II/E-13
EFFECTS OF MICRO-NUTRIENTS ON THE SURVIVAL AND GROWTH OF INDIAN CARP FRY

by

B.C. DAS
Indian Statistical Institute
Calcutta 35, India

Abstract

Effects of micro-nutrients on the survival, growth and biochemistry of Indian carp fry from 3 to 26 days of age have been investigated. Survival is enhanced significantly by treatment with vitamin B complex, yeast, or ruminant stomach extract with cobalt nitrate. The reduction in survival due to density is significantly controlled by treatment with vitamin B complex or yeast. Among the micro-nutrient treatments employed, yeast increases growth most. Vitamin B complex and ruminant stomach extract also significantly increases growth. Based on these results, an equation for projecting yield after a specified period of time has been formulated. Yield is expressed as a function of the initial number of oneday-old fry, the proportion surviving for a given density and treatment, and the expected weight of any fish of a given species and age group. Biochemical determinations of total protein, non-protein nitrogen, acid phosphatase and alkaline phosphatase have been carried out for untreated fry and fry treated with yeast. Treatment with yeast has a statistically significant effect on total protein and non-protein nitrogen. A significant change in total protein, acid phosphatase and alkaline phosphatase occurs with age. The significant regression of weight on total protein decreases with yeast treatment. As yeast treatment decreases the total protein per g dry weight but increases the total weight, it appears to make fry more efficient as weight gaining units.

INFLUENCE DES OLIGO-ELEMENTS SUR LE TAUX DE SURVIE ET DE CROISSANCE DES ALEVINS DE CARPE INDIENNE

Résumé

Les études ont porté sur les effets des oligo-éléments sur la survie, la croissance et la biochimie d'alevins de carpe indienne âgés de 3 à 26 jours. Le taux de survie est sensiblement amélioré par un traitement au complexe vitaminique B, à la levure ou à l'extrait d'estomac de ruminant associé à du nitrate du cobalt. La diminution du taux de survie due à la densité est sensiblement réduite par un traitement au complexe vitaminique B ou à la levure. Parmi les oligo-éléments employés, c'est la levure qui a eu l'effet le plus favorable sur la croissance. Le complexe vitaminique B et l'extrait d'estomac de ruminant ont également sensiblement amélioré la croissance. Partant de ces résultats, on a établi une équation permettant de prévoir la production après un laps de temps déterminé. Dans cette formule, la production est exprimée sous forme de fonction du nombre initial d'alevins d'un jour, de la proportion de survivants calculée pour une densité et un traitement donnés et du poids prévu d'un poisson quelconque d'une espèce donnée selon le groupe d'âge. Le dosage biochimique des protéines totales, de l'azote non protéique, de la phosphatase acide et de la phosphatase alcaline a été effectué pour les alevins non traités et les alevins traités à la levure. Du point de vue statistique, le traitement à la levure a un effet sensible sur la quantité totale de protéines et d'azote non protéique. Avec l'âge, on constate un changement notable des protéines totales, de la phosphatase acide et de la phosphatase alcaline. La régression sensible de la relation poids/protéines totales diminue lorsque les sujets sont traités à la levure. Etant donné que ce traitement a pour effet de diminuer la quantité de protéines par gramme de poids sec mais d'augmenter le poids total, il paraît rendre les alevins plus aptes à prendre du poids.

