivers, lakes and dams15. The fish 3 times a week. of these ecosystems5,12,19. Fish are suitable is omnivorous, feeding on fish, birds, bioindicator animals because they respond frogs, small mammals, reptiles, snails, Collection of samples with great sensitivity to changes in the crabs and other invertebrates. It is also After a depuration period of 1 month aquatic environment3,14,18,19. capable of feeding on seeds and fruit15. the fish were netted, removed from the The Sharptooth catfish (Clarias gariepinus) Numerous chemical pollutants have holding tank and placed in a lateral posi-

100 been detected in tissue samples from tion. Fish were not anesthetised for blood 100 aDepartment of Paraclinical Sciences, Faculty of Veteri- nary Science, University of Pretoria, Private Bag X04, Sharptooth catfish collected in South collection. Blood was collected from the 7 95 Onderstepoort, 0110 South Africa. Africa . Anatomical and histological ab- central vein, located just below the spinal 95 bDepartment of Anatomy and Physiology,Faculty of Veter- normalities of the reproductive organs cord, caudal to the cloaca and using the inary Science, University of Pretoria, Private Bag X04, 75 Onderstepoort, 0110 South Africa. have been observed in Sharptooth catfish lateral line as reference point. The blood 75 cDigital Veterinary Diagnostics, PO Box 41468, Gars- collected from the Rietvlei and Marais was collected with heparinised (Heparin fontein East, 0060 South Africa. Dams near Pretoria, South Africa1. These sodium-fresenius, Intramed) 2 m sy- *Author for correspondence. E-mail: [email protected] aberrations were similar to those attributed ringes (Omnifix 2 m , B Braun Melsungen 25 25 Received: March 2007. Accepted: November 2008. to chronic exposure to aquatic pollutants AG) and 22 G needles (22GX1½’, Terumo).

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0 0 100 100 Table 1: Summary of the Sharptooth catfish clinical chemistry data (95 % confidence and/or 2.5 to 97.5 % percentile reference ranges). 95 95 Approximate normal / Non-normal data distribution or Sexual difference 75 Gaussian data data unable to be normalised by 75 log-transformation Parameter Units Lower Mean Upper Lower Median Upper Difference P-value P-value 95 % CL 95 % CL 2.5 % 97.5 % t-test Willcoxon 25 percentile percentile equal var. Rank 25

5 Length mm 430 550 660 NS 0.190 5 TPP g/ 30.8 46.2 61.6 NS 0.799 0 0 ALB g/ 8.1 12.35 16.7 Sig 0.036 GLOB g/ 21.8 33.1 44.4 NS 0.670 A/G ratio 0.21 0.37 0.54 Sig 0.014 ALT U/ 0 12.7 29 NS 0.345 ALP U/ 0.1 2.3 52 NS 0.849 AST U/ 0 117 285 NS 0.700 CK U/ 200 2370 28052 NS 0.888 GGT U/ 0 6.6 15.0 Sig 0.004 LD U/ 0 450 878 NS 0.959 HBD U/ 0 131 306 NS 0.597 GLD U/ 0 17 55 NS 1.000 T Bil µmol/ 4.4 16.4 38.7 NS 0.919 Urea mmol/ 0.3 1.9 3.4 NS 0.442 Creat µmol/ 0.0 20.5 29.0 NS 0.643

T4 nmol/ 0.0 4.5 7.5 NS 0.642

NS = no significant difference between male and female group data (P ≥ 0.05). Sig = significant difference between male and female group data (P < 0.05).

