- Email: [email protected]
Utilization of body stores in embryonated ova and larvae of two coregonid species (Coregonus lavaretus L. and C. albula L.)
Camp. Biorhem. Physiol. Vol. 79A. No. 3, pp. 329-334. 1984 Printed in Great Britain
0300.9629/84$3.00 + 0.00 ic 1984 Pergamon Press Ltd
UTILIZATION OF BODY STORES IN EMBRYONATED OVA AND LARVAE OF TWO COREGONID SPECIES (COREGONUS LAVARETUS L. AND C. ALBULA L.) K. Institute
of Ichthyobiology
and
DADROWSKI
Fisheries,
and M.
Academy Kortowo,
LUCZYNSKI
of Agriculture Poland
and Technology,
IO-957 Olsztyn-
(Received 6 December 1983) Abstract-l. Developing eggs of whitefish (Coregonus lavaretus L.) and vendace (Coregonus albulu L.) were kept at l-2°C and some eggs taken gradually up to 8°C to provoke mass hatching of embryos. 2. Wet weight, dry matter and the contents of lipid, protein and ash were measured in fish during the
course of experiment. 3. Dry matter content decreased gradually in whitefish eggs from 15.64 to 11.95% during 1 month at l-2°C whereas vendace eggs showed only a slight decrease from 16.27 to 15.53%. 4. In both species protein content decreased but lipid increased when approaching the natural time of hatching. 5. During delayed hatching at low water temperatures protein contributes to catabolism, whereas lipid content decreased only in the later phase of the experiment. 6. Larvae starved for 10 days after hatching lost increasing amounts of dry matter (from 26.1 to 50.3% of body weight) and protein (from 18.7 to 45.9% of body weight) as they remained longer in cold water as embryos. 7. A correspondence was found between assessment of metabolic utilization of body stores based on chemical analysis of fish body and previous work on oxygen consumption and nitrogen excretion.
INTRODUCTION
and the heaviest alevins of Oncorhynchus tshawytcha at the completion of yolk sac absorption in the lowest water temperature. In another salmonid Salvelinus alpinus, weight at hatching was only slightly influenced by the temperature of incubation, but efficiency of yolk utilization was higher at 4°C (34-36x) than at 8°C (11.6-12.6x) (Griiber and Wieser, 1983). Hamor and Garside (1977) showed at lower temperature and 100% oxygen saturation that yolk is used more efficiently by Salmo salur embryos, but after hatching the warmer temperature tends to enhance the efficiency of yolk utilization. This lack of consistency in the pattern of endogenous nutrient utilization among the salmonids may be due to the evolution of interspecific differences in their biology. Howell (1980), Johns and Howell (1980) and Johns et al. (1981) have shown with two marine fish that larvae reared in the range of temperatures or in cyclic temperatures had similar body length and efficiency of yolk utilization at the yolk sac absorption point. Studying the yolk and oil globule absorption rate at several temperatures, Ehrlich and Muszynski (1982) concluded that taking into account Q10 values, protein mobilization was very active at the lowest possible temperatures for the larval grunion (Leurestes tenius), where larvae drastically decreased the metabolism of oil. At the highest temperatures tolerated by larval grunion, both fat and protein utilization decreased, the latter to a much greater extent. In the freshwater cyprinid, Cyprinus carpio, protein and lipid were the main substances utilized during the yolk transformation into fish body, but considering the energetical value of the substances Kamler (1976) suggested equal contribution of both to fish metabo-
The utilization of endogenous stores of lipid and protein in developing eggs of teleosts is fundamental to understanding and eventually predicting the effect of changes in environmental conditions, both in natural spawning grounds and hatcheries. Thus, it seemed essential to evaluate the metabolic effects on developing embryos and larvae of lowered water temperature because this procedure is currently used to delay hatching of coregonid eleutheroembryos. The synchronization of artificially delayed hatching and suitable thermal and feeding conditions for stocking fish in lakes is the way to improve the survival in natural environments (Fhichter, 1980). However, to our knowledge, no attention has so far been given to the vitality of larvae obtained after delayed hatching. The effect of temperature during coregonid embryogenesis has been studied in a number of species (see Dabrowski, 1981), but to determine whether the absorption of endogenous reserves and the efficiency of this process depend on the temperature seemed to be an interesting area to investigate. Extensive information is available on nutritional substances and energy metabolism during embryonic development of teleosts. However, there is no agreement as to the way or “strategy” of embryonic and larval utilization of endogenous materials. For instance in salmonids Wood (1932) suggested temperature independent yolk utilization efficiency between 3 and 12°C whereas Marr (1966) indicated an optimum temperature for this process at 10°C. Heming (1982) and Heming et al. (1982) clearly proved that the yolk sac is most efficiently utilized over the range of 6-12°C
are produced
329
330
K. DADROWSKIand M. LUCZYNSKI
lism. Kaushik et al. (1982) studied oxygen consumption and nitrogen metabolism during early development of carp and found that the fat/protein energy ratio (FPER) was 1.96 during the prehatching stage and increased to 5.0 at larval stage. A similar approach applied to evaluate coregonid metabolism led Dabrowski et al. (1984) to conclude that during post-hatching and early free-swimming stage protein is furthermore utilized as energy, the situation changing during prolonged fasting of larvae. This work deals with changes in protein and lipid content as energy sources during early life history of two coregonid fishes and presents the possibility of using such cumulative data to find out the fate of yolk reserves under particular thermal conditions. MATERIALS AND METHODS
