Annual changes in serum calcium and inorganic phosphate levels and correlation with gonadal status of a freshwater murrel, Channa punctatus (Bloch)

Braz J Med Biol Res, August 1998, Volume 31(8) 1069-1073

Annual changes in serum calcium and inorganic phosphate levels and correlation with gonadal status of a freshwater murrel, Channa punctatus (Bloch)

S.K. Srivastav and A.K. Srivastav

Department of Zoology, University of Gorakhpur, Gorakhpur, India

References

Abstract

Introduction

Material and Methods

Results

^{Correspondence and Footnotes}

Adult Channa punctatus murrels of both sexes (60-80 g) were collected locally from Ramgarh Lake during the second week of every month (10 individuals of each sex/month) throughout the year. Blood samples were collected and analyzed for serum calcium and phosphate levels by the methods of Trinder (1960) and Fiske and Subbarow (1925), respectively. Gonads were fixed to judge the state of maturation of the fish. Males exhibited no change in serum calcium levels throughout the year in correlation with testicular maturation. However, serum phosphate levels exhibited a rise in correlation with the increased gonadosomatic index. Females showed marked seasonal changes in serum calcium and phosphate levels which were associated with ovarian maturation (vitellogenesis).

Key words: calcium, inorganic phosphate, reproduction, teleost, vitellogenesis

Vitellogenesis involves hepatic formation of yolk proteins (vitellogenin) and raises blood vitellogenin content followed by deposition of two forms of yolk in the ovary: yolk vesicles (mucopolysaccharides) and yolk granules (phospholipoproteins). Several authors have correlated the increased blood calcium content with ovarian maturation (1-7). Estradiol has been shown to stimulate hepatic formation of yolk proteins (vitellogenins) and to raise serum vitellogenin levels and the protein-bound fraction of plasma calcium levels (8-19). However, few studies describe the relationship between blood phosphate level and gonadal maturation in fishes (7,20). Oguri and Takada (goldfish; 11), Woodhead (arctic cod; 12) and Singh and Srivastav (catfish; 7) have found no correlation between serum calcium level and testicular maturation.

In the present study an attempt was made to investigate changes in serum calcium and phosphate levels in relation to ovarian maturation (vitellogenesis) and testicular maturation in a freshwater murrel, Channa punctatus.

Ten male and ten female adult Channa punctatus murrels (15-20 cm; 60-80 g) were collected during the second week of every month throughout the year. Body and gonad weights were recorded for each animal to assess the variation in gonadosomatic index (GSI): GSI = gonad weight/body weight x 100.

Blood samples were collected under MS222 anesthesia by sectioning the caudal peduncle. Sera were separated by centrifugation and analyzed for calcium and phosphate levels according to the methods of Trinder (21) and Fiske and Subbarow (22), respectively. For histological studies gonads were fixed in Bouin's fixative. Sections were cut at 6 µm and processed with hematoxylin-eosin (HE).

To test the statistical significance, ANOVA, Student-Newman-Keuls and Dunnett multiple comparison tests were used.

The annual gonadal cycle of Channa punctatus can be divided into five phases, as shown in Table 1.

Males

No change in serum calcium level was recorded throughout the year (Figure 1A) in relation to testicular cycle. However, serum phosphate level (Figure 1B) exhibited a rise corresponding to an increased GSI (Figure 1C).

Females

The lowest serum calcium levels were recorded during the resting phase (March and October), with values increasing thereafter and peaking in June and early January (Figure 1A) when the end of the prespawning phase occurs for both reproductive peaks. During the spawning and postspawning phases, serum calcium levels showed a progressive fall (Figure 1A).

Serum phosphate levels increased with increasing GSI (Figure 1B and C). The serum calcium and phosphate levels of male and female Channa punctatus during different reproductive phases are shown in Figure 2A and B.

ANOVA indicated that the seasonal differences in serum calcium and phosphate values of males were not significant, whereas in females the serum calcium (P<0.03; F = 5.162) and phosphate values (P<0.05; F = 4.74) were significant. According to the Student-Newman-Keuls test, the serum calcium and phosphate levels of males and females showed no significant seasonal differences. According to the Dunnett multiple comparison test (considering resting phase as control), the serum calcium levels of preparatory, prespawning and spawning phases in females were significantly different from controls (P<0.05).