The blood was transferred to lithium hep- using Randox reagents (Randox Labora- allowable D’Agostino limit for skewness), arin blood collection tubes (BD tories) (optimised DGKC recommended the data were subjected to a logarithmic Vacutainer, Becton, Dickinson and Com- method). α-Hydroxybutyrate dehydro- transformation and the transformed pany) immediately after collection. Blood genase (HBD) was determined on an data re-tested for normality. was centrifuged at 1500 g, the plasma col- automated clinical chemistry analyser • Where the logarithmic transformation lected and frozen in sterile vials (1.8 m (Roche/Hitachi Modular Analytics) (opti- did not yield a good approximation of cryotube vials, Nunc A/S). Samples were mised DGKC recommended method). normality or when the D’Agostino submitted to the Clinical Pathology Labo- All enzyme activity assay results were criterion for kurtosis was exceeded,

ratory, Faculty of Veterinary Science, Uni- obtained at 37 °C. Total thyroxine (T4) was non-parametric statistical methods (vide versity of Pretoria for analysis. determined using a Coat-A-Count Total infra) were then applied to those data. 125 T4 solid phase Iodine Radioimmuno- • The reference interval (range) for those Clinical chemistry assay kit (Siemens Medical Solutions data that met the criteria for approxi- The NExCT™ clinical chemistry system Diagnostics). Radioactivity was measured mate normality (both untransformed as (Schiapparelli Biosysytems) was used to by an Auto-Gamma 5000 Series Gamma well as log-transformed) was based on quantify total plasma protein (TPP) Counting system. Owing to sample the 95 % confidence limit for 26 observa- (Biuret principle of Weichselbaum), total quality and/or availability, only 15 sera tions (mean ± 2.056 SD). bilirubin (T Bil) (van den Bergh diazo (8 from male fish sand 7 from female fish) • The reference intervals (ranges) for data principle), albumin (ALB) (BCG dye- were assayed. that were non-normal (even after log- binding method) and urea (enzymatic UV transformation) were calculated accord- method) using NExCT™ reagent kits. Statistical treatment of data ing to the non-parametric method of Creatinine (kinetic Jaffe method), alkaline Using the statistical software package, Rumke and Bezemer13, using the 2.5 % phosphatase (ALP) (pNP substrate and NCSS 2000 Statistical System for Windows and 97.5 % percentile approximation. AMP buffer), aspartate aminotransferase (Copyright J L Hintze, Kaysville, Utah, • In order to determine whether there (AST) (IFCC recommended kinetic UV USA, 1998), the following statistical proce- was a significant difference between method) and γ-glutamyltransferase dures were conducted: male and female fish data for the various (GGT) (optimised kinetic method of Szaz) • All data were inspected for outlier parameters, a Student’s t-test compari- were determined using ACE™ reagent values by both the National Committee son was performed on approximately kits and the ACE™ clinical chemistry sys- for Clinical Laboratory Science (NCCLS) ‘normal’ data and the non-parametric tem. Lactate dehydrogenase (LD) (opti- recommended Dixon algorithm8 as well Wilcoxon Rank-Sum Test was used

100 mised DGKC recommended method), as Horn’s algorithm based on the on ‘non-normal’ data. 100 alanine aminotransferase (ALT) (IFCC Box-Cox transformation16. Only data RESULTS 95 recommended kinetic UV method) and detected as ‘outliers’ by both methods 95 creatine kinase (CK) (NAC-activated were then defined as true outliers. The Laboratory results 75 DGKC recommended method) were de- data distributions were tested for ‘nor- The clinical chemistry results of the 75 termined by the Alfa Wassermann clinical mality’ (or approximation to a Gaussian Sharptooth catfish are summarised in chemistry systems (ACE® & NExCT™) distribution). Table 1 and also compared, in Table 2, using Alfa Wassermann reagents. Gluta- • Where the distribution did not pass the with data from the Channel catfish 25 25 mate dehydrogenase (GLD) was quantified test for normality (by exceeding the (Ictalurus punctatus) and the domestic

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0 0 100 100 Table 2: Clinical chemistry data of the Sharptooth catfish compared with data from the Channel catfish and domestic dog. 95 95 Recommended reference 95 % confidence range in Reference range in domestic dogs11 75 range in Sharptooth catfish 22 acclimated Channel catfish4 75 Derived from mean ± 2 SD Parameter Units Lower Mean Upper Lower Mean Upper Lower Mid-range Upper cutoff cutoff cutoff cutoff cutoff value cutoff 25 25