In November whitefish (Coregonus lavarem L.) and vendace (Gregonus albula L.) eggs were obtained from naturally mature fish from Mazurian lakes (near Olsztyn, Poland). Eggs were kept in a commercial hatchery until the eyed stage and then transferred to the laboratory maintained water at a temperature not exceeding I-2°C. At the time approaching natural hatching in April, the portions of eggs were gradually raised up to 8C to provoke mass hatching of embryos. Sampling of eggs started at 18 March, but the first attempts to hatch the whitefish or vendace were undertaken on 20 April and 8 April, respectively. Then the samples of larvae were reared in tanks for 10 days at a temperature of IO-12°C. Samples of eggs or larvae, 100-200 in number, were weighed after draining. Triplicate samples were used on every occasion to determine the chemical composition, dry matter content, nitrogen, lipid and ash. The methods were essentially the same as used by Dabrowski (1976). Briefly, dry matter was determined at 105°C after 24 hr, nitrogen by the micro-Kjehdal method, protein was calculated as N x 6.25 and ash after combusting samples at 550°C. The exception was the lipid analysis by chloroform-methanol extraction for 8-10 hr. Snedecor’s test was used to examine the significance of the differences; standard deviations were given throughout. Kendall rank correlation coefficients were calculated to check the significance of the trend in time series. RESULTS
Chemical changes during delayed hatching Whitefish embryos released from the egg envelope weighed 6.25 + 0.16 (SD) mg, i.e. 58.6% of egg weight. When expressed in dry matter or protein, respective figures were 63.3 and 62.9%. Vendace embryo weight was 2.74 + 0.14 mg, i.e. 86.1% of egg wet weight, 90.5% of dry weight or 90.0% of protein. These are the average data measured on several occasions. In Table 1 the data of chemical analysis of coregonid eggs is presented. Dry matter content gradually decreased in whitefish eggs during the last two months of artificially delayed hatching, whereas vendate eggs have shown only a slight decrease. Ash content in eggs of both species decreased. Lipid content in C. lavaretus increased during the experiment, but in vendace, lipid level dropped significantly after reaching a maximum value of 37.2%. Naturally, protein content was inversely proportional to lipid content. During the period of nearly two months at l-2”C, the absolute weight of embryonated whitefish ova
331
Utilization of body stores in two coregonid species
0
0
A
A
A
0
0
A
A
AA
0
0
A
0
0
0
0 0
0
&
I .I11 1
A
I
A
A
1
1
29.111
&IV
11
21
A
1
A
I
13..Iv
2o.w
2a.w
26
33
41
A I
4.v 47
t
7.v
A I
l&V 56
Data Number Of
days
Fig. 1. Changes in the content of protein (e), lipid {O) and ash (A) of the embryonated ova of vendace (A) and whitefish (B) during the course of delaying hatching at I-2°C.
from 1.77 to 1.32mg dry matter, whereas vendace varied between 0.509 and 0.590mg, but showed no tendency to decrease. Content of protein, lipid and ash in individual eggs of examined coregonid fish is shown in Fig. 1. Eggs of both species show an increase in lipid content as they are approaching a natural hatching time in the beginning of April, and a visible decrease of lipid content in May samples. Since variation in protein content is the only possible explanation of the resufts, it is interpreted, particularly evident in the case of C. lauaretus, that mobilization of protein was intensified in the beginning of May. Based on the decrease of a protein in whitefish egg during 18-29 March and 4-13 May, the total loss of nitrogen was estimated to be 81 and 150 pg N/l000 fish/hr. Vendace eggs during abrupt decrease of protein content during 18-29 March, must have been losing 52.8 pg N/1000 fish/hr.
decreased
Chemical changes during ,fasting
There was some decrease in the lipid content in starving coregonids, but the decrease in dry matter was the most visible; in vendace it decreased gradually from 15.92 to 9.19% when fasting commenced on
22 April or 3 May. The changes in chemical content of eleutheroembryos at hatching and during fasting are presented in Figs 2 and 3. The loss of dry matter during 10 days of fasting increase rapidly, as embryos remained longer in cold water. Between days 26 and 56 fish were transferred to 8°C to be hatched and subsequently were fasted for the following IO days (Fig. 2), they lost 26.1, 16.6, 36.7, 45.5 and 50.3% of dry weight. Larvae fasted for 10 days when transferred on days 26, 33, 41 and 50 of the trial lost 18.7, 21.8, 34.6 and 45.9% of protein. The test of trend in time series by Kendall rank correlation coefficients revealed that trends show statistical significance, at P -=I 0.05and P < 0.01, respectively. Correlation coefficients were 0.80 and 1.0 with variance 0.16 and 0.24, respectively. Surprisingly, the lipid losses were equal during 10 days fasting independent of the time of transfer. This phenomenon led to a considerable differences in fat/protein energy ratio (FPER) which for vendace larvae transferred to warmer water at day 26,41 and 50 of the trial amounted to 1.61, 0.90 and 0.66, respectively. During prolonged embryonic life in cooling system there was no clear preference of
332
K.
DADROWSKI
21
26
56
10
5 Days
alter
15
20
transfer
Fig. 2. Changes in the content of ash, lipid and protein in the embryonated ova and larvae of vendace. Arrows indicate the time of hatching. During the period of 10 days after hatching fasting larvae were kept at IO-12°C.
protein or lipid to be used, but hatching intensified use of protein proportionally to the delay. During fasting nitrogen loss was consequently increasing and vendace larvae transferred at days 26, 33,41 and 50 depleted 36,48, 72 and 98.5 pg N/l000 fish/hr. Bearing in mind that fish were at lo-12’C it was much less in comparison to the loss during embryogenesis at l-2°C. However, the periods during embryogenesis were arbitrarily chosen during the time of change. During other periods the excretory products can be accumulated within the eggs. Whitefish loss during hatching was much greater than in vendace (Fig. 3) but during fasting nitrogen excretion of fish transferred to warm water at days 33 and 41 of the trial amounted to 59.2 and 19.2pg N/l000 fish/hr. Estimation of protein and lipid usage during fasting resulted in FPER equal to 2.35. DISCUSSION
It is interesting to note that the estimations of nitrogen loss during late embryogenesis and fasting larvae are in good concordance with the previous results of nitrogen excretion rate in C. lavaretus (Dabrowski et al., 1984). Before hatching at 12°C ammonia excretion rate was approximately IOOpg N/l000 fish/hr. However, it has been recently found that among the winter flounder larvae excretory
and M.
