Journal of Aquariculture & Aquatic Sciences Article

Journal of Aquariculture & Aquatic Sciences
Volume 4, Number 4

SOME STUDIES ON THE PROTEIN REQUIREMENT
OF THE GUPPY, POECILIA RETICULATA (PETERS)

Shim Kim Fah and Chua Yan Leng, Department of Zoology, National University of Singapore, Lower Kent Ridge Road, Singapore, O511

ABSTRACT

The dietary protein requirements of the guppy, Poecilia reticulata (Peters), were studied with the specific objective of determining the optimum level for growth, feeding efficiency, gonad development, and carcass composition.

280 six-week-old female guppies were randomly distributed into 14 glass tanks of 60 cm x 35 cm x 30 cm. Seven experimental diets consisting of varying levels of protein from 0 percent to 60 percent at 10 percent intervals were formulated using casein and fish meal as the main dietary protein source. Fish receiving no dietary protein were greatly retarded in growth. Of all the protein levels tested, 30 percent was found to be the optimum for growth (body weight gain). Fish fed with the non-protein diet showed significantly higher feed conversion ratios than fish in the other treatments. The 30 percent and 40 percent dietary protein groups obtained the lowest feed conversion rate.

Exclusion of protein from the diet greatly retarded the development of the fish ovary. Fish kept on the protein diets had significantly heavier and more well-developed gonads than those on the 0 percent protein diet. The greatest mean number of yolky ooctyes present in both ovaries was found in fish given 30 percent and 40 percent dietary protein levels.

INTRODUCTION

Most of the nutritional studies on fish have been carried out on food fish. There has been little work on the nutrition of tropical aquarium fishes.

One of the most intensely researched areas of fish nutrition centers on dietary protein requirements for growth. Most work suggests that fish require two to four times more dietary protein than warm-blooded animal like birds and mammals (Lee et al., 1977; Beeson et al., 1953). This is due to a greater need in fish for the essential amino acids (Mertz, 1969).

A difference in the optimum levels of protein was found between carnivorous and omnivorous fish. Typically, carnivores such as plaice, Pleuronectes platessa Linnaeus (Cowey et al., 1972), and rainbow trout, Salmo gairdneri Richardson (Ogino, et al., 1976), require 40 to 55 percent dietary protein. In contrast, omnivores such a blue tilapia, Sarotherodon aureus (Steindachner) (David and Sickney, 1978) and guppy Poecilia reticulata (Peters) (Dahlgren, 1980) require 35 to 47 percent dietary protein.

Protein is usually the most expensive major constituent in a diet. Information on the optimum level of dietary protein required would thus be useful in formulating an economical and well-balanced feed for the guppy. Knowledge of the optimal protein requirements of the fish would also help to contribute towards high production on fish farms.

It is the aim of this study to determine the optimum dietary protein level for guppies reared in the tropical area and to investigate the effects of varying dietary protein levels have on the gonadal development and body composition of the fish.

MATERIALS AND METHODS

One month old female guppy fry of the tuxedo variety were acclimatized to laboratory conditions for about two weeks. During this period they were fed on commercial dry fish flakes, Litramin.

Fourteen glass tanks, each measuring 60 cm x 35 cm x 30 cm, were set up to contain 35 liters of clean standing tap water. Constant aeration, temperature, pH, and salinity were ensured through the period of experimentation.

At the beginning of the experiment, 20 young, healthy guppies of about 100 mg in body weight were randomly distributed into the 14 tanks. Altogether, seven test diets were used. These contained varying levels of protein from 0 percent to 60 percent at 10 percent intervals. Each treatment consisted of two replicates.

The tanks were cleaned once every ten days by siphoning off the accumulated feces and about three-quarters of the water. The water removed was then replaced with clean standing tap water from a stock fiberglass tank. The average temperature was 28°C.

Fish meal and casein were used as the dietary protein sources in this experiment. the composition of the experimental diets is shown in table 1 and their proximate analysis in table 2.

A fixed amount of the experimental diet was weighed out for each tank of fish each week. sufficient water was added to make the diet into paste which was then reweighed. the paste could be stored in plastic vials in the refrigerator and used for the rest of the week.

The fish were fed to satiation. each time a small amount of feed was dropped into the water and this process was repeated until satiation was observed. at the end of one week, the amount of feed left in each plastic vial was measured and the food consumed was calculated.