EFECTOS DE LOS MICRO-NUTRIENTES SOBRE LA SUPERVIVENCIA Y CRECIMIENTO DE LOS ALEVINES DE CARPA INDIA

Extracto

Han sido investigados los efectos de los micro-nutrientes sobre la supervivencia, crecimiento y bioquímica de los alevines de carpa india de 3 a 26 días de edad. La supervivencia se mejora notablemente mediante al tratamiento con complejo de vitamina B, levaduras o extracto de estómagos de rumiantes con nitrato de cobalto. La reducción de la supervivencia debida a la densidad se limita sensiblemente mediante el tratamiento con complejo de vitamina B o levaduras. Entre los tratamientos con micro-nutrientes empleados, las levaduras son las que determinan un crecimiento mayor. El complejo de vitamina B y los extractos de estómagos de rumiantes también aumentan notablemente el desarrollo. Basada en estos resultados se ha formulado una ecuación para proyectar el rendimiento al cabo de un determinado período de tiempo. El rendimiento se expresa como función del número inicial de alevines de un día, la proporción superviviente para una determinada densidad y tratamiento, y el peso esperado de cualquier pez de una determinada especie y grupo de edad. Se han llevado a cabo determinaciones bioquímicas de la proteína total, nitrógeno no proteínico, fosfatasa ácida y fosfatasa alcalina para alevines sin tratar y tratados con levadura. El tratamiento con esta última tiene un efecto estadísticamente sensible sobre el nitrógeno proteínico y no proteínico total. Con la edad se registra un sensible cambio en la proteína total, la fosfatasa ácida y la alcalina. La notable regresión del peso sobre la proteína total disminuye con el tratamiento de levadura. Como este tratamiento hace descender la proteína total por gramo de peso de materia seca pero aumenta el peso total, hace que los alevines sean más eficaces como unidades ganadoras de peso.

1 INTRODUCTION

The survival and growth of an individual organism are influenced by the conditions characterizing the post-embryonic period of its life. In all species, survival is initially low, that is, the probability that the individual organism will die is high. If the internal and external environment are suitably modified, the likelihood of the individual organism's surviving increases. Development undergoes an initial latent period, after which there is very rapid growth. It is possible to alter the rate of growth during this period by changing the internal and external environment. The sensitivity of the organism during this period to its environment suggests that it may be practicable to determine the optimum conditions for the desired level of survival and rate of growth.

In commercial carp ¹ cultivation in India, as traditionally and generally practised, the newly hatched fish are collected at the river spawning grounds and placed in large earthen vessels. The hatchlings live in the vessels for several days during which they are taken to ponds for cultivation. After only one day in the earthen vessels, counts have shown that only ten to twenty percent of them may be expected to survive. Effects on growth can only be surmised. From the point of view of food production, it is of utmost importance to reduce the wastage of fish fry which results from these traditional practices. Efforts in this direction which have recently been made include transport of fry in oxygenated plastic bags and induced spawning of carp by pituitary injections (e.g. Barrackpore, Central Inland Fisheries Research Institute, 1958).

The Biometry Research Unit of the Indian Statistical Institute has been engaged in an intensive study of the post-embryonic period of life of Indian carp for nearly a decade. Particular attention has been devoted to the identification of micro-nutrients which will significantly increase both the growth and survival of the fry. This paper deals with the effects of micro-nutrients on survival and growth during the post-embryonic period, on projections of yield, and on the biochemistry of fry.

¹ The carps commercially cultivated in India are Catla catla, Labeo rohita and Cirrhina mrigala. In this paper these species are considered together, except where specifically mentioned.

2 SURVIVAL OF FRY

2.1 Procedures

2.1.1 Laboratory

The laboratory contained a large number of experimental units for cultivation and observation of the fry. Each experimental unit consisted of an earthen bowl, 40 cm in diameter, which contained 9 litres of water and was covered with screening. The water of each unit was changed every 24 hours by a siphoning technique. Fresh pond water, from the same source, was added and the water maintained at the 9 litre level throughout the experiment. Temperature and pH of the water were recorded daily. Fluorescent tube lights were arranged to ensure uniform lighting throughout the laboratory 12 hours daily, and fans were arranged to keep the air circulating throughout the laboratory. Experiments were conducted yearly during the spawning season from June to August.

2.1.2 Supply and feeding of fry

Indian fresh-water carp were obtained from the river spawning grounds one day after hatching from supplies for commercial carp cultivators. The carp were brought to the laboratory on the same day in the earthen vessels in which they were supplied. They were immediately allocated to the experimental units by randomly assigning one teaspoonful of carp (containing approximately 600 carp) to each experimental unit. Exact enumeration was not possible due to the minute size of the carp.

