Liver histology and hematological parameters of female Rhamdia quelen fed different lipid sources

Cleonice Cristina Hilbig Nivaldo Ferreira do Nascimento Ana Caroline Sousa Campos Leandro Freitas Martins Arlene Sobrinho Ventura Laura Satiko Okada Nakaghi Robie Allan Bombardelli About the authors

ABSTRACT

The objective with this study was to evaluate the liver histology and hematological parameters of female Rhamdia quelen fed diets supplemented with different oils: 5% marine fish oil, 5% refined palm oil, 5% soybean oil, and a combination of the three. The lipid vacuolization (steatosis) in the liver was analyzed according to a score of vacuolization when: 0 = absence, 1 = reduced, 2 = intermediate, and 3 = intense. At the end of the experiment, females (n = 10) were selected from each treatment (two per cage), and the blood was collected for erythrocyte and biochemical analysis. Lower vacuolization indices in the liver were observed at the beginning of the experiment. However, with time, the presence of vacuoles was more evident but presented similar morphology among all treatments. The blood parameters were also not influenced by the different diets, except for glucose levels, which was higher in the treatment with the mixture of oils. The absence of differences regarding liver morphology (steatosis) and hematological parameters indicates that the replacement of fish oil by vegetable oils could be performed without damage to the health of R. quelen females.

Keywords:
blood; fatty acids; fish; nutrition

Introduction

In fish farming, the diet represents the highest cost (Babalola and Apata, 2012Babalola, T. O. and Apata, D. F. 2012. Impact of palm oil and extracted fish meal on haematological parameters, serum constituents and histology of African catfish (Heterobranchus longifilis) fingerlings. Scientific Journal of Review 1:70-83.), especially when its composition is derived from animal source, such as meal and fish oil. However, due to scarcity and high cost of such resources, studies are being developed with its partial or total replacement by vegetable-origin products (Ng and Wang, 2011Ng, W.-K. and Wang, Y. 2011. Inclusion of crude palm oil in the broodstock diets of female Nile tilapia, Oreochromis niloticus, resulted in enhanced reproductive performance compared to broodfish fed diets with added fish oil or linseed oil. Aquaculture 314:122-131. https://doi.org/10.1016/j.aquaculture.2011.01.034
https://doi.org/10.1016/j.aquaculture.20...
; Ng, 2004Ng, W. K. 2004. Palm oil as a novel dietary lipid source in aquaculture feeds. Palm Oil Developments 41:14-18.; Ayisi et al., 2018Ayisi, C. L.; Zhao, J. and Wu, J.-W. 2018. Replacement of fish oil with palm oil: Effects on growth performance, innate immune response, antioxidant capacity and disease resistance in Nile tilapia (Or eochromis niloticus). Plos One 13:e0196100. https://doi.org/10.1371/journal.pone.0196100
https://doi.org/10.1371/journal.pone.019...
).