Figure 1
- Seasonal changes in serum calcium (A) and phosphate (B) levels and gonadosomatic index (C) of male and female Channa punctatus. Each value represents the mean ± SD of ten specimens.

Figure 2
- Serum calcium (A) and phosphate (B) levels of male and female Channa punctatus during different reproductive phases. Each value represents the mean ± SD of ten specimens. RP = Resting phase; PP = preparatory phase; PS = prespawning phase; SP = spawning phase; PO = postspawning phase.

In the present study, in agreement with reports by others, no correlation was found between serum calcium level and testicular maturation (6,7,9,11,20). In contrast to these reports, Woodhead and Woodhead (10) and Woodhead (12) have suggested a positive correlation between blood calcium level and testicular maturation in arctic cod and sea cod. Moreover, administration of testosterone propionate to male goldfish and killifish had no effect on blood calcium levels (23,24).

Bjornsson et al. (15) have reported that female rainbow trouts have higher levels of protein-bound calcium than males from September to February. Balbontin et al. (20), however, recorded fluctuations in the serum calcium levels of both sexes of hake (Merluccius gayi gayi) throughout the year and reported that calcium levels remained relatively constant from December to August in killifish. The authors suggested that this difference between male hake and killifish may correspond to different patterns of sex hormone secretion.

In male Channa punctatus, serum phosphate levels seemed to increase with increasing GSI even though the statistical results were not significant. A similar correlation has also been reported earlier by Balbontin et al. (20) for killifish and by Singh and Srivastav (7) for Heteropneustes fossilis.

A marked seasonal variation in serum calcium level was observed in female Channa punctatus, associated with ovarian maturation (i.e., different phases of vitellogenesis), in agreement with earlier observations (5-7,9-12,15,20,25). Bjornsson and Haux (26) reported that free plasma calcium levels are not affected, but the increase in total plasma calcium is due to the appearance of the calcium-containing yolk protein precursor vittellogenin in plasma.

Nagler et al. (27) have reported a linear relationship between increasing levels of indirect indicators such as serum total phosphoprotein phosphorus, alkali-labile phosphoprotein phosphorus, total calcium and increasing level of serum vitellogenin in mature female rainbow trout (Salmo gairdneri). The enhanced secretion of estrogen during the sexual maturation of females increases the calcium level (6,28). Bailey (23), Ho and Vanstone (8), Fleming et al. (9), Chan and Chester Jones (14) and Woodhead (13) detected increased serum calcium levels in female fish after the administration of estradiol. In female fish estradiol administration increases serum calcium level, as also observed in males of Carassius auratus (23), Fundulus kansae and Fundulus catenatus (9), Oncorhynchus nerka (8), Gadus morhua (13) and Fundulus heteroclitus (24). Serum calcium levels fell during the spawning and postspawning phases in Channa punctatus, in agreement with the earlier investigations of Fontaine et al. (25), Swarup et al. (6) and Singh and Srivastav (7).

We conclude that there is no correlation between the serum calcium levels of male Channa punctatus and testicular maturation; however, serum phosphate increased with increasing GSI. In females, the serum calcium and phosphate levels exhibited a progressive increase with ovarian maturation.

Address for correspondence: S.K. Srivastav, Department of Zoology, University of Gorakhpur, Gorakhpur 273 009, India.

S.K. Srivastav is the recipient of a fellowship from CSIR, New Delhi. Received August 6, 1997. Accepted May 19, 1998.

