Haematology and blood chemistry parameters have generally been studied in fish species commonly used in research or cultured as a food source. For species such as salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio), catfish (Clarias gariepinus) and tilapia (Oreochromis Hybrid), reference ranges have been established(Reference Sandnes, Lie and Waagbø1–Reference Hrubec, Cardinale and Smith6) and are used to evaluate clinical status. Haematology data for ornamental fish species are limited and often restricted to common cyprinid species such as the goldfish and koi carp(Reference Groff and Zinkl7, Reference Tripathi, Latimer and Burnley8). Parameters for a couple of large aquarium species, such as the red pacu (Piaractus brachypomus) and the Acadian redfish (Sebastes fasciatus), have now been documented(Reference Sakamoto, Lewbart and Smith9, Reference Innis, Davis and Mandelman10). There is a vast number of aquarium-held ornamental fish, and while these two fish may provide representative baseline data for both Characiformes and Scorpaeniformes, data are still lacking for other major groups of ornamental fish.
The red top ice blue cichlid (Metriaclima greshakei formerly Pseudotropheus greshakei) is a tropical freshwater fish belonging to the Cichlidae family and is one of the most commonly kept ornamental cichlids; the most commonly cultured cichlids are tilapia (Oreochromis spp.), which are important as a food fish species. M. greshakei, often known as mbuna (rock dwelling) cichlids, are endemic to Lake Malawi, East Africa and are used as models of evolutionary speciation because of their diversity(Reference Genner and Turner11). In the aquarium trade, many cichlid fish are highly valued and important species, which include discus, angelfish and oscars. In this study, M. greshakei was used as a model ornamental cichlid to establish a range of blood haematology and chemistry parameters, for use as a diagnostic tool.
Materials and methods
The study was reviewed and approved by the WALTHAM® Ethical Review Committee and complied with Home Office regulations.
Animals and diet
The first generation of male red top ice blue cichlids bred from parent cichlids was used in this research study (on 31 July 2007 and 11 September 2007), at an age of 28 months. This study included only male fish due to volume requirements for blood sampling. These fish were maintained individually in 50-litre glass aquaria, because of their aggressive behaviour. These aquaria were an integral component of a tropical re-circulating system held at 24·8 ± 0·1°C with 12:12 h photoperiod. Chemical water parameters were maintained at a pH of 7·3 (se 0·1), 0 mg/l ammonia, 9·2 (se 5·7) mg/l nitrate and 0 mg/l nitrite, and were measured using a HACH DR/2500 spectrophotometer. The cichlids were fed to satiation with a combination of AQUARIAN® tropical flake and pellets (MARS Fishcare, Chalfont, PA, USA) by automatic feeders, 4–5 times a day. Fish were not fed on the day of sampling.
Blood sampling and analysis
Blood samples of sixteen cichlids, with a mean weight of 99·7 (se 4·1) g, were taken on two occasions, 6 weeks apart. On the first occasion eight fish were sampled for plasma chemistry and eight for haematology and this order was reversed on the second occasion. Immediately following capture, individual fish were supported with a foam rubber cradle upside down in a water-filled bowl. The head of the fish was covered with a moist towel, to reduce stress. Blood was withdrawn from the caudal vein using a 1 ml sterile pre-heparinzed syringe with a 22G × 1′ needle and was divided into two lithium heparin tubes. One tube was spun at 4 °C, 200 g for 10 min using a Jouan BR4i centrifuge and the plasma was frozen at (80 °C until analysis. Plasma was analysed for the following biochemical parameters using an Olympus AU400 Clinical Chemistry Autoanalyzer (Olympus, Tokyo, Japan): protein, albumin, glucose, P, Ca, Na, K, Cl, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholesterol, urea and creatinine. Globulins were calculated from the difference between total protein and albumin values. A separate whole-blood sample was drawn into a heparinized capillary tube and the haematocrit level was determined after centrifugation for 5 min at 12 000 g. Hb was measured using the HemoCue blood haemoglobin system. Absolute cell counts and their differentials were calculated by a modified method of Inoue(Reference Inoue12) using a FACSCalibur flow cytometer. The mean cell volume, mean cell Hb and mean cell Hb concentration were calculated by standard formulae. Blood smears were made using anticoagulated blood and were stained with Wright–Giemsa for cell cytology observations.
Data analysis
Data from previous ‘in-house’ studies using ornamental fish generated sufficiently small values for standard errors, when using a sample size of fifteen fish. Therefore, the number of fish used in this study was considered statistically adequate, and compares favourably with similar studies reported in the literature(Reference Sandnes, Lie and Waagbø1, Reference Bailey13–Reference Yang and Chen15). Reference intervals were established following the guidelines proposed by the National Committee for Clinical Laboratory Standards(16). Values were ranked, and the highest and lowest 2·5 % values were discarded, no outliers were identified in this data set. The range of the remaining values was the reference interval. A t test was used to statistically compare the groups of eight fish between time points for plasma chemistry and haematology (P < 0·05) using Statgraphics Centurion XVI.