Among the lipid sources employed in the replacement of fish oil, soybean and palm oil (Turchini et al., 2009Turchini, G. M.; Torstensen, B. E. and Ng, W. K. 2009. Fish oil replacement in finfish nutrition. Reviews in Aquaculture 1:10-57. https://doi.org/10.1111/j.1753-5131.2008.01001.x
https://doi.org/10.1111/j.1753-5131.2008...
) can be highlighted. Soybean oil represents 19 to 30% of oleic acid (C18:1n-9); 44 to 62% of linoleic acid (C18:2n-6), the main representative of the omega-6 fatty acids series; and 4 to 11% of linolenic acid (C18:3n-3), the main representative of the omega-3 series (Brasil, 1999Brasil. 1999. Agência Nacional de Vigilância Sanitária. Resolução RDC, n. 482 de 23 de setembro de 1999. Republicada 20 jun. 2000. Brasília, DF. Available at: <http://www.anvisa.gov.br/legis/resol/482_99.htm>. Accessed on: Dec. 08, 2018.
http://www.anvisa.gov.br/legis/resol/482...
). Palm oil, on the other hand, is considered a superior source of energy because it contains a large amount of saturated fatty acids (48%), represented mainly by palmitic acid (C16:0), and monounsaturated acids (42%), represented by oleic acid (C18:1n-9) (Ng, 2004Ng, W. K. 2004. Palm oil as a novel dietary lipid source in aquaculture feeds. Palm Oil Developments 41:14-18.; Ng and Wang, 2011Ng, W.-K. and Wang, Y. 2011. Inclusion of crude palm oil in the broodstock diets of female Nile tilapia, Oreochromis niloticus, resulted in enhanced reproductive performance compared to broodfish fed diets with added fish oil or linseed oil. Aquaculture 314:122-131. https://doi.org/10.1016/j.aquaculture.2011.01.034
https://doi.org/10.1016/j.aquaculture.20...
). Therefore, vegetable oils can be used for partial or complete replacement of fish oil in fish diets (Babalola et al., 2011Babalola, T. O.; Apata, D. F.; Omotosho, J. S. and Adebayo, M. A. 2011. Differential effects of dietary lipids on growth performance, digestibility, fatty acid composition and histology of African catfish (Heterobranchus longifilis) fingerlings. Food and Nutrition Sciences 2:11-21.; Ayisi et al., 2018Ayisi, C. L.; Zhao, J. and Wu, J.-W. 2018. Replacement of fish oil with palm oil: Effects on growth performance, innate immune response, antioxidant capacity and disease resistance in Nile tilapia (Or eochromis niloticus). Plos One 13:e0196100. https://doi.org/10.1371/journal.pone.0196100
https://doi.org/10.1371/journal.pone.019...
; Ayisi and Zhao, 2014Ayisi, C. L. and Zhao, J. L. 2014. Recent developments in the use of palm oil in aquaculture feeds: a review. International Journal of Scientific & Technology Research 3:259-264.; Ng, 2002Ng, W. K. 2002. Potential of palm oil utilisation in aquaculture feeds. Asia Pacific Journal of Clinical Nutrition 11:S473-S476. https://doi.org/10.1046/j.1440-6047.11.s.7.7.x
https://doi.org/10.1046/j.1440-6047.11.s...
).

Histological changes in the liver are easily detectable when the feedings are not suitable (Tacon, 1992Tacon, A. G. 1992. Nutritional fish pathology: morphological signs of nutrient deficiency and toxicity in farmed fish. FAO Fisheries Technical Paper No. 330. FAO, Rome.), and some studies show that the replacement of fish oil by vegetable oils can induce the occurrence of steatosis in fish (Tessaro et al., 2014Tessaro, L.; Toledo, C. P. R.; Neumann, G.; Krause, R. A.; Meurer, F.; Natali, M. R. M. and Bombardelli, R. A. 2014. Animal performance and reproductive aspects of female Rhamdia quelen fed on different levels of digestible energy. Aquaculture Research 45:1425-1433. https://doi.org/10.1111/are.12087
https://doi.org/10.1111/are.12087...
; Caballero et al., 2004Caballero, M. J.; Izquierdo, M. S.; Kjørsvik, E.; Fernandez, A. J. and Rosenlund, G. 2004. Histological alterations in the liver of sea bream, Sparus aurata L., caused by short-or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source. Journal of Fish Diseases 27:531-541. https://doi.org/10.1111/j.1365-2761.2004.00572.x
https://doi.org/10.1111/j.1365-2761.2004...
). Consequently, the analysis of liver histology is essential in such types of study, which can be used as an indicator of the nutritional status of the individuals (Caballero et al., 2004Caballero, M. J.; Izquierdo, M. S.; Kjørsvik, E.; Fernandez, A. J. and Rosenlund, G. 2004. Histological alterations in the liver of sea bream, Sparus aurata L., caused by short-or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source. Journal of Fish Diseases 27:531-541. https://doi.org/10.1111/j.1365-2761.2004.00572.x
https://doi.org/10.1111/j.1365-2761.2004...
; Raskovic et al., 2011Raskovic, B. S.; Stankovic, M. B.; Markovic, Z. Z. and Poleksic, V. D. 2011. Histological methods in the assessment of different feed effects on liver and intestine of fish. Journal of Agricultural Sciences 56:87-100. https://doi.org/10.2298/JAS1101087R
https://doi.org/10.2298/JAS1101087R...
). Additionally, lipid changes in the diet could also influence blood parameters, an indicator of the physiological equilibrium of the animal (Babalola et al., 2009Babalola, T. O.; Adebayo, M. A.; Apata, D. F. and Omotosho, J. S. 2009. Effect of dietary alternative lipid sources on haematological parameters and serum constituents of Heterobranchus longifilis fingerlings. Tropical Animal Health Production 41:371. https://doi.org/10.1007/s11250-008-9199-1
https://doi.org/10.1007/s11250-008-9199-...
).