LUCZYNSKI
products primary amines are equal to 50% of the ammonia nitrogen. Furthermore, other unidentified nitrogenous substances constituted more than 50% of the total N loss (Cetta and Capuzzo, 1982). So, although there is a good corroboration of the results of nitrogen loss in the early life of coregonids by two methods, to assure the conclusion some more studies are needed to clarify the point for other nitrogenous substances, i.e. primary amines, in freshwater fish larvae excretory products. Lipids were used predominantly during prolonged fasting of whitefish (Dabrowski, 1976), leading to an FPER of 4.5. It seems that lipid usage is increased during prolonged fasting, as previously indicated (Dabrowski et al., 1984) when FPER increased from 0.86 to 1.99 from pre-hatching to free swimming stage of whitefish. Vendace behave very differently in this respect. Lipid metabolism seemed to be inhibited and protein mobilization served as the main energy resource in this species. Kamler (1976) found FPER almost equal to 1.0 for common carp during embryogenesis and larval stage. However, Kaushik et al. (1982) postulated that during the larval stages of carp lipid metabolism predominated, the FPER being as high as 4-5. So, there are differences among species but also environmental factors or the composition of the ova (Kamler and Malczewski, 1982; Eldridge et ul., 1982) may influence directions of embryo and larval metabolism. According to Boulekbache (1981) the sequential utilization of energy substrates by trout embryos entirely neglected protein. This author points towards glycolysis being dominated in early organogenesis and lipid utilization in late embryogenesis up to hatching. Plaice and herring unfed larvae used protein as the main energy source (Ehrlich, 1974) whereas grunion larvae used three times more calories from lipid stores than from protein. Ehrlich and Muszynski (1982) found an even more complex pic-
1.0
p1.0 z g
1.0
5
10 Days
after
15
20
transfer
Fig. 3. Changes in the content of ash, lipid, protein and dry matter in the embryonated ova and larvae of whitefish. Arrows indicate the time of hatching. During the period of 10 days after hatching fasting larvae were kept at IO- 12 ‘C.
Utilization of body stores in two coregonid species ture of mobilization for protein and lipid in grunion larvae. Since selected temperatures were 5-7°C higher for feeding than unfed larvae, even after 1 day of fasting of the latter, larvae exhibited specific metabolism in different temperatures. It was noticed in the present study that an increase in lipid content during the late embryogenesis of two coregonids might be associated with prolonged period of cold water. A similar phenomena was reported by Cetta and Capuzzo (1982) where they found an increase of lipid in winter flounder eggs from 2.6 to 7.3 pg per egg. Biosynthesis of lipids in the embryonated fish ova is well documented (Turner, 1979). On the other hand, in Sciuenops ocelluta, Vetter et al. (1983) observed a decrease in lipid of 30% and glycogen of 53% during embryonic life, but the amount of the former was 100 times that of the latter. Glycogen reserves were not assessed in the present study on coregonids, although glycogen depletion rate from larval fish liver can be a precise indicator of fluctuation of feeding conditions (O’Connell and Paloma, 1981). An apparent increase of protein content in coregonid eggs was attributed to a variation in egg size of samples. Sibers and Rosenthal (1977) measured influx of amino acids to the developing herring eggs and estimated that it can cover approximately 1% of the enerav reauired. So. an increase in drv matter and protein in developing eggs of Sciaenops bcellata (Vetter et al..‘l983) can be internreted as only apparent. On the’ other hand, nitrogen loss &r&g embryogenesis or larval period recalculated from the available data in the literature give figures of the same order of magnitude as found in coregonids. Winter flounder larvae at 7°C lost 2.4 pg N/l000 fish/hr (Cetta and Capuzzo, 1982), whereas striped bass larvae at 24°C 15.2pg N/l000 fish/hr (Rogers and Westin, 1981). It seems that in the latter species where an oil globule contributes 50% of egg’s dry weight, lipid is the main source of energy rather than protein. Eldrich et al. (1982) working with striped bass larvae, showed that fasting larvae conserved the oil glubule, whereas those fed progressively higher concentrations of exogenous food consumed oil energy at a faster rate. This phenomenon was most probably responsible for the differences reported among three species of marine larvae by Houde and Schekter (1983). The initial high assimilation efficiency in sea bream larvae (75%) and subsequent drop was due to the initial utilization of the yolk-sac reserves. Larvae of bay anchovy, which lost 46% of body weight between the yolk-sac and feeding stages, had the lowest assimilation efficiency. Since polyunsaturated fatty acids are selectively consumed during fish embryogenesis and there is transesterification between triglyceride and phospholipid within the yolk-sac lipoprotein (Nagakawa and Tsuchiya, 1976) it still remains to be understood what this processes means for the assimilation of the first exogenous food by larvae. First food was found to result in the negative nitrogen budget of stomachless common carp larvae (Kaushik and Dabrowski, 1983) and the lipid role in this specific period was not investigated. During embryonic and early larval life examined in the present study, whitefish lost 40-48 y0 of protein and 39943% lipid, while vendace lost 30-55x of
333
protein and 27-65x of lipid. Hayes (1949) reported at the end of yolk 40% of protein and 75% of lipid is used by salmon embryo and larvae. Freshwater carp consumed 62% of protein and 70% of lipid from the commencement of embryonic development to 6-7 days after hatching (Kamler, 1976). The present study in a “cooling system” combines the effect of low and high temperature effects on fish embryo. Firstly, it is advantageous to let embryos develop in low temperature to increase the efficiency of yolk utilization and body size at hatching (Luczynski, 1981). However, extension of this period creates the condition of body reserves exhaustion at the beginning of free swimming stage-leaving the high temperature effect. It seems inevitable that hatched larvae are more susceptible to fasting proportionally to the delay of hatching. It is, however, necessary to mention that these samples of vendace and whitefish put to suitable food conditions have shown very high survival (86-89x for whitefish and 58-72x for vendace up to 15 and 28 days of rearing, respectively). The mean instantaneous rate of net biomass gain did not differ among the lots subjected to delay of hatching (Luczynski and Kolman, 1983). Acknowledgements-We would like to thank C. A. Mills (River Laboratory, Wareham, Dorset) for critical comments on the manuscript, S. Kaushik for statistical advice, and R. Bardega for technical work.