Table I

Mean percentage survival of Indian carp fry at 11 days of age *

* Based on six experiments

Table II

Expected percentage survival of Indian carp fry at 7 days of age in relation to initial density

Following the Government of India recommended cultural practices for Indian carp fry (Chopra, 1951), the carp were fed live Daphnia cultivated in a richly fertilized pond. Each experimental unit received 10 ml of a solution of five parts Daphnia by volume to 95 parts of water daily. This ration was considered sufficient as some Daphnia always remained unconsumed.

2.1.3 Micro-nutrients

The effects of five micro-nutrients were studied. The micro-nutrients and their daily dosage levels are as follows:

1. B₁₂: crystalline vitamin B₁₂, 25 μg; British Drug Houses Ltd.

2. RSE + Co(NO₃)_2. 6H₂O: ruminant stomach extract, 10 ml, and cobalt nitrate, 1 μg. The ruminant stomach extract consisted of the juice expressed from the entire contents of the four stomachs of a freshly slaughtered goat; no further water was added. The cobalt nitrate was obtained in powder form and dissolved in distilled water. The molarity was 3.43 × 10^-6 and total impurities present did not exceed 0.045 percent.

3. Co(NO₃)₂. 6H₂O: cobalt nitrate, 1 μg.

4. B complex: vitamin B complex, one tablet containing 10 mg thiamine hydrochloride, 3 mg riboflavin, 2 mg pyridoxine hydrochloride, 15 mg nicotinic acid amide, and 3 mg calcium pantothenate; Teddington Chemical Factory, Bombay.

5. Yeast: dried brewer's yeast, one tablet containing 0.324 g yeast (prior to 1963) or 0.5 g yeast (1963 onwards); British Drug Houses Ltd.

2.1.4 Collection of survival data

To obtain the survival data, the dead carp were completely enumerated for each experimental unit daily. The dead carp settled to the bottom of the experimental unit after a centrifugal current was induced in the water, and were withdrawn with a glass pippette, placed on blotting paper and counted. A fresh pippette was used for each experimental unit each day. At the end of the experiment, the live carp were counted and the number initially placed in the experimental units was reconstructed by adding the total number dead to the number alive at the end of the experiment.

2.2 Statistical design

Randomized block designs were employed for each experiment. Using a randomized block design, the experimental units were placed in blocks and randomly assigned to the several treatments. This design permits a test of the significance of block effects (indicating position within the laboratory) as well as treatment effects and distributes the treatments randomly throughout the laboratory. Tests of significance were carried out by analysis of variance (Cochran and Cox, 1957).

2.3 Results: Percentage survival

Table I presents the mean percentage surviving (column (2)) under the different micro-nutrient treatments, along with the mean initial density of the fry, in terms of ml water per fish (column (3)) and number of fish per litre (column (4)). The value of 30.33 percent for the untreated control is comparable to figures reported for carp nurseries (Barrackpore, Central Inland Fisheries Research Institute, 1962). For marine nurseries, Halliday (1965) has reported 52 percent survival. Treatment with certain micro-nutrients significantly enhances survival over that of the untreated control. Sixty percent survive with vitamin B complex, yeast, or ruminant stomach extract with cobalt nitrate. Some increase in survival also occurs with vitamin B₁₂ and with cobalt nitrate. It was hypothesized that the cobalt nitrate permitted synthesis of vitamin B₁₂ by the fry, either from their regular diet or from the ruminant stomach extract (Das, 1959; 1960 a, b; 1965 b; Das and Krishnamurthy, 1959).

2.4 Results: Density

The effect of density on survival has been widely observed (Lack, 1954; Andrewartha and Birch, 1954); increasing density being associated with decreasing survival. The density-survival relationship for Indian carp fry was characterized mathematically for the first time by Das (1961 a). A hyperbolic equation with Y = percentage survival, Z = number surviving, X = initial number, and a and b being the fitted constants, characterizes the density-survival relationship during the post-embryonic period of life. For the hyperbolic equation fitted to the effect of density on survival of untreated control carp at the end of the first week, a = 1.1434 and b = 0.0060. Similarly, for carp treated with vitamin B complex, a = 1.0143 and b = 0.0010. Comparison of the values for the constant a shows that vitamin treatment immediately enhanced survival. Comparison of the values of the constant b shows that vitamin B complex alleviated the effect of density on survival. These results are illustrated by Table II. Expected percentage survival, computed from the hyperbolic equation, is reported in terms of density as initial volume of water available per fish as well as initial number of fish per litre of water. Expected values for treated and untreated control carp show that at low density, and even more at high density, vitamin B complex treatment enhances survival.