The silver catfish, Rhamdia quelen (Quoy and Gaimard, 1824), are classified as an omnivorous species (Hernández et al., 2009Hernández, D. R.; Pérez Gianeselli, M. and Domitrovic, H. A. 2009. Morphology, histology and histochemistry of the digestive system of South American catfish (Rhamdia quelen). International Journal of Morphology 27:105-111. https://doi.org/10.4067/S0717-95022009000100019
https://doi.org/10.4067/S0717-9502200900...
), and some previous studies have demonstrated that this species can efficiently convert C18 into polyunsaturated fatty acids when feed diets contain vegetable oils (Vargas et al., 2015Vargas, R. J.; Garcia, A. S.; Baggio, S. R.; Cargnin-Ferreira, E. and Fracalossi, D. M. 2015. A combination of plant oils promotes adequate growth of the freshwater catfish Rhamdia quelen (Quoy & Gaimard 1824). Aquaculture Research 46:3046-3055. https://doi.org/10.1111/are.12466
https://doi.org/10.1111/are.12466...
). Therefore, the objective of this study was to histologically analyze the liver and the blood parameters of Rhamdia quelen females fed different lipid sources.

Material and Methods

The experiment was carried out in Toledo, Paraná, Brazil (24°46'48.31" S; 53°43'25.77" W) from May 2013 to April 2014. All the procedures involving animals were conducted in accordance with the approved protocol of institutional committee on animal use (case number 6728/12).

Four isoproteic diets were formulated, containing 38% crude protein and 5% lipids: 5% of marine fish oil, 5% palm oil, 5% soybean oil, and a combination of the three (Mix) (Table 1). The experimental diets were extruded using an Ex-micro (Exteec) and dried under forced ventilation at 55 °C for 12 h. After weighing, the oil was added with a rotation mixer for 30 min, resulting in pellets of 4 mm.

Table 1
Formulation and mean values of the composition of experimental diets

The experimental design was completely randomized with four treatments and five replicates. Two thousand fish were distributed in 20 concrete tanks with a total area of 12 m2. Each tank received 100 juveniles of R. quelen with initial mean weight of 24.50±0.78 g. The fish remained in this system and were fed the experimental diets for five months until sex identification was possible. In October 2013, 20 females (total = 400) from each concrete tank were selected and transferred to twenty cages (4×2×1 m). Feeding rate was established to be 2% of body weight.day−1, and fish were fed twice a day, scheduled at 10:00 and 16:00 h.

Water temperature was monitored twice daily, in the morning (25.5±1.59 °C) and in the afternoon (27.2±1.16 °C) with a YSI 550 A. Dissolved oxygen (YSI 550 A) and pH (Tecnal® Tec 5) were recorded weekly, determined at 06.00 h (5.36±1.64 mg L−1 and 7.18±0.33) and at 16.00 h (7.64±2.73 mg L−1 and 8.60±0.93), respectively. Ammonia levels were checked monthly (0.03±0.02) by the colorimetric method (Koroleff, 1976Koroleff, F. 1976. Determination of ammonia. p.126-133. In: Methods of seawater analysis. Grashoff, E. and Kremling, E., eds. Verlag Chemie Weinhein, German.).

Liver sampling (central portion) was performed in October 2013, December 2013, February 2014, and April 2014, after five, seven, nine, and eleven months of feeding, respectively. Two females were randomly collected from each cage, euthanized (0.75 mg L−1 benzocaine), and dissected for removal of the liver. Fragments of the organ (∼0.5 cm) were fixed in Bouin solution for 24 h. Afterwards, the samples were washed in water and maintained in 70% ethanol until processing. Paraffin blocks were cut into 5-µm sections and stained with PAS (Periodic acid-Schiff) (Tolosa et al., 2003Tolosa, E. M. C.; Rodrigues, C. J.; Behmer, O. A. and Freitas Neto, A. G. 2003. Manual de técnicas para histologia: normal e patológica. Manole, Barueri.).