TPP g/ 34.4 46.2 58.0 22 34 46 54 64 75 5 5 ALB g/ 9.04 12.35 15.65 6 10 14 23 27 31 ALT U/ 0.1 12.7 25.2 0.0 2.6 5.8 10 60 109 0 0 ALP U/ 0.2 2.3 25.1 0.0 66 134 1 58 114 AST U/ 0 117 285 0.0 91 193 13 14 15 CK U/ 355 2370 15811 0.0 3156 6565 52 210 368 GGT U/ 0.0 6.6 13.2 0.0 2.4 6.4 LD U/ 0 450 878 0.0 177 499 0 118 236 T Bil µmol/ 4.4 16.4 38.7 0.0 3.4 6.8 0.0 2.6 5.1 Urea mmol/ 0.6 1.9 3.1 1.39 2.96 4.53 2.9 6.5 10.0 Creat µmol/ 0.0 20.5 29.0 0.0 61.8 132.6 44.2 97.3 150.3 4 T4 nmol/ 0.0 4.5 8.8 15.6 25.4 35.2 19.3 35.4 51.5

dog. Table 3 records the data for the and these data items were excluded ranges computed by the methods de- plasma chemistry constituents where a from further statistical calculations. scribed above. statistically significant difference be- However, the rest of the data from these DISCUSSION tween the sexes was found. fish were retained in the data set. • This left the data set with 13 male fish as Outliers Outlier rejection well as 13 female fish with just 2 addi- Although the view exists that outlier The inspection of data for outlier data tional data points deleted (a globulin data derived from clinically normal subjects values revealed that: value in a male fish and a GGT value in a should be retained in a dataset that is • A female fish (B02) was very signifi- female fish). intended to reflect the ‘normal’ or ‘healthy’ cantly different from the other fish in reference range, the NCCLS has accepted the study (being considerably smaller Data distributions that it is often not possible to identify with a length of 24.7 cm, compared with Using the d’Agostino criteria for skew- sub-clinical illness or pathology9 and the 95 % reference range of 38.1–69.8 cm). ness and kurtosis, ALP,AST,CK, LD, HBD, therefore recommends the elimination of Furthermore, this fish had outlier values GLD and T Bil were found to be too outliers (using appropriate statistical for plasma proteins (albumin and globu- severely skewed to be accepted as ‘ap- techniques, vide supra) if their inclusion lin) and total bilirubin as well as zero proximately normal’. In addition, length, could lead to establishing a reference

value results for ALT,ALP,AST,LD, HBD creatinine and T4 were found to show more range that loses its diagnostic sensitivity and creatinine. On the basis of these kurtosis than would be acceptable. Log- through being too ‘wide’. This is particu- findings, this fish’s data were excluded transformations were applied to the larly a threat when dealing with relatively from the data used for further statistical skewed data and ALP and CK were found small numbers of subjects in the sample treatment. to transform sufficiently well to approxi- from the population, as is the case here. • A female fish (B31) had outlier values mate normality. Those 2 parameters The NCCLS recommendation is that at for ALT, AST, LD and HBD as well as a were, thereafter, manipulated in their least 120 subjects should be included in zero value for GGT. It was decided that log-transformed state for statistical analy- the sample. In this study only 28 fish were this fish was probably not ‘normal’ (i.e. sis and only the final data were trans- used and the authors accept that, as a normal in terms of health) and should formed back to non-log (non-exponential) consequence, the derived reference not be included in the calculation of data. Consequently, length, AST, LD, ranges can, at present, only be provisional

reference ranges that purported to HBD, GLD, T Bil, creatinine and T4 data and that, in time, they will be re-evaluated reflect clinical chemistry data in healthy were handled by non-parametric statistical through the accumulation of more data catfish. approaches. from healthy catfish. In the interim, how- • Furthermore, the globulin value of ever, the authors believe that the proposed Fish B01 and GGT value of Fish B11 Reference ranges (derived) reference ranges will provide a were identified as outliers (by both the Table 1 reflects the 95 % confidence useful set of criteria to assist in using the Dixon as well as Box-Cox approaches) and/or 2.5 to 97.5 % percentile reference catfish as a bioindicator species.