2.5 Discussion

Results of experiments conducted in earthen bowls, which simulate equipment used by commercial carp cultivators, show that micro-nutrients can enhance survival and reduce the effect of density. It should be noted that even at very low densities, treated carp are more likely to survive than untreated carp. Projected values for the effects of such treatment on a large scale will be presented in section 4.

3 GROWTH OF FRY

3.1 Post-embryonic growth

3.1.1 Collection of data

Laboratory, supply and feeding of fry and micro-nutrients have been described in sections 2.1.1, 2.1.2, and 2.1.3. Growth data were collected by withdrawing and weighing samples consisting of ten living fry daily from the experimental units. Samples were withdrawn from half of the replications for all treatments on even-numbered days of age and from the other half on odd-numbered days of age. Each experimental unit was sampled every other day throughout the experimental period. Samples were kept separate from each other throughout the drying and weighing process so that weight data would be available for individual experimental units.

The drying and weighing procedure followed the same time schedule every day. After being withdrawn from an experimental unit, a sample of ten fry was placed in 70 percent alcohol and then the ten fry were dried separately on blotting paper. After this, the sample was placed in a drying chamber at 32°C for six hours and then at 60°C for the next 18 hours. It was then placed in a dessicator for one hour, after which it was immediately weighed on a chemical balance.

3.1.2 Results

Table III gives the dry weight per individual carp fry at 3, 11, and 19 days of age under different micro-nutrient treatments. The initial equivalence of the fry is illustrated by column (2). While at 11 days the fry still are nearly equal in weight, by 19 days of age clear differences (P<0.01) are apparent. In the order of effect on growth rate, from most to least effective, the micro-nutrients are yeast, vitamin B complex, ruminant stomach extract with cobalt nitrate, and vitamin B₁₂. These results suggest that i) vitamin B₁₂ is not a carp growth factor, ii) yeast contains a growth factor not present in standard preparations of vitamin B complex, and iii) ruminant stomach extract with cobalt nitrate has an effect not unlike that of vitamin B complex. The results of this and other experiments (Das, 1960 b, 1965 b) support the hypothesis that growth in the post-embryonic period of life can be enhanced when the internal and external environment are modified by micro-nutrients. Furthermore, results with varying yeast dosage levels (Das, 1965 b) suggest that growth is enhanced by the protein added to the diet by yeast.

3.2 Juvenile growth

3.2.1 Collection of data

Three ponds on the premises of the Indian Statistical Institute were used for this investigation. Two months before the fish were to be placed in the ponds, all fish present were removed and the water was fertilized with mahua seed cakes. The length, width, and depth, in meters, of the three ponds were 46.0, 27.5, and 4.5; 26.0, 24.5, and 4.0; and 27.5, 23.0, and 4.0 respectively.

Three hundred fry each of Catla catla and Labeo rohita were placed in each of the ponds when they were twenty days old. No additional food, beyond that existing naturally in the ponds, was given after the fish were introduced. To obtain the growth rate data, samples of fish were withdrawn for weighing at regular intervals throughout a two-year period. A sample was defined as all fish obtained in the first two nettings of a pond. Each fish caught was weighed individually on an accurate balance and put back in the pond (Das, 1960c).

3.2.2 Results

Table IV gives the mean weights in grams of Catla catla and Labeo rohita specimens according to age. A mean weight may be interpreted as the expected weight for an individual fish. Using mean wet weights for the post-embryonic period and the mean weights of Table IV, linear equations have been fitted to age in days. The fitted equations are: W'_c = -12.0880 + 1.3682 a for Catla catla and W'_r = 16.1597 + 0.2596 a for Labeo rohita, where W' = expected weight in grams and a = age in days. The more rapid growth of Catla is illustrated by these equations and also by the values reported in Table IV. The equations permit projections of yield which will be discussed in section 4.