The lipid vacuolization (steatosis) in the liver was analyzed according to a score of vacuolization (0 = lack of vacuoles, 1 = reduced, 2 = intermediate, 3 = intense), according to Caballero et al. (2004)Caballero, M. J.; Izquierdo, M. S.; Kjørsvik, E.; Fernandez, A. J. and Rosenlund, G. 2004. Histological alterations in the liver of sea bream, Sparus aurata L., caused by short-or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source. Journal of Fish Diseases 27:531-541. https://doi.org/10.1111/j.1365-2761.2004.00572.x
https://doi.org/10.1111/j.1365-2761.2004...
and Tessaro et al. (2014)Tessaro, L.; Toledo, C. P. R.; Neumann, G.; Krause, R. A.; Meurer, F.; Natali, M. R. M. and Bombardelli, R. A. 2014. Animal performance and reproductive aspects of female Rhamdia quelen fed on different levels of digestible energy. Aquaculture Research 45:1425-1433. https://doi.org/10.1111/are.12087
https://doi.org/10.1111/are.12087...
, with the use of a light microscope (400X) (Leica, DM 2500).

At the end of the experiment (April 14), two females were collected from each cage (10 females/treatment), anesthetized (75 mg L−1 of benzocaine), and the blood collected (1.5 mL) by a caudal punction with the aid of a syringe (without coagulant). Afterwards, 0.5 mL of the blood was poured into a tube containing 25 µL of anticoagulant (EDTA 10%) for erythrocyte analyses: erythrocyte count in Neubauer chamber; hematocrit determination, using the micro-hematocrit method; and hemoglobin measurements using the cyanomethemoglobin method.

An additional 1 mL of the blood was poured into a tube without coagulant and centrifuged at 2060 × g (Baby I 206 BL FANEM®). The serum was sent for biochemical analysis (total cholesterol, triglycerides, total proteins, and glucose), performed by spectrophotometry using a commercial kit (Analisa®).

Data were checked for homogeneity using Levene's test (Brown and Forsythe, 1974Brown, M. B. and Forsythe, B. F. 1974. Robust test for the equality of variances. Journal of the American Statistical Association 69:364-367. https://doi.org/10.2307/2285659
https://doi.org/10.2307/2285659...
), and for normality using the Cramer-von Mises test (Darling, 1957Darling, D. A. 1957. The Komogorov-Smirnov, Cramer-von mises tests. Annals of Mathematical Statistics 28:823-838. https://doi.org/10.1214/aoms/1177706788
https://doi.org/10.1214/aoms/1177706788...
). Afterwards, the data were analyzed by ANOVA, followed by Tukey's test using the software STATISTICA 7.0. Significance was considered at P = 0.05.

Results

At the beginning of the analysis, lower indices of vacuolization were observed (Table 2). With time, the presence of vacuoles was more evident but occurred homogeneously in all experimental diets (Table 2, Figure 1). In the blood parameters (Table 3), no difference among treatments were observed for erythrocytes (P = 0.53), hematocrit (P = 0.65), hemoglobin (P = 0.99), total proteins (P = 0.91), cholesterol (P = 0.94), and triglycerides (P = 0.95). However, for glucose levels, lower values (P = 0.03) were observed for palm oil treatment (51.17±3.82 mg dL−1), especially when compared with the mix treatment (74.46±4.81 mg dL−1).

Table 2
Histological alterations of the liver of R. quelen females fed different lipid sources
Figure 1
Small change in cell size (vacuolization score = 1), palm oil treatment/October 2013 (A); hepatocytes-like adipocytes; smaller vacuoles (vacuolization score = 2), mix treatment/December (B). (PAS, 400X).
Table 3
Mean ± standard error of the erythrocyte and biochemical variables of the blood of R. quelen females fed different lipid sources

Discussion

In the present study, it is observed that vegetable oils do not significantly interfere with the histology and hematological parameters of female Rhamdia quelen. The vacuolization observed in the current study were homogenous in fish fed all experimental diets and does not indicate an intense pattern of steatosis. Therefore, such injury cannot be attributed to the inclusion of 5% of vegetable oils in the diet. However, other authors, such as Tessaro et al. (2014)Tessaro, L.; Toledo, C. P. R.; Neumann, G.; Krause, R. A.; Meurer, F.; Natali, M. R. M. and Bombardelli, R. A. 2014. Animal performance and reproductive aspects of female Rhamdia quelen fed on different levels of digestible energy. Aquaculture Research 45:1425-1433. https://doi.org/10.1111/are.12087
https://doi.org/10.1111/are.12087...
with females R. quelen and Bombardelli et al. (2009)Bombardelli, R. A.; Hayashi, C.; Natali, M. R. M.; Sanches, E. A. and Piana, P. A. 2009. Desempenho reprodutivo e zootécnico e deposição de lipídios nos hepatócitos de fêmeas de tilápia-do-nilo alimentadas com rações de diversos níveis energéticos. Revista Brasileira de Zootecnia 38:1391-1399. https://doi.org/10.1590/S1516-35982009000800001
https://doi.org/10.1590/S1516-3598200900...
with Nile tilapia, observed an increased level of vacuolization with high levels of energy associated with vegetable oils.