REFERENCES
Boulekbache H. (1981) Energy metabolism in fish development. Am. 2001. 21, 377-389. Cetta C. M. and Capuzzo J. M. (1982) Physiological and biochemical aspects of embryonic and larval development of the winter flounder Pseudopleuronectes americanus. Mar. Biol. 71, 327-337. Dabrowski K. (1976) An attempt to determine survival time for starving fish larvae. Aquaculture 8, 169-173. Dabrowski K. (1981) Early life history of pollan (Coregonus pollan Thompson) from Lough Neagh, Northern Ireland. ht. Rev. ges. Hydrobiol. 66, 299-326. Dabrowski K., Kaushik S. J. and Luquet P. (1984) Metabolic utilization of body stores during early life of whitefish (Coregonus lavaretus L.). J. Fish. Biol. Ehrlich K. F. (1974) Chemical changes during growth and starvation of herring larvae. In The Early Life History of Fish (Edited by Blaxter J. H. S.), pp. 301-323. Springer, Berlin. Ehrlich K. F. and Muszynski G. (1982) Effects of temperature on interactions of physiological and behavioural capacities of larval California grunion: adaptation to the planktonic environment. J. exp. Mar. Biol. Ecol. 60, 223-244. Eldrich M. B., Whipple J. A. and Bowers M. J. (1982) Bioenergetics and growth of striped bass, Morone saxatilis, embryos and larvae. Fish. Bull. 80, 461474. Fliichter J. (1980) Reviews of the present knowledge of rearing whitefish (Coregonidae) larvae. Aquaculture 19, 191-208. Grtiber K. and Wieser W. (1983) Energetics of development of the alpine charr, Salvelinus alpinus, in relation to temperature and oxygen. J. camp. Physiol. 149, 485493. Hamor T. and Garside E. T. (1977) Size relations and yolk utilization in embryonated ova and alevins of Atlantic salmon Salmo salar L. in various combinations of temperature and dissolved oxygen. Can. J. Zool. 55, 1892-1898. Hayes F. R. (1949) The growth, general chemistry, and
334
K. DADROWSKI and M. LUCZYNSKI
temperature relations of salmonid eggs. Q. Rev. Biol. 24, 281-308. Heming T. A. (1982) Effect of temperature on the utilization of yolk by chinook salmon (Oncorhynchus tshawytscha) eggs and alevins. Can. J. Fish. aquaf. Sci. 39, 184-190. Heming T. A., McJnemey J. E. and Alderdice D. F. (1982) Effect of temperature on initial feeding in alevins of chinook salmon (Oncorhynchus tshawytscha). Can. J. Fish. uquut. Sci. 39, 1554-1562. Houde E. D. and Schekter R. C. (1983) Oxygen uptake and comparative energetics among eggs and larvae of three subtropical marine fishes. Mar. Biol. 72, 283-293. Howell W. H. (1980) Temperature effects on growth and yolk utilization in yellowtail flounder (Limundu firrugineu) yolk sac larvae. Fish. Bull. 78, 731-736. Johns D. M. and Howell W. H. (1980) Yolk utilization in summer flounder (Parulichthys dentutus) embryos and larvae reared at two temperatures. Mar. Ecol. Progr. Ser. 2, 1-8. Johns D. M., Howell W. H. and Klein-MacPhee G. (1981) Yolk utilization and growth to yolk-sac absorption in summer flounder (Purulichthys dentutus) !arvae at constant and cyclic temperatures. Mar. Biol. 63, 301-308. Kamler E. (1976) Variability of respiration and body composition during early developmental stages of carp. Pol. Arch. hydrobiol. 23, 43 l-485. Kamler E. and Malczewski B. (1982) Quality of carp eggs obtained by induced breeding. Pal. Arch. hydrobiol. 29, 599-606. Kaushik S. J. and Dabrowski K. (1983) Postprandial metabolic changes in juvenile carp (Cyrinus curpio L.). Reprod. Nutr. Develop. 23, 223-234. Kaushik S. J., Dabrowski K. and Luquet P. (1982) Patterns of nitrogen excretion and oxygen consumption during ontogenesis of carp (Cyprinus curpio L.). Can. J. Fish. u9uur. Sci. 39, 1095-t 105. Luczynski M. (1981) The influence of the incubation
temperature of Coregonus ulbulu L. eggs on the embryogenesis rate and on the survival and morphology of eleutheroembryos and larvae. Ph.D. thesis. Inland Fisheries Institute, Olsztyn, Poland. Luczynski M. and Kolman R. (1983) Survival and growth rates of Coregonus albula (L.) larvae of experimentally delayed hatching. S.I.L. XXI Congress, Lyon, 21-28 Aug. (France). Marr D. H. A. (1966) Influence of temperature on the efficiency of growth of salmonid embryos. Nature, Lond. 212, 957-959. Nakagawa H. and Tsuchiya Y. (1976) Studies on rainbow trout egg (Sulmo guirdneri irideus). VI. Changes of lipid composition in yolk during development. J. Fuc. Fish. Anim. Husb. Hiroshima Univ. 15, 35-46. O’Connell C. P. and Paloma P. A. (1981) Histochemical indications of liver glycogen in samples of emaciated and robust larvae of the northern anchovy, Engraulis mordax. Fish. Bull. 79, 806812. Rogers B. A. and Westin D. T. (1981) Laboratory studies on effects of temperature and delayed initial feeding on development of striped bass larvae. Trans. Am. Fish. Sot. 110, too-1 10. Siebers D. and Rosenthal H. (1977) Amino acid absorption by developing herring eggs. Helgoliinder wiss. Meeresunters. 29, 464472. Turner C. (1979) Metabolism and energy conversion during early development. In Fish Physiology (Edited by Hoar W. S., Randall D. J. and Brett J. R.), pp. 261-278. Academic Press, New York. Vetter R. D., Hodson R. E. and Arnold C. (1983) Energy metabolism in a rapidly developing marine fish, egg, the red drum (Sciuenops ocellufa). Can. J. Fish. Aguar. Sci. 40, 627-634. Wood A. H. (1932) The effect of temperature on the growth and respiration of fish embryos (Sulmofurio). J. exp. Biol. 9, 271-276.