Table 3: Data for the plasma chemistry constituents where a statistically significant difference between the sexes was found (ALB, A/G ratio and GGT).

100 100 Reference range for all fish in 95 % Confidence data for 95 % Confidence data for female this study (both sexes) male Sharptooth catfish Sharptooth catfish 95 95 Parameter Units Lower Mean Upper Lower Mean Upper Lower Mean Upper 75 95 % CL 95 % CL 95 % CL 95 % CL 95 % CL 95 % CL 75

ALB g/ 8.1 12.35 16.7 10.5 11.5 12.5 11.9 13.2 14.5 A/G ratio 0.21 0.37 0.54 0.30 0.34 0.38 0.37 0.41 0.46

25 GGT U/ 0.0 6.6 15.0 7.5 8.9 10.2 1.5 4.3 7.0 25

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The elimination of the 1st female fish T4 levels were very much lower in this such as C. batrachus are not always in 95 95 (B02) from the study can be justified on study than those reported in the same agreement, with specimens from 6 75 the basis that this was a very small speci- species during the breeding phase. How- studied apparently having a functional 75 men compared with the other fish (being ever, they were similar to, but still lower urea cycle10. only half the average length). Whether than, the values found during the quies- The level of plasma creatinine, a sensitive this fish’s small size was attributable to cent phase in the previous study. and reliable index of GFR in mammals11, 25 age or disease is not known (it did not Reports on clinical chemistry parame- suggests that it may not be as useful in 25 appear to be clinically ill and no age deter- ters in other species of fish include a Sharptooth catfish. However, Channel 5 minations were made). Furthermore, the limited number on catfish, although not catfish appear to have a serum creatinine 5 finding that this fish had the lowest TPP, Clarias spp. One study, on Channel catfish range that is similar to that found in 0 0 albumin and globulin values and zero (Ictalurus punctatus)4 is possibly the most mammals and it is reasonable to assume values for 5 plasma enzyme activities as substantial in terms of range of chemistry that, in that species, it would be an equally well as the creatinine concentration, con- parameters as well as number of fish useful index of GFR. In fish creatine stitute reasonable grounds for believing sampled. When the data on Channel predominates over creatinine. Creatine is that this specimen was not a typical mem- catfish from that publication are com- excreted through the kidneys, not the ber of an otherwise reasonably homoge- pared with the data in this study, there gills, and forms more than half of the neous group of healthy fish. are some very striking differences and urinary nitrogen excretion. It should be The elimination of the other female similarities. When, in addition, these data mentioned that GFR in fish is affected by fish (B31) is justified on the basis that it from fish are compared with those from the salinity of the water in which the fish represented 4 of the 7 identified ‘outlier’ domestic mammals (the canine range are kept, which would affect the useful- parameter values in the dataset. These 4 being used for illustrative purposes), ness of creatine as an index of GFR. relatively high plasma enzyme values there are further striking issues that beg to Although there are considerable differ- constitute reasonable grounds for believ- be addressed in future studies. These data ences in the plasma enzyme activities ing that, although it was not clinically ill, are reflected in Table 2. between Sharptooth catfish and Channel this fish may have harboured a sub- Sharptooth catfish appear to have a catfish, it is difficult to evaluate their clinical illness. slightly higher TPP range than Channel