3.3 Discussion

The results reported in Table III demonstrate that micro-nutrients enhance the growth of Indian carp fry. As the growth of domesticated avian and mammalian species has also been found to be enhanced by micro-nutrients, particularly vitamins, this result is not unexpected. The present contribution is in identifying the effective micro-nutrients and their optimum dosage levels, with particular attention to feasibility for commercial carp cultivation. In the experiment reported in Table III, fry treated with yeast weighed the most. While yeast, vitamin B complex and ruminant stomach extract with cobalt nitrate were equally effective in enhancing survival, yeast was superior to vitamin B complex and the extract in increasing growth. Yeast shares the survival-promoting and density-controlling factors of vitamin B complex and the extract, and in addition contains a growth-promoting factor. The dosage employed has been 0.5 g per 9 litres, approximately 56 mg per litre of water. This dosage can be economically and easily administered in large-scale carp cultivation. Some projections of the yield which might be expected by implementation of yeast treatment at the post-embryonic stage are discussed in section 4.

Table III

Mean dry weight in milligrams of Indian carp fry at 3, 11, and 19 days of age *

* Based on one experiment with 6 replications

Table IV

Mean wet weight in grams at known age of the Indian carp Catla catla and Labeo rohita reared in ponds

Observations on the growth of pond-reared fry have been summarized in Table IV. The conditions of cultivation were those typically encountered in commercial carp cultivation in the region around Calcutta, India. The observations may reflect the usual pattern of growth. It may be noted that growth studies on Indian carp are generally based on catches from rivers, and hence the results may not reflect the pattern of growth in ponds. Finally, it may be mentioned that the results in Table IV are for carp of known age and not age as inferred from scale readings or length-frequency distributions.

4 PROJECTIONS OF YIELD

Yield of any crop depends on two basic components, the number of productive units and the values of the productive units (Das, 1963). In the context of fisheries, the number of productive units is the number of fish of a given species and age group, and the values of the productive units are their weights. The expected value for any productive unit taken at random is the mean of the weights. Yield is defined as the total wet weight of the catch of a given species and age group, and can be regarded as the product of the number of fish and the expected weight of any fish taken at random, i.e., the mean weight of all fish in the catch.

The data presented in the preceding sections provide information about both the number of fish and their expected weight. Section 2 describes the effects of density and vitamin treatment on the number of fish, and section 3 presents expected values for pondreared fish of two species at varying ages. These findings permit a more detailed statement of the components of yield. Let

be the true yield after a time period t, given the initial number n₀, density d and treatment Tr in the post-embryonic period, and let

be its estimate. Then

when

n_o is the initial number of one-day-old fry,

p_d,Tr is the proportion surviving under specified density and treatment in the post-embryonic period, and

w_t,Tr is the mean weight after time period t for all fish receiving treatment Tr.

Table V gives projected values of Y'(t/n_o, d, Tr) at t = 1 year for n_o = 100,000, using the values of p_{d, Tr} and w_{t, Tr} from Tables II and IV. In Table V, percentage survival at 7 days gives 100p_d,Tr. For t = 1 year and Tr = 0 (untreated control), Table IV gives w_{t, Tr} = 0.49 kg for Catla catla and w_{t, Tr} = 0.11 kg for Labeo rohita, which are the values used to compute Y'(t/n_o, d, Tr) shown as kg wet weight yield.