0300.9629/84$3.00 + 0.00 ic 1984 Pergamon Press Ltd
UTILIZATION OF BODY STORES IN EMBRYONATED OVA AND LARVAE OF TWO COREGONID SPECIES (COREGONUS LAVARETUS L. AND C. ALBULA L.) K. Institute
of Ichthyobiology
and
DADROWSKI
Fisheries,
and M.
Academy Kortowo,
LUCZYNSKI
of Agriculture Poland
and Technology,
IO-957 Olsztyn-
(Received 6 December 1983) Abstract-l. Developing eggs of whitefish (Coregonus lavaretus L.) and vendace (Coregonus albulu L.) were kept at l-2°C and some eggs taken gradually up to 8°C to provoke mass hatching of embryos. 2. Wet weight, dry matter and the contents of lipid, protein and ash were measured in fish during the
course of experiment. 3. Dry matter content decreased gradually in whitefish eggs from 15.64 to 11.95% during 1 month at l-2°C whereas vendace eggs showed only a slight decrease from 16.27 to 15.53%. 4. In both species protein content decreased but lipid increased when approaching the natural time of hatching. 5. During delayed hatching at low water temperatures protein contributes to catabolism, whereas lipid content decreased only in the later phase of the experiment. 6. Larvae starved for 10 days after hatching lost increasing amounts of dry matter (from 26.1 to 50.3% of body weight) and protein (from 18.7 to 45.9% of body weight) as they remained longer in cold water as embryos. 7. A correspondence was found between assessment of metabolic utilization of body stores based on chemical analysis of fish body and previous work on oxygen consumption and nitrogen excretion.
INTRODUCTION
and the heaviest alevins of Oncorhynchus tshawytcha at the completion of yolk sac absorption in the lowest water temperature. In another salmonid Salvelinus alpinus, weight at hatching was only slightly influenced by the temperature of incubation, but efficiency of yolk utilization was higher at 4°C (34-36x) than at 8°C (11.6-12.6x) (Griiber and Wieser, 1983). Hamor and Garside (1977) showed at lower temperature and 100% oxygen saturation that yolk is used more efficiently by Salmo salur embryos, but after hatching the warmer temperature tends to enhance the efficiency of yolk utilization. This lack of consistency in the pattern of endogenous nutrient utilization among the salmonids may be due to the evolution of interspecific differences in their biology. Howell (1980), Johns and Howell (1980) and Johns et al. (1981) have shown with two marine fish that larvae reared in the range of temperatures or in cyclic temperatures had similar body length and efficiency of yolk utilization at the yolk sac absorption point. Studying the yolk and oil globule absorption rate at several temperatures, Ehrlich and Muszynski (1982) concluded that taking into account Q10 values, protein mobilization was very active at the lowest possible temperatures for the larval grunion (Leurestes tenius), where larvae drastically decreased the metabolism of oil. At the highest temperatures tolerated by larval grunion, both fat and protein utilization decreased, the latter to a much greater extent. In the freshwater cyprinid, Cyprinus carpio, protein and lipid were the main substances utilized during the yolk transformation into fish body, but considering the energetical value of the substances Kamler (1976) suggested equal contribution of both to fish metabo-
The utilization of endogenous stores of lipid and protein in developing eggs of teleosts is fundamental to understanding and eventually predicting the effect of changes in environmental conditions, both in natural spawning grounds and hatcheries. Thus, it seemed essential to evaluate the metabolic effects on developing embryos and larvae of lowered water temperature because this procedure is currently used to delay hatching of coregonid eleutheroembryos. The synchronization of artificially delayed hatching and suitable thermal and feeding conditions for stocking fish in lakes is the way to improve the survival in natural environments (Fhichter, 1980). However, to our knowledge, no attention has so far been given to the vitality of larvae obtained after delayed hatching. The effect of temperature during coregonid embryogenesis has been studied in a number of species (see Dabrowski, 1981), but to determine whether the absorption of endogenous reserves and the efficiency of this process depend on the temperature seemed to be an interesting area to investigate. Extensive information is available on nutritional substances and energy metabolism during embryonic development of teleosts. However, there is no agreement as to the way or “strategy” of embryonic and larval utilization of endogenous materials. For instance in salmonids Wood (1932) suggested temperature independent yolk utilization efficiency between 3 and 12°C whereas Marr (1966) indicated an optimum temperature for this process at 10°C. Heming (1982) and Heming et al. (1982) clearly proved that the yolk sac is most efficiently utilized over the range of 6-12°C
are produced
329
330
K. DADROWSKIand M. LUCZYNSKI
lism. Kaushik et al. (1982) studied oxygen consumption and nitrogen metabolism during early development of carp and found that the fat/protein energy ratio (FPER) was 1.96 during the prehatching stage and increased to 5.0 at larval stage. A similar approach applied to evaluate coregonid metabolism led Dabrowski et al. (1984) to conclude that during post-hatching and early free-swimming stage protein is furthermore utilized as energy, the situation changing during prolonged fasting of larvae. This work deals with changes in protein and lipid content as energy sources during early life history of two coregonid fishes and presents the possibility of using such cumulative data to find out the fate of yolk reserves under particular thermal conditions. MATERIALS AND METHODS
