A PHYSICO-CHEMICAL ANALYSIS OF RIPE, UNSPAWNED EGGS OF SOME FRESHWATER

TELEOSTS *

BY REHAN HASAN AND A. K . JAFRI

(Department of Zoology, Aligarh Muslim University, Aligarh, India) Received February 29, 1964

(Communicated by Dr. B. S. Bhimachar, F.A.SC.)

INTRODUCTION

ALTHOUGH a large body of information has accumulated on the chemical composition of fish (Jowett and Davies, 1938; Williams, 1951; Kuppu- swamy, 1958; Krishnamurthi, 1958; Idler et al., 1958 a and b, 1959; Love et al., 1959, Chang et al., 1960 a and b; P lacke t al., 1961 ; and Borgstrom, 1960, little attention has been paid to hard roes and eggs. Besides the obser- vations of Idler and Bitners (1960) on the roes of Oncorhynchus nerka no adequate knowledge exists about the gross chemical composition and physical properties of fish eggs. In the present communication we propose to present a comparative analysis, of eggs belonging to 19 species of freshwater teleosts.

MATERIAL AND METHODS

Fully ripe and almost running fishes were obtained from the local fish market and their ovaries dissected out. The eggs were then removed from the ovaries and Sampled for various determinations. For each item usually duplicate determinations were made either separately or on pooled samples from not less than three individuals.

Colour of eggs was studied by comparing it with Jenson and Nicholson's colour chart for cement paints. Ova diameter records were made on eggs fixed in 10~ formalin for about an hour. Specific gravity was determined by direct displacement of distilled water. Moisture was estimated by oven- drying weighed samples of eggs in crucibles for about 14 hours at 100 ° C. For fat extraction the method of Saha and Guha (1939) was followed. For protein, total nitrogen was determined colorimetrically after digesting 0.1 gm.

* This work was carried out while one of the authors (R. H.) was a temporary lecturer in the Department of Zoology, A.M.U., Aligarh.

BI 1

2 REIIAN HASAN AND A. K. JAFRI

of fresh eggs in 5 ml. of 1 : I sulphuric acid usin~ saturated potassium persul- phate as oxidizing agent (Wong; 1923). The figure 6.25 was used as a protein factor. Calcium, iron (ferric) and total phosphorus were analysed according to the methods suggested by Clark and Collip (1925), Kennedy (1927) and Fiske and Subbarow (1925) respectively.

PHYSICAL PROPERTIES

Colour.--The colour of eggs of a particular species was fairly specific. However, it varied slightly in certain individuals and with minor differences in the condition of ripeness. The general colour scheme of eggs of all the species under investigation is given in Table I.

Specific gravity.--There is not much variation in the specific gravity of eggs of different species. Most of them have a specific gravity approxi- mately 1-1 (Table I). Mystus v~ttatus and Mystus cavasfus have the heaviest eggs, almost one and a half times as heavy as water. The lowest specific gravity is encountered generally in those species whose eggs have a very high fat content. For example the eggs of Mug~l corsuta which have 35~ fat content are only 1.033 times heavier than distilled water.

Although it seems fairly reasonable to presume an inverse relationship between the two parameters (fat content and specific gravity) our observations do not reveal such a relationship to be strictly applicable. Species having the same specific gravity may sometimes differ considerably in the fat content of their eggs (Table I). When fat percentage on a dry matter basis is plotted against specific gravity of eggs an interesting relationship is obtained (Fig. 1).

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FAT AS p f l l H : i ~ f A 6 l OP DRy MAT1ER

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I~G. I. Distribution of specific gravity of the eggs against their fat content as percentage of dry matter. - 1-19refer to species as enumerated in Table I.

The lack of proportionate decrease in the specific gravity with the increase of fat content is probably due to the nature of lipids present. Further investi- gations are needed on the chemical composition, the specific gravity and

Physico-Chemical Analysis of Eggs of Some Freshwater Teleosts 3

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the degree of saturation of fatty constituents of eggs of different species before a satisfactory explanation of such a relationship can be put forward.