Table V

Effect of density and treatment in the post-embryonic period on the estimated yield in kg of one-year-old Indian carp reared in ponds

Table VI

Mean biochemical values for Indian carp fry treated with 0,0.5 and 2.0 g yeast daily

The effects of density and of treatment in the post-embryonic period on projections of yield after one year are illustrated by the values in Table V. At high density (10 ml/fry), yeast or vitamin treatment triples projected yield. At medium density (40 ml/fry), treatment doubles projected yield, and increases it by a lower factor at low density (100 ml/fry). If density cannot be reduced, yeast or vitamin treatment increases survival, hence Pd, Tr yield. Without treatment, reduction in density from 10 ml/fry to 40 ml/fry nearly triples projected yield, and reduction from 10 ml/fry to 100 ml/fry nearly quadruples projected yield. The detrimental effects of density on survival are greatly reduced by yeast or vitamin treatment, and for treated fry, a change in density from 10 ml/fry to 100 ml/fry does not even double projected yield.

Equation [3] is a simplified statement of the factors entering into yield. It does, however, make explicit the role of post-embryonic conditions in projections of yield at a later date, and is based entirely upon experimental and observational results. It assumes no further reduction in survival after the post-embryonic period and hence will overestimate yield to the extent that predators and other environmental factors are hostile to the fish being cultivated.

As used in Table V, w_{t, Tr} has only been computed for pond-reared fish not treated with vitamins at the post-embryonic stage (Tr = 0). The results given in Table III suggest that growth is also affected by yeast treatment. If it can be demonstrated that w_{t, Tr}, at t = 1 year, differs significantly for treated (Tr = 1) and untreated (Tr = 0) fry, then values of Y'(t/n_o, d, Tr) can be computed for both sets of values of w_{t, Tr}. Further investigations are underway in this laboratory to determine whether or not post-embryonic treatment affects w_{t, Tr} at a later date.

The importance of the post-embryonic period for yield of fish as a commercial crop has been shown by equation [3]. From the point of view of commercial pond fish culture, neglect of the fry at the post-embryonic period is clearly wasteful. From a purely biological point of view, investigations to determine p_{d, Tr} and w_{t, Tr} under varying conditions and for various species, should be of interest, but in turn could also have practical value.

5 MICRO-NUTRIENTS AND BIOCHEMISTRY OF FRY

5.1 Procedures

5.1.1 Laboratory

Laboratory and supply and feeding of fry have been described in sections 2.1.1 and 2.1.2.

5.1.2 Micro-nutrients

Biochemical investigations have been carried out for fry treated with 0.5 and 2.0 g yeast (British Drug Houses Ltd.) daily. The untreated control is designated as 0 g yeast.

5.1.3 Biochemical determinations

Samples of fry were dried and weighed according to the procedure described in section 3.1.1. Each sample consisting of ten fry was treated separately in the bio-chemical analysis. A dried and weighed sample of fry was crushed with mortar and pestle, dissolved in 10 ml distilled water, and centrifuged at 2500 rpm for 10 minutes. Ten ml of the supernatant fluid was analysed for total protein, non-protein nitrogen, acid phosphatase and alkaline phosphatase, using standard laboratory methods (King and Wooten, 1956). The biochemical determinations were recorded in g/ml for total protein, mg/ml for non-protein nitrogen, and KA units/ml for acid phosphatase and alkaline phosphatase. By dividing each biochemical value by the dry weight in g, the base of the unit of measurement was changed from one ml to one g dry weight. Conversion of the values was necessary to permit comparison of samples differing in weight.

Table VII

Biochemical values per g dry weight for Indian carp fry treated with 0, 0.5, and 2.0 g yeast daily

Table VIII

Prediction of dry weight for Indian carp fry by biochemical determinations

** P<0.01

* P<0.05

5.2 Statistical design and analysis

A set of six latin-squares, with each experimental unit being represented only once in one latin-square, was designed (Cochran and Cox, 1957). Each latin-square had three rows and three columns. The three treatments were replicated three times in each latin-square, with any one treatment assigned only once to any row or column. This design permitted statistical control of position effects within the laboratory on experimental data. Effects of treatment and age, within a six-day period, could be statistically examined by the analysis of variance. The experiment was conducted for 24 days, permitting the set of six latin-squares to be replicated four times. Fry were tested daily from 3 to 26 days of age. Analyses of variance were carried out to test the significance of the effects of treatment, age, rows, and columns. The analyses were carried out separately for total protein (g/g dry weight), non-protein nitrogen (mg/g dry weight), acid phosphatase (KA units/g dry weight), and alkaline phosphatase (KA units/g dry weight). The matrix of correlation coefficients for the four biochemical determinations and weight was computed separately for 0, 0.5, and 2.0 g yeast treatment groups. For each of the three matrices so obtained, multiple regression equations were computed for the prediction of weight by the four biochemical determinations.