In November whitefish (Coregonus lavarem L.) and vendace (Gregonus albula L.) eggs were obtained from naturally mature fish from Mazurian lakes (near Olsztyn, Poland). Eggs were kept in a commercial hatchery until the eyed stage and then transferred to the laboratory maintained water at a temperature not exceeding I-2°C. At the time approaching natural hatching in April, the portions of eggs were gradually raised up to 8C to provoke mass hatching of embryos. Sampling of eggs started at 18 March, but the first attempts to hatch the whitefish or vendace were undertaken on 20 April and 8 April, respectively. Then the samples of larvae were reared in tanks for 10 days at a temperature of IO-12°C. Samples of eggs or larvae, 100-200 in number, were weighed after draining. Triplicate samples were used on every occasion to determine the chemical composition, dry matter content, nitrogen, lipid and ash. The methods were essentially the same as used by Dabrowski (1976). Briefly, dry matter was determined at 105°C after 24 hr, nitrogen by the micro-Kjehdal method, protein was calculated as N x 6.25 and ash after combusting samples at 550°C. The exception was the lipid analysis by chloroform-methanol extraction for 8-10 hr. Snedecor’s test was used to examine the significance of the differences; standard deviations were given throughout. Kendall rank correlation coefficients were calculated to check the significance of the trend in time series. RESULTS
Chemical changes during delayed hatching Whitefish embryos released from the egg envelope weighed 6.25 + 0.16 (SD) mg, i.e. 58.6% of egg weight. When expressed in dry matter or protein, respective figures were 63.3 and 62.9%. Vendace embryo weight was 2.74 + 0.14 mg, i.e. 86.1% of egg wet weight, 90.5% of dry weight or 90.0% of protein. These are the average data measured on several occasions. In Table 1 the data of chemical analysis of coregonid eggs is presented. Dry matter content gradually decreased in whitefish eggs during the last two months of artificially delayed hatching, whereas vendate eggs have shown only a slight decrease. Ash content in eggs of both species decreased. Lipid content in C. lavaretus increased during the experiment, but in vendace, lipid level dropped significantly after reaching a maximum value of 37.2%. Naturally, protein content was inversely proportional to lipid content. During the period of nearly two months at l-2”C, the absolute weight of embryonated whitefish ova
331
Utilization of body stores in two coregonid species
0
0
A
A
A
0
0
A
A
AA
0
0
A
0
0
0
0 0
0
&
I .I11 1
A
I
A
A
1
1
29.111
&IV
11
21
A
1
A
I
13..Iv
2o.w
2a.w
26
33
41
A I
4.v 47
t
7.v
A I
l&V 56
Data Number Of
days
Fig. 1. Changes in the content of protein (e), lipid {O) and ash (A) of the embryonated ova of vendace (A) and whitefish (B) during the course of delaying hatching at I-2°C.
from 1.77 to 1.32mg dry matter, whereas vendace varied between 0.509 and 0.590mg, but showed no tendency to decrease. Content of protein, lipid and ash in individual eggs of examined coregonid fish is shown in Fig. 1. Eggs of both species show an increase in lipid content as they are approaching a natural hatching time in the beginning of April, and a visible decrease of lipid content in May samples. Since variation in protein content is the only possible explanation of the resufts, it is interpreted, particularly evident in the case of C. lauaretus, that mobilization of protein was intensified in the beginning of May. Based on the decrease of a protein in whitefish egg during 18-29 March and 4-13 May, the total loss of nitrogen was estimated to be 81 and 150 pg N/l000 fish/hr. Vendace eggs during abrupt decrease of protein content during 18-29 March, must have been losing 52.8 pg N/1000 fish/hr.
decreased
Chemical changes during ,fasting
There was some decrease in the lipid content in starving coregonids, but the decrease in dry matter was the most visible; in vendace it decreased gradually from 15.92 to 9.19% when fasting commenced on
22 April or 3 May. The changes in chemical content of eleutheroembryos at hatching and during fasting are presented in Figs 2 and 3. The loss of dry matter during 10 days of fasting increase rapidly, as embryos remained longer in cold water. Between days 26 and 56 fish were transferred to 8°C to be hatched and subsequently were fasted for the following IO days (Fig. 2), they lost 26.1, 16.6, 36.7, 45.5 and 50.3% of dry weight. Larvae fasted for 10 days when transferred on days 26, 33, 41 and 50 of the trial lost 18.7, 21.8, 34.6 and 45.9% of protein. The test of trend in time series by Kendall rank correlation coefficients revealed that trends show statistical significance, at P -=I 0.05and P < 0.01, respectively. Correlation coefficients were 0.80 and 1.0 with variance 0.16 and 0.24, respectively. Surprisingly, the lipid losses were equal during 10 days fasting independent of the time of transfer. This phenomenon led to a considerable differences in fat/protein energy ratio (FPER) which for vendace larvae transferred to warmer water at day 26,41 and 50 of the trial amounted to 1.61, 0.90 and 0.66, respectively. During prolonged embryonic life in cooling system there was no clear preference of
332
K.
DADROWSKI
21
26
56
10
5 Days
alter
15
20
transfer
Fig. 2. Changes in the content of ash, lipid and protein in the embryonated ova and larvae of vendace. Arrows indicate the time of hatching. During the period of 10 days after hatching fasting larvae were kept at IO-12°C.
protein or lipid to be used, but hatching intensified use of protein proportionally to the delay. During fasting nitrogen loss was consequently increasing and vendace larvae transferred at days 26, 33,41 and 50 depleted 36,48, 72 and 98.5 pg N/l000 fish/hr. Bearing in mind that fish were at lo-12’C it was much less in comparison to the loss during embryogenesis at l-2°C. However, the periods during embryogenesis were arbitrarily chosen during the time of change. During other periods the excretory products can be accumulated within the eggs. Whitefish loss during hatching was much greater than in vendace (Fig. 3) but during fasting nitrogen excretion of fish transferred to warm water at days 33 and 41 of the trial amounted to 59.2 and 19.2pg N/l000 fish/hr. Estimation of protein and lipid usage during fasting resulted in FPER equal to 2.35. DISCUSSION
It is interesting to note that the estimations of nitrogen loss during late embryogenesis and fasting larvae are in good concordance with the previous results of nitrogen excretion rate in C. lavaretus (Dabrowski et al., 1984). Before hatching at 12°C ammonia excretion rate was approximately IOOpg N/l000 fish/hr. However, it has been recently found that among the winter flounder larvae excretory
and M.