Table 1. composition of experimental diets (g/00 g feed)

Percentage Protein in Diet

Ingredients 0 10 20 30 40 50 60

Casein 0.00 2.83 14.32 25.82 37.32 48.82 60.32

Fish meal 0.00 14.50 16.00 17.50 19.00 20.50 22.00

Dextrin 66.00 51.67 41.50 31.68 21.68 11.68 1.68

Corn oil 8.00 7.00 6.00 5.00 4.00 3.00 2.00

Cod liver oil 2.00 2.00 2.00 2.00 2.00 2.00 2.00

Tapioca 5.00 5.00 5.00 5.00 5.00 5.00 5.00

Vitamin Premix* 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Mineral Premix** 18.00 16.00 14.00 12.00 10.00 8.00 6.00

Metabolic Energy (ME) (kcal/g) 3.14 3.04 3.07 3.11 3.15 3.18 3.22

Protein: Energy ratio (mg protein/kcal ME) 0.00 32.89 65.15 96.46 126.98 157.23 186.34

*to provide per kg dry diet: vitamin a 2000 i.u., a-tocopherol 30 mg, menaphthone 80 mg, thiamin 10 mg, riboflavin 20 mg, niacin 150 mg, pyridoxine 10 mg, pantothenic acid 40 mg, folic acid 5 mg, biotin 1 mg, vitamin b12 0.02 mg, inositol 400 mg, choline 3,000 mg, ascorbic acid 100 mg.

**to provide per 100 g dry diet: calcium tetrahydrogen orthophosphate 68.62 g, magnesium carbonate 9.06 g, sodium chloride 7.97 g, white limestone 5.45 g, potassium chloride 4.98 g, ferrous sulphate 2.99 g, zinc sulphate 0.398 g, manganese sulphate 0.27 g, cobalt sulphate 0.0997 g, copper sulphate 0.097 g, calcium iodate 0.0245 g, aluminum sulphate 0.0199 g.

The fish were weighed bi-weekly. weighing was done about 24 hours after the fish were last fed in order to reduce the error and variability due to stomach contents.

At the end of eight weeks feeding experiment, eight fish were taken from each tank and individually weighed. each fish was quickly decapitated to kill it. it was then dissected and its ovaries removed and weighed. the gonadosomatic index (gsi) was calculated.

At the beginning of the experiment, 40 fish were taken, weighed, and subjected to proximate analysis (aoac, 1975). this was done to determine the moisture, crude protein, crude fat, and ash contents of the carcasses. at the end of the experiment, the remaining fish from two tanks of each treatment were grouped together and subjected to proximate analysis to determine the body composition.

The data of the mean body weight gains of the fish at the end of the feeding experiment and the gonadosomatic index (gsi) values were subjected to logarithmic transformation because of the heterogeneity of variances and then to one-way analysis of variance followed by the student-newman-keuls multiple range test (sokal and rohlf, 1969).

RESULTS AND DISCUSSION

Growth. The fish fed on the protein diets showed significantly greater mean body weight gains than fish fed on a non-protein diet (Table 3). Fish given the non-protein diet showed abnormal and stunted growth. There was a small overall mean body weight gain of 0.02 during the experiment. Protein is absolutely essential for the buildup of new body tissue. If protein is excluded from the diet, no growth of the fish might be expected. In fact, a decrease in body weight has been reported by many workers for fish given a non-protein diet (Dabrowski, 1977; Jauncey, 1982). A slight increase in weight, however, was reported by Sen et al. (1978). Ogino et al. (1976) proposed that the slight weight gain in carp (Cyprinus carpio Linneaus) fed on a protein-free diet may be due to the deposition of lipids in the body, the lipids being derived mainly from carbohydrates. This seems to be the case in the present experiment. The protein-free diet is also the one with the highest carbohydrate level. Carbohydrates could have been converted into body fat, thus giving the very high fat content in the fish of this group and resulting in an increase weight.

Among the fish fed protein diets, those on 20 percent and higher levels of protein had significantly greater gains in mean body weight than fish on a 10 percent protein diet. The 30 percent diet also proved to be significantly better than the 20 percent protein diet. No significant differences in the mean body weight gain were detected among the fish given the 20, 40, 50, and 60 percent diets.

Table 2. Proximate analysis of experimental diets.

Diets with different protein levels (%) Percentage on wet weight basis

Moisture Dry Matter Crude Protein Crude Fat Ash

0 9.83 90.17 0.00 7.52 13.70

10 9.31 90.69 11.35 9.80 14.56

20 9.10 90.90 21.65 8.84 15.31

30 9.50 90.50 30.55 9.08 14.99

40 9.49 90.51 39.45 10.53 13.86

50 9.14 90.86 49.68 11.94 12.91

60 9.44 90.56 59.79 10.98 13.96

The results indicate that raising the level of dietary protein up to 30 percent gave increasingly better growth in the female guppy. However, further increases in the dietary protein level beyond 30 percent did not give significantly greater weight gains. Fish fed on the 30 percent protein diet in the experiment thus showed the best for growth.