potential usefulness (in either species) as catfish (differing by some 10 to 12 g/ ). appropriate markers of tissue (organ) Reference ranges Compared with most mammals, how- damage. In mammals, an enzyme such The authors could find no previous data ever, this range is still 10 to 20 g/ lower. as Sorbitol (Iditol) dehydrogenase can on clinical chemistry reference ranges The difference between the 2 species of reflect very severe hepatic pathology 17 (with the exception of T4, see below) for catfish appears to lie in the globulin level, when it is elevated above 5 U/ , whereas this species and therefore it is difficult to as the fish do not differ substantially in CK in cattle for instance, can rise to values comment on whether these data meet terms of ALB. The ALB ranges found in in the thousands (even tens of thousands) any specific expectations. both Sharptooth catfish and Channel as a marker of myopathy. This demon- A somewhat disturbing feature is the catfish are substantially lower than that strates that the actual numeric value of number of zero activity results for enzyme found in mammals (6 to 15 g/ compared plasma enzyme activity is very poorly data. With ALT, ALP and GGT this is per- with 23 to 30 g/ ). In mammals a concen- correlated with the degree of tissue haps not surprising as the ranges are quite tration below 15 g/ is consistent with damage. Consequently, the fact that low and the modal values are not far from fluid loss from the plasma into body cavi- plasma ALT in Channel catfish is approxi- zero (20, 8 and 9 U/ , respectively). How- ties (such as ascitic transudate) and mately one third to one fourth of that ever, it is surprising to find zero values for interstitium (oedema). However, it is found in Sharptooth catfish and the AST, LD and HBD, as the ranges are set well-known that plasma protein concen- plasma ALT activity in the latter species is much higher and the modal values are, trations in fish are lower than in higher about 20 % of what is commonly encoun- relatively speaking, very high (316, 630 vertebrates5. tered in carnivores, may not offer any and 250 U/ , respectively). In addition, the A striking difference in clinical chemistry useful information about the potential finding of zero enzyme activity values parameters between these 2 species of usefulness of this enzyme as a marker of appears to recur within the same fish catfish and dogs relates to the renally hepatocellular pathology. In mammals, (plasma sample) and could suggest that cleared nitrogenous waste products, the transaminases ALT and AST as well as there may have been some sample or plasma urea and creatinine. The catfish the bile-duct-associated ALP and GGT sample-handling effect. Illustrating this is urea values appear to lie around the 1 to tend to be useful markers of hepato- the finding that each zero activity in LDH 5 mmol/ level, whereas mammals tend to cellular pathology and biliary pathology (and there are 4 such results) is correlated have plasma urea values in the 3 to respectively11. There are, however, impor- with at least 2 other enzyme zero activity 10 mmol/ range. Plasma urea is a fair and tant differences in their respective sensi- results. In the 30-year laboratory experience commonly used indicator of glomerular tivity. ALT is more liver specific than AST of one of the authors (FR), this is a very filtration (GFR) in dogs11 but could be a in carnivores than it is in ruminants, while unusual finding and could suggest the relatively poor (insensitive) index in plasma AST activity is almost the oppo- presence of inhibitors or analytical prob- Sharptooth catfish. Ip and co-workers10 site, being more sensitive in ruminants.