LUCZYNSKI
products primary amines are equal to 50% of the ammonia nitrogen. Furthermore, other unidentified nitrogenous substances constituted more than 50% of the total N loss (Cetta and Capuzzo, 1982). So, although there is a good corroboration of the results of nitrogen loss in the early life of coregonids by two methods, to assure the conclusion some more studies are needed to clarify the point for other nitrogenous substances, i.e. primary amines, in freshwater fish larvae excretory products. Lipids were used predominantly during prolonged fasting of whitefish (Dabrowski, 1976), leading to an FPER of 4.5. It seems that lipid usage is increased during prolonged fasting, as previously indicated (Dabrowski et al., 1984) when FPER increased from 0.86 to 1.99 from pre-hatching to free swimming stage of whitefish. Vendace behave very differently in this respect. Lipid metabolism seemed to be inhibited and protein mobilization served as the main energy resource in this species. Kamler (1976) found FPER almost equal to 1.0 for common carp during embryogenesis and larval stage. However, Kaushik et al. (1982) postulated that during the larval stages of carp lipid metabolism predominated, the FPER being as high as 4-5. So, there are differences among species but also environmental factors or the composition of the ova (Kamler and Malczewski, 1982; Eldridge et ul., 1982) may influence directions of embryo and larval metabolism. According to Boulekbache (1981) the sequential utilization of energy substrates by trout embryos entirely neglected protein. This author points towards glycolysis being dominated in early organogenesis and lipid utilization in late embryogenesis up to hatching. Plaice and herring unfed larvae used protein as the main energy source (Ehrlich, 1974) whereas grunion larvae used three times more calories from lipid stores than from protein. Ehrlich and Muszynski (1982) found an even more complex pic-
1.0
p1.0 z g
1.0
5
10 Days
after
15
20
transfer
Fig. 3. Changes in the content of ash, lipid, protein and dry matter in the embryonated ova and larvae of whitefish. Arrows indicate the time of hatching. During the period of 10 days after hatching fasting larvae were kept at IO- 12 ‘C.
Utilization of body stores in two coregonid species ture of mobilization for protein and lipid in grunion larvae. Since selected temperatures were 5-7°C higher for feeding than unfed larvae, even after 1 day of fasting of the latter, larvae exhibited specific metabolism in different temperatures. It was noticed in the present study that an increase in lipid content during the late embryogenesis of two coregonids might be associated with prolonged period of cold water. A similar phenomena was reported by Cetta and Capuzzo (1982) where they found an increase of lipid in winter flounder eggs from 2.6 to 7.3 pg per egg. Biosynthesis of lipids in the embryonated fish ova is well documented (Turner, 1979). On the other hand, in Sciuenops ocelluta, Vetter et al. (1983) observed a decrease in lipid of 30% and glycogen of 53% during embryonic life, but the amount of the former was 100 times that of the latter. Glycogen reserves were not assessed in the present study on coregonids, although glycogen depletion rate from larval fish liver can be a precise indicator of fluctuation of feeding conditions (O’Connell and Paloma, 1981). An apparent increase of protein content in coregonid eggs was attributed to a variation in egg size of samples. Sibers and Rosenthal (1977) measured influx of amino acids to the developing herring eggs and estimated that it can cover approximately 1% of the enerav reauired. So. an increase in drv matter and protein in developing eggs of Sciaenops bcellata (Vetter et al..‘l983) can be internreted as only apparent. On the’ other hand, nitrogen loss &r&g embryogenesis or larval period recalculated from the available data in the literature give figures of the same order of magnitude as found in coregonids. Winter flounder larvae at 7°C lost 2.4 pg N/l000 fish/hr (Cetta and Capuzzo, 1982), whereas striped bass larvae at 24°C 15.2pg N/l000 fish/hr (Rogers and Westin, 1981). It seems that in the latter species where an oil globule contributes 50% of egg’s dry weight, lipid is the main source of energy rather than protein. Eldrich et al. (1982) working with striped bass larvae, showed that fasting larvae conserved the oil glubule, whereas those fed progressively higher concentrations of exogenous food consumed oil energy at a faster rate. This phenomenon was most probably responsible for the differences reported among three species of marine larvae by Houde and Schekter (1983). The initial high assimilation efficiency in sea bream larvae (75%) and subsequent drop was due to the initial utilization of the yolk-sac reserves. Larvae of bay anchovy, which lost 46% of body weight between the yolk-sac and feeding stages, had the lowest assimilation efficiency. Since polyunsaturated fatty acids are selectively consumed during fish embryogenesis and there is transesterification between triglyceride and phospholipid within the yolk-sac lipoprotein (Nagakawa and Tsuchiya, 1976) it still remains to be understood what this processes means for the assimilation of the first exogenous food by larvae. First food was found to result in the negative nitrogen budget of stomachless common carp larvae (Kaushik and Dabrowski, 1983) and the lipid role in this specific period was not investigated. During embryonic and early larval life examined in the present study, whitefish lost 40-48 y0 of protein and 39943% lipid, while vendace lost 30-55x of
333
protein and 27-65x of lipid. Hayes (1949) reported at the end of yolk 40% of protein and 75% of lipid is used by salmon embryo and larvae. Freshwater carp consumed 62% of protein and 70% of lipid from the commencement of embryonic development to 6-7 days after hatching (Kamler, 1976). The present study in a “cooling system” combines the effect of low and high temperature effects on fish embryo. Firstly, it is advantageous to let embryos develop in low temperature to increase the efficiency of yolk utilization and body size at hatching (Luczynski, 1981). However, extension of this period creates the condition of body reserves exhaustion at the beginning of free swimming stage-leaving the high temperature effect. It seems inevitable that hatched larvae are more susceptible to fasting proportionally to the delay of hatching. It is, however, necessary to mention that these samples of vendace and whitefish put to suitable food conditions have shown very high survival (86-89x for whitefish and 58-72x for vendace up to 15 and 28 days of rearing, respectively). The mean instantaneous rate of net biomass gain did not differ among the lots subjected to delay of hatching (Luczynski and Kolman, 1983). Acknowledgements-We would like to thank C. A. Mills (River Laboratory, Wareham, Dorset) for critical comments on the manuscript, S. Kaushik for statistical advice, and R. Bardega for technical work.