Lim et al. (1979) attributed the slightly lower weight gains of milkfish, Chanos chanos (Forsskal), fed diets with 50 percent and 60 percent protein compared to those fed a 40 percent protein diet to insufficient no-protein energy in the diets. Prather and Lovell (1973) even indicated that diets with high levels of protein and low amount of non-protein energy may be toxic to channel catfish, Ictalurus punctatus (Rafinesque).

The diets used in the experiment were approximately isocaloric. At the higher dietary protein levels, a higher proportion of the energy available in the diet was supplied by protein instead of carbohydrates or lipid. Excess protein might be deaminated in the liver. This could explain why the 40 percent to 60 percent diets supported less efficient growth of the fish than the diet with only 30 percent protein.

Jauncey (1982) postulated that the slight decrease in specific growth rate at protein levels above the optimum for juvenile tilapia, Sarotherodon mossambicus (Peters), may be due to less dietary energy available for growth due to the energy needed to daminate and excrete excess absorbed amino acids. This could be the second reason why protein levels above 30 percent gave lower weight gains in the present investigation.

In an experiment where channel catfish fingerlings were fed diets with varying protein to energy rations, Garling and Wilson (1976) showed that diets containing about 88 mg protein/kcal and adequate energy levels produced fish with optimum weight gains. In pond studies, for the same fish, Prather and Lovell (1973) found the optimum ration to be between 131.6 and 146.7 mg protein/kcal. In the present study, the protein/energy ratios of the tests diets ranged o 186.34 mg protein/kcal metabolize energy (ME)(Table 1). Optimum weight gains of the fish were obtained with the 30 percent protein diet which had a protein/energy ratio of 96.46 mg protein/kcal ME.

Feed Conversion. The mean feed conversion rates for female guppies fed different levels of dietary protein are shown in Figure 1. The feed conversion values of fish fed on the non-protein diet had a very high value. This group of fish had only a very small increase in mean body weight throughout the experimental period. No matter how much the amount of feed they consumed, the amount of body weight gained was still very small. As a result, a high feed conversion value was obtained.

Table 3. Mean body weight gain and gonadal development of female guppy fed varying dietary levels of protein.

Dietary Protein Level (%) Mean body weight gain (mg) Mean weight of ovary (x10-4g) Mean GSI % Mean number of yolky oocytes

0 20^a 19.2^a 1.73^a 1^a*

10 130^b 149.9^b 7.97^bc 5^b

20 180^c 152.8^b 6.23^b 7^b

30 270^d 352.9^c 8.21^c 12^c

40 225^cd 290.7^c 8.72^c 12^c

50 205^cd 201.9^b 6.15^b 6^b

60 240^cd 194.6^b 6.47^b 9^bc

*Treatment means in each column having the same superscript are not statistically different at P(less than)0.05.

Figure 1. Mean feed conversion (of our two-week periods) of female guppy fed on varying dietary protein levels

The fish fed on all the protein diets had significantly lower feed conversion values than fish fed on a non-protein diet. With the presence of protein in the diet, true growth could occur, resulting in a lowering of the feed conversion values. However, no significant difference was detected among the feed conversion values of fish fed on the different protein diets. Despite the lack of significance, the 30 percent and 40 percent protein levels both gave the lowest mean feed conversion value of 4.04 suggesting that feed was most efficiently utilized at these two protein levels.

Gonadal Development. It is shown in Table 3 that fish fed on the 10 percent to 60 percent protein diets had significantly higher GSI values than fish on the non-protein diet. There were no significant differences among the GSI values of fish fed on the 10, 30, and 40 percent protein diets. In spite of the lack of significance, the 40 percent diet resulted in the highest mean GSI.

Most of the fish sampled for the zero protein diet had not reached the yolky oocyte stage. The majority of oocytes in the four fish were in the early stages of development although the oocytes at the yolk vesicle stage were present. It seems that the absence of protein in the diet greatly hindered the oocyte development in the ovary.

Despite the greater variation in the sizes and weights of ovaries of fish fed on the protein diets, practically all the fish sampled had oocytes which had already developed up to the yolk granule stage. However, the perinucleolar and the yolk vesicle stages were also observed. Yolk consists mainly of phospholipoproteins. Protein is thus a very important constituent of the yolk and the presence of dietary protein consequently supports good oocyte development. A larger number of oocytes at the early developmental stages was observed in the ovary of fish fed on the 10 percent protein diet than in those given the higher protein levels.