Regardless of the oil source, digestible energy levels must be considered in diet formulation. The results of this study showed that until 5% of replacement of fish oil for soybean oil and palm oil, it does not cause significant changes in liver histology. The steatosis could be characterized by the excess of lipid ingestion that exceeds the oxidation capacity of the liver, which results in the deposition of lipid in the hepatocytes (vacuoles) (Caballero et al., 2004Caballero, M. J.; Izquierdo, M. S.; Kjørsvik, E.; Fernandez, A. J. and Rosenlund, G. 2004. Histological alterations in the liver of sea bream, Sparus aurata L., caused by short-or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source. Journal of Fish Diseases 27:531-541. https://doi.org/10.1111/j.1365-2761.2004.00572.x
https://doi.org/10.1111/j.1365-2761.2004...
). Therefore, the results suggest that different diets using vegetable oils do not overload the liver.

The results of blood parameters evaluated also indicated a normal pattern expected for the species, presenting no significant influence of the diets. Mature erythrocytes transport oxygen and carbon dioxide through hemoglobin (Ranzani-Paiva et al., 2013Ranzani-Paiva, M. J. T.; Páuda, S. B.; Tavares-Dias, M. and Egami, M. I. 2013. Métodos para análise hematológica em peixes. Eduem, Maringá.). In the present study, the diets did not influence the number of erythrocytes, and the values are within the normal range established by Tavares-Dias et al. (2002)Tavares-Dias, M.; Melo, J. F. B.; Moraes, G. and Moraes, F. R. 2002. Características hematológicas de teleósteos brasileiros. IV. Variáveis do jundiá Rhamdia quelen (Pimelodidae). Ciência Rural 32:693-698. https://doi.org/10.1590/S0103-84782002000400024
https://doi.org/10.1590/S0103-8478200200...
, 1.55 to 2.92 × 106/μL, for R. quelen. As hemoglobin, the hematocrit is employed to categorize anemia when the values are below the normal pattern. The diets also did not influence the values of hemoglobin and hematocrit; however, there were increased values of hematocrits in relation to other reports for R. quelen (Tavares-Dias et al., 2002Tavares-Dias, M.; Melo, J. F. B.; Moraes, G. and Moraes, F. R. 2002. Características hematológicas de teleósteos brasileiros. IV. Variáveis do jundiá Rhamdia quelen (Pimelodidae). Ciência Rural 32:693-698. https://doi.org/10.1590/S0103-84782002000400024
https://doi.org/10.1590/S0103-8478200200...
), as well as when evaluating diets containing different protein levels (Camargo et al., 2005Camargo, S. O.; Pouey, J. L. and Martins, C. 2005. Parâmetros eritrocitários do jundiá (Rhamdia quelen) submetido à dieta com diferentes níveis de proteína. Ciência Rural 35:1406-1411. https://doi.org/10.1590/S0103-84782005000600028
https://doi.org/10.1590/S0103-8478200500...
; Melo et al., 2006Melo, J. F. B.; Tavares-Dias, M.; Lundestedt, L. M. and Moraes, G. 2006. Efeito do conteúdo de proteína na dieta sobre os parâmetros hematológicos e metabólicos do bagre sul americano Rhamdia quelen. Revista de Ciências Agroambientais 1:43-51.).