REFERENCES
Boulekbache H. (1981) Energy metabolism in fish development. Am. 2001. 21, 377-389. Cetta C. M. and Capuzzo J. M. (1982) Physiological and biochemical aspects of embryonic and larval development of the winter flounder Pseudopleuronectes americanus. Mar. Biol. 71, 327-337. Dabrowski K. (1976) An attempt to determine survival time for starving fish larvae. Aquaculture 8, 169-173. Dabrowski K. (1981) Early life history of pollan (Coregonus pollan Thompson) from Lough Neagh, Northern Ireland. ht. Rev. ges. Hydrobiol. 66, 299-326. Dabrowski K., Kaushik S. J. and Luquet P. (1984) Metabolic utilization of body stores during early life of whitefish (Coregonus lavaretus L.). J. Fish. Biol. Ehrlich K. F. (1974) Chemical changes during growth and starvation of herring larvae. In The Early Life History of Fish (Edited by Blaxter J. H. S.), pp. 301-323. Springer, Berlin. Ehrlich K. F. and Muszynski G. (1982) Effects of temperature on interactions of physiological and behavioural capacities of larval California grunion: adaptation to the planktonic environment. J. exp. Mar. Biol. Ecol. 60, 223-244. Eldrich M. B., Whipple J. A. and Bowers M. J. (1982) Bioenergetics and growth of striped bass, Morone saxatilis, embryos and larvae. Fish. Bull. 80, 461474. Fliichter J. (1980) Reviews of the present knowledge of rearing whitefish (Coregonidae) larvae. Aquaculture 19, 191-208. Grtiber K. and Wieser W. (1983) Energetics of development of the alpine charr, Salvelinus alpinus, in relation to temperature and oxygen. J. camp. Physiol. 149, 485493. Hamor T. and Garside E. T. (1977) Size relations and yolk utilization in embryonated ova and alevins of Atlantic salmon Salmo salar L. in various combinations of temperature and dissolved oxygen. Can. J. Zool. 55, 1892-1898. Hayes F. R. (1949) The growth, general chemistry, and
334
K. DADROWSKI and M. LUCZYNSKI
temperature relations of salmonid eggs. Q. Rev. Biol. 24, 281-308. Heming T. A. (1982) Effect of temperature on the utilization of yolk by chinook salmon (Oncorhynchus tshawytscha) eggs and alevins. Can. J. Fish. aquaf. Sci. 39, 184-190. Heming T. A., McJnemey J. E. and Alderdice D. F. (1982) Effect of temperature on initial feeding in alevins of chinook salmon (Oncorhynchus tshawytscha). Can. J. Fish. uquut. Sci. 39, 1554-1562. Houde E. D. and Schekter R. C. (1983) Oxygen uptake and comparative energetics among eggs and larvae of three subtropical marine fishes. Mar. Biol. 72, 283-293. Howell W. H. (1980) Temperature effects on growth and yolk utilization in yellowtail flounder (Limundu firrugineu) yolk sac larvae. Fish. Bull. 78, 731-736. Johns D. M. and Howell W. H. (1980) Yolk utilization in summer flounder (Parulichthys dentutus) embryos and larvae reared at two temperatures. Mar. Ecol. Progr. Ser. 2, 1-8. Johns D. M., Howell W. H. and Klein-MacPhee G. (1981) Yolk utilization and growth to yolk-sac absorption in summer flounder (Purulichthys dentutus) !arvae at constant and cyclic temperatures. Mar. Biol. 63, 301-308. Kamler E. (1976) Variability of respiration and body composition during early developmental stages of carp. Pol. Arch. hydrobiol. 23, 43 l-485. Kamler E. and Malczewski B. (1982) Quality of carp eggs obtained by induced breeding. Pal. Arch. hydrobiol. 29, 599-606. Kaushik S. J. and Dabrowski K. (1983) Postprandial metabolic changes in juvenile carp (Cyrinus curpio L.). Reprod. Nutr. Develop. 23, 223-234. Kaushik S. J., Dabrowski K. and Luquet P. (1982) Patterns of nitrogen excretion and oxygen consumption during ontogenesis of carp (Cyprinus curpio L.). Can. J. Fish. u9uur. Sci. 39, 1095-t 105. Luczynski M. (1981) The influence of the incubation
temperature of Coregonus ulbulu L. eggs on the embryogenesis rate and on the survival and morphology of eleutheroembryos and larvae. Ph.D. thesis. Inland Fisheries Institute, Olsztyn, Poland. Luczynski M. and Kolman R. (1983) Survival and growth rates of Coregonus albula (L.) larvae of experimentally delayed hatching. S.I.L. XXI Congress, Lyon, 21-28 Aug. (France). Marr D. H. A. (1966) Influence of temperature on the efficiency of growth of salmonid embryos. Nature, Lond. 212, 957-959. Nakagawa H. and Tsuchiya Y. (1976) Studies on rainbow trout egg (Sulmo guirdneri irideus). VI. Changes of lipid composition in yolk during development. J. Fuc. Fish. Anim. Husb. Hiroshima Univ. 15, 35-46. O’Connell C. P. and Paloma P. A. (1981) Histochemical indications of liver glycogen in samples of emaciated and robust larvae of the northern anchovy, Engraulis mordax. Fish. Bull. 79, 806812. Rogers B. A. and Westin D. T. (1981) Laboratory studies on effects of temperature and delayed initial feeding on development of striped bass larvae. Trans. Am. Fish. Sot. 110, too-1 10. Siebers D. and Rosenthal H. (1977) Amino acid absorption by developing herring eggs. Helgoliinder wiss. Meeresunters. 29, 464472. Turner C. (1979) Metabolism and energy conversion during early development. In Fish Physiology (Edited by Hoar W. S., Randall D. J. and Brett J. R.), pp. 261-278. Academic Press, New York. Vetter R. D., Hodson R. E. and Arnold C. (1983) Energy metabolism in a rapidly developing marine fish, egg, the red drum (Sciuenops ocellufa). Can. J. Fish. Aguar. Sci. 40, 627-634. Wood A. H. (1932) The effect of temperature on the growth and respiration of fish embryos (Sulmofurio). J. exp. Biol. 9, 271-276.