The 30 percent and 40 percent protein diets appeared to be the best level of protein for gonadal development since it resulted in the greatest mean ovary weight and in the largest mean number of yolky oocytes in the ovary.

Body composition. In general, fish contain approximately 80 to 85 percent water, extreme values ranging from 53 to 89.3 percent. This water content is higher than that in birds (about 70 percent) and mammals (about 75 percent) (Vinogradov, 1953). From Table 4, the body moisture of the fish before and after the experiment ranged from 70.63 to 74.35 percent, well within the limits defined above for fish. The moisture content of fish was higher before the experiment than in any of the treatments after the experiment. The higher moisture contents of younger animals is quite a well-known fact. All the seven experimental diets had about the same moisture content, ranging from 9.10 to 9.83 percent. Thus the seven treatments did not affect the fish moisture content to any marked extent. This is in congruence with the results of Dabrowski (1977) who found no change in the body moisture content of grass carp, Ctenopharyngodon idella Valenciennes, fry when the dietary protein level was varied.

There is a general trend of an increase in the body protein of the female guppy as the dietary protein level is increased. Fish receiving 10 percent or more of dietary protein had more than 50 percent of their body dry matter in the form of protein whilst in those fish on the zero protein diet, crude protein form only 47.06 percent of the dry body weight. Ogino and Saito (1979) reported the linear relation between protein content of diet and the body protein of young carp using casein as the protein source, within the dietary protein levels from 4.6 to 38.2 percent. Satia (1974) showed a general increase in the protein content in the carcass of rainbow trout in relation to the amount present in the diet. The increase in protein content of fish as dietary protein level is increased has also been reported by Nose and Arai (1972), Cowey et al. (1972), and Luquet and Sabut (1973).

Comparing the figures for crude fat in the fish before and after the experiment, fish fed the 0 to 30 percent protein diets appeared to have accumulated fat. The fat content of those given the 40 to 60 percent protein diets did not appear different from the initial fact content of the fish. Fish on the non-protein diet showed very marked accumulation of fat and loss of protein. Rainbow trout have been found to lose both fat and protein on a high carbohydrate and low protein diet whilst goldfish, Carassius auratus (Linnaeus), accumulate fat and lose protein (Nose, 1961). The guppy resembles the goldfish more closely than rainbow trout in that there was an accumulation of fat and loss of protein in fish given the protein-free diet.

TABLE 4. Carcass analysis of female guppy

Fish fed on diets with different protein levels (%) Percentage on wet weight basis Percentage on dry weight basis

Moisture Dry Matter Crude Protein Crude Fat Ash

Beginning of experiment

74.35 25.65 58.01 22.01 18.02

End of experiment

0 71.07 28.93 47.06 36.25 15.04

10 72.80 27.20 52.89 30.32 15.78

20 72.72 27.78 54.66 25.32 17.16

30 70.73 29.27 55.39 26.81 16.08

40 70.63 29.37 55.71 22.23 17.09

50 73.61 26.39 55.24 21.45 17.84

60 73.38 26.62 58.47 22.88 17.43

Body lipid content was found to be positively correlated with dietary lipid levels in rainbow trout but with carbohydrate levels in carp (Cyprinus carpio Linnaeus) (Ogino et al., 1976). Referring to Tables 2 and 4, it can be seen that fish which had high crude fat content were actually those which had received diets containing high carbohydrate. This is similar to the situation found in carp. It could be possible that excess carbohydrate was converted into body fat for storage.

The body ash level of the fish in all seven treatments fell within a narrow rank of 15.04 to 17.84 percent. It appeared to be unaffected by the different dietary protein levels. The mineral premix and fish meal are the sources of ash in the diet. Fish meal is a good source of calcium and phosphorous (Allen, 1970). In the formulation of the experimental diets, the proportion of mineral premix in the diet was successively lowered whilst that of fish meal was progressively raised as the dietary protein level was increased (Table 1). As a result, all the diets had similar ash contents. This could perhaps be why all seven treatments gave fish rather similar proportions of body ash.

Based on the present study, it may be suggested that 30 to 40 percent dietary protein should be the optimal level for feeding guppies.

ACKNOWLEDGEMENTS

The project was supported by a research grant awarded by the National University of Singapore (NUS) (RP 79/82). The authors wish to thank Mr. Chua Whye Leng of Nutrition Laboratory, Department of Zoology, NUS for the proximate analysis of the fish foods and carcasses. The authors also wish to express their appreciation to Miss Tan Hiang Boon for typing the manuscript and Mrs. Yap Oi Yee for preparing the graph.

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