Since river or pond water may have slightly higher specific gravity than distilled water due to dissolved substances, eggs having specific gravity of 1.1 can neithter be truly pelagic nor demersal. This idea is supported by the observations of Alikunhi and Nagaraja Rao (1951) on the eggs of Cirrht'na reba and of Khan (1943) on Cirrhina mrigala. Alikunhi and Nagaraja Rao (1951) have found that the eggs of C. reba are demersal only in stagnant water while Khan has noted that the eggs of C. mrigala are non-floating. Probably they remain partially suspended well above the bottom and may therefore be considered as semidemersal: Since oxygen concentration is low at the bottom this feature seems to be an adaptation to ensure optimum supply of oxygen during embryo- nic development of these species. The eggs of Mystus vittatus and M. cavasius seem to be completely demersal because they are much heavier than water. Eggs of other species having a specific gravity lower than 1.08 may be con- sidered as pelagic, for example those of Mugil corsula. The observations of Pakrasi and Alikunhi (1952) on the eggs of Mugil corsula reveal them to be really so. They are semi-pelagic and remain suspended below the surface of water.

Size.--Present observations on the maximum diameter of eggs of all the species under investigation correspond fairly well with the maximum diameter for some species noted by Qasim and Qayyum (1961). Earlier observations on the maximum size of eggs of H. fossilis, M. corsula and M. vittatus as de- scribed by Ghosh and Karr (1952), Jones and Sujansingani (1954) and Prabhu (1956)respectively are also in conformity with our results (Table I).

Largest eggs were found in Notopterus notopterus, Rita rita and Masta- cembelus armatus and the smallest in Gadusia chapra, Amblypharyngodon mola and Mystus vittatus. In view of the smaller space available for the development of roes in Notopterus notopterus the very large size of eggs (3 mm.) would considerably reduce the reproductive potential of the species. This is possibly compensated by some sort of brood vigilance on the part of the parents. In an allied species, namely N. chitala parents are reported to giaard the nest (Alikunhi, 1957) and a similar beh&viour might also be present in N. notopterus.

PROTEIN CONTENT

The percentage of protein in all the species under investigation varied between 20 and 35~o on fresh weight basis (Table I). Earlier observations on freshwater as well as marine fish of this country reveal that muscle which is

Physico-Chemical Analysis of Eggs of Some Freshwater Teleosts 5

supposed to be the richest source of protein contains only 9-25~ of protein (Kuppuswamy et al., 1958). Similar results were obtained on some Atlantic species (Mannan et al., 1961 a and b). Biochemical investigations carried out in our own laboratory on various tissues of freshwater fishes indicate that the highest protein values for muscle which are obtained in prespawning months reach only to a maximum of 25~o. Ripe eggs have a considerably higher protein content. In migrating sockeye salmon Idler and Bitners (1960) have noted that the roes show a protein nitrogen value of 4"13~o (approx. 25.8~ protein) when they are 715 miles upstream. Corresponding values for muscle are much lower, approximately only half as much (Idler and Bitners, 1958). Since the egg yolk is a reserve of lipoproteins to be uti- lized in the development of embryo, high protein values are to be expected. Fish eggs contain more protein than hen's egg which has on an average only 12~o protein when fresh .(Kuppuswamy et aL, 1958).

The percentage of protein in fish eggs seems to vary according to groups. Carps have a higher mean value (34~o). Then come the cat-fishes (31~o). The spiny-eels have a mean value of only 26~o. Eggs having low protein value (less than 30~o) generally compensate their energy reserve by accumu- lating more fat. This can be seen clearly by comparing protein and fat figures for fishes like Ophicephalus punctatus, Mugil corsula and Gadusia chapra with those obtained for other species, especially cat-fishes and carps.

MOISTURE CONTENT

Fish eggs generally have a moisture content of 62~ or less. The low moisture content of fish eggs in comparison to those of amniotes is under- standable since they are laid in an aquatic environment from where absorp- tion of water during embryonic development may take place according to r.equirements. Cat-fishes have relatively more water content (average 64-99~0) than the carps (average 60~) and spiny-eels have only on an average 54.6~. Lesser figures are obtained in fish like Ophicephalus punctatus and M. corsula (Table I).