5.3 Results

5.3.1 Analysis of variance

Table VI gives the means for the four biochemical determinations for Indian carp fry from 15 to 26 days of age according to yeast treatment. For the last set of six latin-squares (age 21 to 26 days), the results of the analyses of variance can be briefly summarized as entries in the two-way table below:

Table VII presents the means for the biochemical values per g dry weight. The results of the analyses of variance for these values in the last set of six latin-squares can also be summarized as entries in a two-way table.

The differences between these two-way tables can be attributed to the relationships between dry weight and the biochemical determinations. The treatment effect on acid phosphatase and alkaline phosphatase can be attributed to the influence of dry weight; when that influence is partialled out, only the age effect remains significant. Similarly, total protein per fish does not charge significantly with age, but when the influence of dry weight is partialled out, age is found to significantly affect total protein. Non-protein nitrogen per carp fry is apparently unaffected by age or treatment; partialling out the influence of dry weight reveals that non-protein nitrogen is affected by treatment but not by age. Age, in these analyses, refers to changes during a six-day period, which is characterized by rapid growth in carp fry.

5.3.2 Multiple regression analysis

The preceding analyses of variance compare the effects of the micronutrients and age on one biochemical determination at a time. Subsequent discussion will deal with multiple regression analysis of the biochemical determinations (Walker and Lev, 1953). The objective of the analysis is to determine the regression of dry weight, which may be interpreted as net yield, on the four biochemical determinations. This analysis has been carried out separately for the three levels of micro-nutrient treatment to determine whether treatment alters the regression on the biochemical factors. Table VIII gives the standard partial regression coefficients, multiple correlations, and multiple regression equations for the prediction of yield, as dry weight. The standard partial regression coefficients are pure numbers which indicate the regression on each biochemical factor taken by itself, for yield, after partialling out the influence of the other factors. The relative contribution of each factor, compared with other factors and under different treatments, is shown by the standard partial regression coefficients. For these data, total protein is most important under 0 g (P<0.01) and 0.5 g (P< 0.05) yeast treatment, but is not statistically significant under 2.0 g yeast treatment. Non-protein nitrogen is important (P<0.05) under 0.5 g yeast treatment. These results suggest that, among the four biochemical factors, total protein per g dry weight, is most related to yield under untreated control conditions, but its relationship decreases with yeast treatment. The negative sign of the total protein regression coefficient means that, the greater the weight, the smaller the total protein per g weight. The multiple correlation coefficient is significant only for the untreated control, which indicates that the regression of dry weight on the four biochemical determinations is significantly altered by yeast treatment. Multiple regression equations for the prediction of yield, as dry weight, for Indian carp fry 15 to 26 days of age, are also given in Table VIII.

5.4 Discussion

Four biochemical factors which may underly yield have been explored: total protein, non-protein nitrogen, acid phosphatase and alkaline phosphatase. Total protein refers to the body protein and non-protein nitrogen measures the product of tissue metabolism and protein digestion. Acid and alkaline phosphatase are esterases involved in digestion and respiration; their optimum pH values are 5 for acid phosphatase and 9 for alkaline phosphatase. By means of the analysis of variance, the effects of micro-nutrients and age on each biochemical factor have been tested, and by means of multiple regression analysis, the regression of yield on the biochemical factors has been explored. The original biochemical determinations, expressed in terms of ml, were converted to the unit of measurement per g dry weight. Statistically significant relationships between the biochemical factors and age and weight for Indian carp fry 15 to 26 days old can be summarized as follows:

It has been demonstrated that the micro-nutrients have a statistically significant effect on dry weight, and on total protein and non-protein nitrogen per g dry weight. In the latter two cases, the nature of this effect is to reduce the amount as compared with the untreated control. The regression of weight on total protein is greatest in the untreated control, and decreases with micro-nutrient treatment, to nil in 2.0 g yeast treatment (see Table VIII). These results suggest that micro-nutrient treatment makes the fish more efficient as a productive unit, i.e., it requires less total protein and removes less non-protein nitrogen per g dry weight. This may be important in considering the merits of micro-nutrient treatment at the initial stage in pond fish culture.