Among the biochemical variables analyzed, only a difference in glucose levels was observed, which was significantly lower in females fed diets containing palm oil. Similar results were observed by Babalola et al. (2009)Babalola, T. O.; Adebayo, M. A.; Apata, D. F. and Omotosho, J. S. 2009. Effect of dietary alternative lipid sources on haematological parameters and serum constituents of Heterobranchus longifilis fingerlings. Tropical Animal Health Production 41:371. https://doi.org/10.1007/s11250-008-9199-1
https://doi.org/10.1007/s11250-008-9199-...
, who tested four sources of saturated fat (palm oil, karite butter, swine fat, and poultry fat) and two sources of oil, from animal (cod liver oil) and vegetable (sunflower oil) origin, in catfish (Heterobranchus longifilis) juveniles. The authors observed that lower levels of glucose were observed for fish fed diets containing fish oil, poultry fat, and karite butter, followed by palm oil. Therefore, as glucose is commonly used as an indicator of stress in fish (Martínez-Porchas et al., 2009Martínez-Porchas, M.; Martínez-Córdova, L. R. and Ramos-Enriquez, R. 2009. Cortisol and glucose: reliable indicators of fish stress. Pan-American Journal of Aquatic Sciences 4:158-178.), this specific aspect needs to be evaluated in the future.

Conclusions

Diets using vegetable oils or their mixture do not interfere with the steatosis and hematological parameters of R. quelen females, making them an interesting source to replace fish oil.

Acknowledgments

This study received financial support from the Auxílio à Pesquisa of the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2012/23089-4) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through the doctoral scholarship (142751/2011-1). We would also like to thank Michael James Stablein of the University of Illinois Urbana-Champaign (IL, USA) for his translation services and review of this work.

References

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    » https://doi.org/10.1371/journal.pone.0196100
  • Ayisi, C. L. and Zhao, J. L. 2014. Recent developments in the use of palm oil in aquaculture feeds: a review. International Journal of Scientific & Technology Research 3:259-264.
  • Babalola, T. O.; Adebayo, M. A.; Apata, D. F. and Omotosho, J. S. 2009. Effect of dietary alternative lipid sources on haematological parameters and serum constituents of Heterobranchus longifilis fingerlings. Tropical Animal Health Production 41:371. https://doi.org/10.1007/s11250-008-9199-1
    » https://doi.org/10.1007/s11250-008-9199-1
  • Babalola, T. O. and Apata, D. F. 2012. Impact of palm oil and extracted fish meal on haematological parameters, serum constituents and histology of African catfish (Heterobranchus longifilis) fingerlings. Scientific Journal of Review 1:70-83.
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    » https://doi.org/10.1590/S1516-35982009000800001
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    » http://www.anvisa.gov.br/legis/resol/482_99.htm
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    » https://doi.org/10.2307/2285659
  • Caballero, M. J.; Izquierdo, M. S.; Kjørsvik, E.; Fernandez, A. J. and Rosenlund, G. 2004. Histological alterations in the liver of sea bream, Sparus aurata L., caused by short-or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source. Journal of Fish Diseases 27:531-541. https://doi.org/10.1111/j.1365-2761.2004.00572.x
    » https://doi.org/10.1111/j.1365-2761.2004.00572.x
  • Camargo, S. O.; Pouey, J. L. and Martins, C. 2005. Parâmetros eritrocitários do jundiá (Rhamdia quelen) submetido à dieta com diferentes níveis de proteína. Ciência Rural 35:1406-1411. https://doi.org/10.1590/S0103-84782005000600028
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  • Hernández, D. R.; Pérez Gianeselli, M. and Domitrovic, H. A. 2009. Morphology, histology and histochemistry of the digestive system of South American catfish (Rhamdia quelen). International Journal of Morphology 27:105-111. https://doi.org/10.4067/S0717-95022009000100019
    » https://doi.org/10.4067/S0717-95022009000100019
  • Koroleff, F. 1976. Determination of ammonia. p.126-133. In: Methods of seawater analysis. Grashoff, E. and Kremling, E., eds. Verlag Chemie Weinhein, German.
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  • Melo, J. F. B.; Tavares-Dias, M.; Lundestedt, L. M. and Moraes, G. 2006. Efeito do conteúdo de proteína na dieta sobre os parâmetros hematológicos e metabólicos do bagre sul americano Rhamdia quelen Revista de Ciências Agroambientais 1:43-51.
  • Ng, W.-K. and Wang, Y. 2011. Inclusion of crude palm oil in the broodstock diets of female Nile tilapia, Oreochromis niloticus, resulted in enhanced reproductive performance compared to broodfish fed diets with added fish oil or linseed oil. Aquaculture 314:122-131. https://doi.org/10.1016/j.aquaculture.2011.01.034
    » https://doi.org/10.1016/j.aquaculture.2011.01.034
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Publication Dates

  • Publication in this collection
    13 July 2020
  • Date of issue
    2020

History

  • Received
    13 Nov 2018
  • Accepted
    22 Apr 2020
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