FAT CONTENT

The fat content also seems to be group specific. While it is very high in certain species like M. corsula, O. punctatus, M. armatus and G. chapra, the carps have an average of 3.4~. In cat-fishes with the exception of Rita rita, it ranges between 0" 68~ and 1.7~o. The average for all cat-fishes irtolud- hag Rita rita is only 1 "9~0.

6 Pd3HAN HASAN AND A. K. JAFRI

It is known that fat has a higher calorific value than protein. A higher fat content would thus mean more energy available in terms of calories for development. It was surmised that a higher fat content might have some effect on the hatching time of various species. Observations of Khan (1925, 1943) and Jones and Sujansingani (1954) on the hatching time of Labeo genius, Cirrhina, mrigala, Wallagonia attu, and Mugil corsula do not support this idea. The eggs of C. mrigala take almost the same time to hatch as the fat rich-eggs of M. corsula.

MOISTURE AND FAT RELATIONSHIP

A negative correlation between fat and water content was observed by Brandes and Dietrich (1958), Brandes (1954) and Mikicinska (1954). They found that in certain species the added value of water and fat (F +W) is a constant. This is true not only for the fish as a whole but for various organs as well. The increase or decrease in fat is associated with a corres- ponding and negative increase or decrease in moisture content. The formulae for fat-water relationship developed by Brandes and others hold good only for particular species. Since the composition of eggs of a particular species should be expected to remain more or less constant a similar treatment of fat and water content is not possible. However, it was considered worthwhile to investigate whether eggs of different species followed any general principle with regard to these fractions.

Figure 2 represents plotted values of fat against moisture for the 19 species examined. Although points for Callichrous pabda, Rite rita and Gadusia chapra lie outside the general range, those for other species under investigation show a general pattern. Fishes which have a fat content of less than 5~ have an F + W ranging from 62 to 66. The mean F + W for cat-fishes excluding Rita rita is 63"878, while for carps it is 63.578. The figures show that most of the species of carps and eat-fishes lie in one group as regards their F + W values. In those fishes which have a fat content higher than 10~ the added value of fat and water ranges between 68 and 78 (Table I). It is interesting to note that in Oncorhynchus nerka during spawning migration when the ovaries show a fat content of 10.72~ the F + W value comes to 73-I2 (Idler and Bitners, 1960). In fact these fishes 715 miles upstream may be treated as freshwater species.

In spite of the fact that F + W values of eggs of all the species analysed here do not seem to follow a particular relationship with mathematical accu- racy, the generalization that fat increases at the expense of water seems fairly valid.

Physico.Chemical Analysis of Eggs of Some Freshwater Teleosts 7

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DRY MATTER AND ASH

An estimate of dry matter has been made on the basis of loss of moisture on drying and it is lowest in fishes having highest percentage of water.

The values of ash obtained for eggs fall in close agreement with those reported in fish muscle by earlier authors (Saha and Guha, 1939; Alexander, 1955). In all the species examined ash content varied within a narrow range of 1.0 to 2"270; higher values being encountered in Notopterus notopterus, M. corsula and the two species of Mystus. The ash content is not dependent on dry matter since most of the constituents are completely oxidizable and leave no ash.

PHOSPHORUS

The phospholipids (lecithins) are a major constituent of egg yolk. These compounds considerably increase the phosphorus content of eggs as com- pared to flesh. Figures lower than 0"6~0 have been obtained by Saha and Guha 0939) for the muscle of L. rohita, C. mrigala, IV. attu and O. punctatus. Corresponding phosphorus values for eggs of those species as given in Table I are higher than 0.7~,.

8 REHAN HASAN AND A. K. JAFRI

On the average cat-fish eggs have slightly higher phosphorus content (1.0~o) than those of carps (0.915~). It is interesting to note that fishes which have higher phosphorus values do not necessarily have more fat and vice versa. This leads to the conclusion that not all phosphorus is associated with fat in the form of lecithin. It may be present in the form of inorganic phosphate or as a constituent of nucleic acids and high energy phosphate compounds. The fact that very high fat content also does not show a pro- portionately high phosphorus makes it clear that besides being a consti tuent of lecithin lipids are deposited as oil or in some other form as well.