The effect of age, within the narrow period of rapid post-embryonic growth, is important in considering the biological basis of fish culture. Moving averages computed for the data in Table VII show that total protein, acid phosphatase and alkaline phosphatase increase with age, and may be regarded as biochemical concomitants or correlates of growth. While non-protein nitrogen changes significantly with age, the pattern of change is irregular. This experiment constitutes the first published effort on the biochemistry of Indian carp fry. The biochemistry of the blood and serum has also been investigated for juvenile Indian carp (Das, 1961 b; 1965 a). Further biochemical experiments are in progress in this laboratory which have as their dual objective the increasing of yield and determining the mechanisms of growth in the post-embryonic period.

6 REFERENCES

Andrewartha, H.C. and L.C. Birch, 1954 The distribution and abundance of animals. Chicago, University of Chicago Press, 782 p.

Barrackpore, 1958 Central Inland Fisheries Research Institute, Rep.cent.Inld.Fish.Res.Inst., Barrackpore, 1957–1958

Barrackpore, 1962 Rep.cent.Inld.Fish.Res.Inst., Barrackpore, 1961–1962

Cochran, W.G. and G.M. Cox, 1957 Experimental designs. 2nd Ed. New York, John Wiley, 611 p.

Chopra, B.N., 1951 (Ed.), Handbook of Indian fisheries. New Delhi, Ministry of Agriculture, 129 p.

Das, B.C., 1959 Comparative effects of vitamin B₁₂, cobalt nitrate and ruminant stomach extract on the survival rate of Indian carp during the first three weeks of life. Indian J.Fish., 6(1):211–21

Das, B.C., 1960a Effectiveness of yeast, vitamin B₁₂, and alternative treatments on survival of Indian carp during the early period of life. Canad.J.Biochem.Physiol., 38(5):453–8

Das, B.C., 1960b Differential growth and survival of post-embryonic carp under treatments with yeast and vitamin B complex. J.exp.Zool., 144(2):159–64

Das, B.C., 1960c Growth rates of Catla catla and Labeo rohita in confined waters. Sci.Cult., 26(2):94–6

Das, B.C., 1961a Effect of density on survival of Indian fresh-water carp during the first two weeks of life. Proc.nat.Inst.Sci.India, 27B(3):91–103

Das, B.C., 1961b A comparative study of the blood biochemistry of several species of Indian carp. Trans.Amer.Fish.Soc., 90(1):1–5

Das, B.C., 1963 Effects of β-napthoxyacetic acid on tillering and grain production in rice. Bot.Gaz., 124(4):270–4

Das, B.C., 1965a Age-related trends in the blood chemistry and hematology of the Indian carp (Catla catla). Gerontologia, 10(1):47–64

Das, B.C., 1965b Yeast dosage level and the post-embryonic survival and growth of the carp Labeo rohita (Ham.). J.exp.Zool., 159(3):405–12

Das, B.C. and H. Krishnamurthy, 1959 Comparative effects of yeast, vitamin B complex and vitamin B₁₂ on the survival rate of Indian minor carp during the first two weeks of life. Proc.nat.Inst.Sci.India, 25B(6):300–6

Halliday, R., 1965 Five acres of fish. New Scient., 27(455):330–1

King, E.J. and I.D.P. Wooten, 1956 Microanalysis in medical biochemistry. 3rd Ed. London, J. and A. Churchill. 292 p.

Lack, D., 1954 The natural regulation of animal numbers. Oxford, Clarendon Press. 343 p.

Walker, H. and J. Lev., 1953 Statistical inference. New York, Holt, Rinehart and Winston. 510 p.