Results
No statistically significant differences were noted between sample periods for any parameters. Results of the plasma chemistry are shown in Table 1, one sample was lost due to clotting; therefore values for fifteen fish are reported. Haematology results are shown in Table 2. Data obtained for urea have not been included in this data set as they were under the dynamic range of the assay, and therefore deemed unreliable. Identification of specific leucocyte types was difficult because of the low numbers of these cells and the clarity of the images obtained. However, it was possible to identify several small lymphocytes, some with pseudopodia, a potential large monocyte, due to its abundant blue-grey cytoplasm and a round nucleus, and a G1 (type 1) granulocyte due to its irregular, non-lobed nucleus.
Table 1 Plasma chemistry reference intervals for Metriaclima greshakei (n 15)*
ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.
* All data are expressed within 95 % reference intervals.
Table 2 Haematology reference intervals for Metriaclima greshakei (n 16)*
MCV, mean cell volume; MCH, mean cell Hb; MCHC, mean cell Hb concentration.
* All data are expressed within 95 % reference intervals.
Discussion
There were no statistically significant differences between sampling periods for haematological or plasma chemistry, indicating that the 6-week period between sampling was sufficient for blood volume and parameter recovery. Low circulating plasma urea levels were expected since this teleost fish is ammonotelic. This species of cichlid remains calm when out of water, therefore anaesthetic was not required while sampling, and so excludes any potential anaesthetic effect on blood parameters(Reference Velišek, Stejskal and Kouril17, Reference Smit and Hattingh18). Glucose levels were within normal ranges, when compared with previously published data for cichlids(Reference Hrubec, Cardinale and Smith6, Reference Terao and Ogawa19), indicating that sampling was undertaken in the absence of a stress response. It should be noted that all the fish used in this study were male, particularly as variations in haematological parameters have been noted between sexes of the same species of fish(Reference Summerfelt20, Reference Gabriel, Ezeri and Opabunmi21).
Cl, Na, K, creatinine, cholesterol and total protein levels were consistent with values obtained for other tropical freshwater fish(Reference Sandnes, Lie and Waagbø1, Reference Hrubec, Cardinale and Smith6, Reference Sakamoto, Lewbart and Smith9, Reference Kori-Siakpere, Ake and Idoge22, Reference Stoskopf23). Albumin level was lower and globulin level was higher with median values of 9·5 and 29 g/l v. 24 and 8 g/l in goldfish(Reference Bailey13), although similar to those for tilapia(Reference Hrubec, Cardinale and Smith6). A fall in serum albumin can indicate liver disease and is associated with a fall in Ca(Reference Casillas, Myers and Rhodes24), which was not reflected in this study. Plasma enzymes are known to be variable between fish species(Reference Luskova25) and levels in this study are comparable to common carp(Reference Yang and Chen15) and tilapia(Reference Hrubec, Cardinale and Smith6) but are low compared with sturgeon(Reference Shi, Li and Zhuang26) and Atlantic salmon(Reference Sandnes, Lie and Waagbø1). When comparing published data, values are often reported as a mix of both plasma and serum, which are not reflective of each other; it has been suggested that plasma values should be used preferentially(Reference Hrubec and Smith27).
Haematology values for Hb, haematocrit, erythrocyte, mean cell volume, mean cell Hb, mean cell Hb concentration and cytology are in alignment with those for other fish species, including the cultured tilapia(Reference Hrubec, Cardinale and Smith6, Reference Kori-Siakpere, Ake and Idoge22, Reference Hrubec, Smith and Robertson14). However, values for P, leucocytes and lymphocytes are lower. These differences may be attributable to water quality and low stocking densities, as fish exposed to higher stocking densities are exposed to poorer water quality and higher bacterial loads, which could induce immune stress. Similar discrepancies have been noted for tilapia and striped bass(Reference Hrubec, Cardinale and Smith6, Reference Hrubec, Smith and Robertson28). In addition to reporting the first set of blood values for M. Greshakei, to our knowledge, this study highlights the similarities between ornamental and cultured cichlids, indicating that previously established data may be utilized for fish belonging to the same family.
Information about the clinical pathology of freshwater tropical fishes is scarce, despite many species having been kept by hobbyists for years. Many factors need to be taken into consideration when comparing reference intervals between fish species, including water quality, stocking density and the sampling methods used, as these can all influence blood indices.
Acknowledgements
There are no conflicts of interest to disclose. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Principal investigator, data collection and manuscript prepared by D. L. S. and intellectual input and support from L. G. A.