CALCIUM

In the eggs of N. notopterus and H. fossilis calcium is fairly high reaching up to 64 to 67 rag. per I00 gm. of fresh tissue. Next to these come the two closely related eat-fishes, 14I. attu and C. pabda. Both of these species have 42 rag. of calcium per 100 gm. Most of the cat-fishes analysed during the present investigation had a higher calcium value in comparison to carps. Average values for both the groups are 35.733 mg. and 12' 285 nag. per 100 gm. respectively. All these figures for calcium are much less as compared to those mentioned by earlier workers (Saha and Guha, 1939) for muscle. Nevertheless, calcium is an important costituent of eggs.

IRON

During ontogeny the foundations of a vascular system are laid down much before the embryo is hatched. In many cases considerable time is lapsed even after hatching during which the larva is solely dependent on the yolk sac. A reasonable supply Of iron reserve in the ovum is therefore necessary to meet the demands of the developing embryo for the formation of respiratory pig- ment, haemoglobin. A fair amount of iron, ranging from 17.5 to 32" 5 mg. per 100 gm., is present in all fish eggs. The variations are possibly specific for each species. Eggs of four species of Labeo that have been investigated show a variation between 21.5 and 30.0rag. per 100gm. The amount of iron present in the eggs does not seem to have any relationship with the iron content of the adult fish and is always lower than the muscle. The eggs of 1t. fossilis contain only 33.0 mg. of iron per 100 gm. of fresh eggs. The same fish according to Saha and Guha (1939) shows highest iron values (226 rag. per 100 grn.) in the muscle.

SIGNIFICANCE OF PERCENTAGE COMPOSITION

On the basis of present investigations one can visualize that the port ~entage composition of eggs of closely related species is approximately ,the

Physico-Chemical Analysis o f Eggs o f Some Freshwater Teleosts 9

same. A comparison of values obtained for various species of Labeo and Mystus serves to illustrate this point. In a more general way it is a group characteristic. Groups of fish like carp and cat fish show within the group a conformity of pattern in the percentage chemical composition of their eggs.

SUMMARY

An analysis of some physical as well as chemical aspects of ripe eggs belonging to 19 species of freshwater fish has been made.

Apart from the eolour, eggs of closely related species resemble each other in their specific gravity, diameter and chemical composition.

Protein, fat and phosphorus content is always higher as compared to muscle.

Moisture content is usually less than 63~0 and decreases with the increase of fat in various species. A higher or lower fat content does not in any way affect the time required for embryonic development and hatching.

Calcium and iron are lower in comparison to the muscle.

ACKNOWLEDGEMENTS

We are grateful to Prof. M. A. Basir, Head of the Department of Zoology, for facilities and encouragement; to Prof. S. Z. Qasim of the Central Insti- tute of Fisheries Education, Bombay, and Prof. G. S. Kennington of the University of Wyoming, Laramie, for criticism and many valuable suggestions.

Thanks are due to Dr. D. R. Idler, Director of the Technological Research Laboratory, Fisheries Research Board of Canada, Halifax, for going through the manuscript.

One of us (A. K. J.)is indebted to the Indian Council of Agricultural Research, New Delhi, for a research assistantship.

AliknjIhi~ K.H. ..

and Nagaraja Rao, S...

Alexander, K.M. . .

Borptrom, G,

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10 ~ HASAN Ah'D A. K. JAFRI

*Brandes, C. I-L

* and Dietrich, R . . . .

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, Tsuyuki, H. and Idler, D .R .

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- - , Ronald, A. P. and Schmidt, P.J.

and Bitners, I.

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Phys ico -Chemica l Analys i s o f E g g s o f S o m e Freshwater Teleosts 11

Khan, Harold

Krishnamunhi, K. P.

Kuppuswamy, S., Srinivasan, M. and Subrahmanyan, V.

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*Mikicinska, J.

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Prabhu, M.S. ..

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* Not co,~.ul~d ia o r i g .