100 lems. This reservation should be borne in contended in an intriguing study that This difference is probably related to the 100 mind when using these recommended ammonia detoxification in C. gariepinus difference in the preferred metabolic path- 95 reference ranges for C. gariepinus, because does not follow the classical ‘urea cycle’ way for gluconeogenesis between carni- 95 low plasma enzyme activities may not (Krebbs Hensleit cycle) and that instead vores and ruminants. The ability to 75 bear any relationship to pathology. High C. gariepinus ‘actively excretes’ ammonia. anticipate the relative usefulness of a 75 enzyme activities, however,would never- The literature in this regard, however, serum enzyme activity is confounded by theless suggest increased release from appears somewhat contradictory, accord- another factor,namely its plasma half-life. damaged cells, increased enzyme induc- ing to these authors, and reports on urea A good illustration of this is seen in carni- 25 25 tion or delayed removal. cycle enzyme activity in related species vores where, in dogs, the half-life is long

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0 0 100 100 enough (2 to 3 days) to allow ALT to sensi- studies but may also, in the short term, during breeding and quiescent periods. 95 95 tively reflect, among other conditions, prove to be of value in monitoring the General and Comparative Endocrinology 129: 146–151 hepatobilliary pathology. In cats, how- health of farmed African Sharptooth cat- 75 7. HeathRGM,Claassen M 1999 An overview 75 ever, the half-life is only about 6 hours, fish. In this case, these parameters could of the pesticide and metal levels present in and consequently the plasma activity also be used as biomarkers to assess the populations of the larger indigenous fish only reflects very severe and sustained re- impact of pollution in their aquatic system. species of selected South African rivers. 25 lease (severe, ongoing pathology) but is In addition, the data from this study Water Research Commission Report, #428/1/99 25 generally insensitive to milder pathol- were all obtained from fish that had been 8. Horn P S, Feng L, Li Y, Pesce A J 2001 Effect 11 of outliers and nonhealthy individuals on 5 ogy . The very substantial difference in kept in fairly stable conditions. In the 5 reference interval estimation. Clinical Chem- ALP activity between Channel and study on Channel catfish referred to 0 istry 47: 2137–2145 0 Sharptooth catfish may therefore reveal above, which compared serum analytes 9. Horn P S, Pesce A J 2003 Reference inter- very little about its relative sensitivity as a of normal and acutely stressed channel vals: an update. Clinica Chimica Acta 334: marker of hepatobilliary pathology. catfish4, it was reported4 that laboratory- 5–23 The above observations imply that, over acclimatised and production pond catfish 10. Ip Y K, Subaidah R M, Liew P C, Loong A M, Hiong K C, Wong W P, Chew S F 2004 and above the establishment of reference exhibited differences in most serum African Sharptooth catfish Clarias gariepinus ranges (the focus of this study), an addi- chemistry parameters measured. The does not detoxify ammonia to urea or tional aspect of plasma clinical chemistry authors concluded that perceived normal amino acids but actively excretes ammonia in catfish, especially in relation to enzyme serum chemistry ranges are influenced by during exposure to environmental ammo- activity, would involve studies on tissue the diet, season, and environmental nia. Physiological and Biochemical Zoology 77: 242–254 activity and plasma half-lives as well as stress. It is likely that this applies to C. 11. Latimer K S, Mahaffey E A, Prasse K W 2003 empirical studies of enzyme activities gariepinus as well, and it is therefore im- Duncan and Prasses’s veterinary laboratory during or associated with specific, known portant that future studies on Sharptooth medicine (4th edn). Iowa State University organ pathology. catfish clinical chemistry also attempt to Press 12. Rodriguez-Cea A, Linde Arias A R, T4 levels were one-ninth of those found define the effect of age, growth stage, season and environmental conditions in Fernandez de la Campa M R, Costa Moreira in dogs. As dogs already have relatively J, Sanz-Mendel A 2006 Metal speciation of low levels when compared with humans, this species to improve the usefulness of metallothionein in white sea catfish, this places a special responsibility on clinical chemistry parameters when this Netuma barba and pearl cichlid, Geophagus investigators to inform the laboratory of fish species is used as a bioindicator. brasiliensis, by orthogonal liquid chroma- this fact so that appropriate calibration tography coupled to ICP-MS detection. Talanta 69: 963–969 and standardisation adjustments can be ACKNOWLEDGEMENTS This study was supported by a grant 13. Rumke C L, Bezemer P D 1972 Method for made to the assay method (generally the determination of normal values. II. developed for human samples) to avoid from the Norwegian Council for Higher New method. Nederlands Tijdschrift voor invalid results. Education’s Programme for Develop- Geneeskunde 116: 1559–1568 ment, Research and Education (NUFU 14. Siroka Z, Drastichova D 2004 Biochemical 08/02). markers of aquatic environment contami- Sex differences nation-Cytochrome P450 in fish. A review. Statistically significant differences be- REFERENCES Acta Veterinaria Brno 73: 123–132 tween the sexes were found for some of 1. Barnhoorn I E, Bornman M S, Pieterse G M 15. Skelton P,2001 A complete guide to the freshwa- the plasma chemistry constituents (Table 1999 Histological evidence of intersex in ter fishes of southern Africa. Southern Book feral sharptooth catfish (Clarias gariepinus) Publishers, Halfway House 3; see also Table 1 for the P-values). How- 16. Solberg E H, Lahti A 2005 Detection of ever, the use of only 13 specimens per sex from an estrogen-polluted water source in Gauteng, South Africa. Environmental outliers in reference distributions: perfor- raises doubt as to whether these data Toxicology 19: 603–608 mance of Horn’s algorithm. Clinical Chemis- accurately represent sex-specific refer- 2. 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