Discussion

• 1. Hess AF, Bills CE, Weinstock M & Rivkin H (1928). Differences in calcium level of the blood between male and female cod. Proceedings of the Society for Experimental Biology and Medicine, 25: 349-350.
• 2. Pora EA (1935). Differences minerales dans la composition du sang suivant le sexe chez Cyprinus carpio. Comptes Rendus des Séances de la Société de Biologie et de ses Filiales, 119: 373-375.
• 3. Pora EA (1936). Sur les differences chimiques et physico-chimiques du sang, suivant les sexes chez Labrus bergyeta. Comptes Rendus des Séances de la Société de Biologie et de ses Filiales, 121: 102-105.
• 4. Scott AP, Bye VJ & Baynes SM (1980). Seasonal variation in sex steroids of female rainbow trout (Salmo gairdneri Richardson). Journal of Fish Biology, 17: 587-592.
• 5. Bromage NR, Whitehead C & Breton B (1982). Relationship between serum levels of gonadotropin, oestradiol-17B and vitellogenin in the control of ovarian development in rainbow trout. II. The effects of alterations in environmental photoperiod. General and Comparative Endocrinology, 47: 366-376.
• 6. Swarup K, Srivastav SP & Srivastav Ajai K (1986). Seasonal changes in the structure and behaviour of Stannius corpuscles and serum calcium level of Clarias batrachus in relation to the reproductive cycle. Zoologischer Anzeiger, 217: 402-408.
• 7. Singh S & Srivastav Ajai K (1990). Changes in the serum calcium and phosphate levels in relation to the annual reproductive cycle of the freshwater catfish, Heteropneustes fossilis. Boletim de Physiologie Animale, 14: 81-86.
• 8. Ho FCW & Vanstone WE (1961). Effect of estradiol monobenzoate on some serum constituents of maturing sockeye salmon (Oncorhynchus nerka). Journal of the Fisheries Research Board of Canada, 18: 859-864.
• 9. Fleming WR, Stanley JG & Meyer AH (1964). Seasonal effects of external calcium, estradiol and ACTH on the serum calcium and sodium levels of Fundulus kansae General and Comparative Endocrinology, 4: 61-67.
• 10. Woodhead PMJ & Woodhead AD (1964). Seasonal Changes in the Physiology of the Sea Cod in Relation to its Environment. I. Seasonal Changes in the Physiological Reactions of Barentzsea Cod, Gadus morhua L. Particularly Affecting Migration and Maturation. IONAF Envir. Symp. Ser. No. 117-175. Academic Press, New York and London.
• 11. Oguri M & Takada N (1967). Serum calcium and magnesium levels of goldfish with special reference to the gonadal maturation. Bulletin of the Japanese Society of Scientific Fisheries, 33: 161-166.
• 12. Woodhead PMJ (1968). Seasonal changes in the calcium content of the blood of arctic cod. Journal of Marine Biological Association of the United Kingdom, 48: 81-91.
• 13. Woodhead PMJ (1969). Effects of oestradiol and thyroxine upon the plasma calcium content of a shark, Scylorhinus conicula. General and Comparative Endocrinology, 13: 310-312.
• 14. Chan DKO & Chester Jones I (1968). Regulation and distribution of plasma calcium and inorganic phosphate in the European eel (Anguilla anguilla L.). Journal of Endocrinology, 32: 109-117.
• 15. Bjornsson B Th, Haux C, Forlin L & Deftos LJ (1986). The involvement of calcitonin in the reproductive physiology of the rainbow trout. Journal of Endocrinology, 108: 17-23.
• 16. Norberg B, Bjornsson B Th, Brown CL, Wichardt V, Deftos LJ & Haux C (1989). Changes in plasma vitellogenin, sex steroids, calcitonin and thyroid hormones related to sexual maturation in female brown trout (Salmo trutta) General and Comparative Endocrinology, 75: 316-326.
• 17. Persson P, Sundell K & Bjornsson B Th (1994). Estradiol-17B-induced calcium uptake and resorption in juvenile rainbow trout, Oncorhynchus mykiss. Fish Physiology and Biochemistry, 13: 379-386.
• 18. Persson P, Takagi Y & Bjornsson B Th (1995). Tartarate resistant acid phosphatase as a marker for scale resorption in rainbow trout, Oncorhynchus mykiss. Effects of estradiol-17B treatment and refeeding. Fish Physiology and Biochemistry, 14: 329-339.
• 19. Yeo IK & Mugiya Y (1997). Effects of extracellular calcium concentration and calcium antagonists on vitellogenin induction by estradiol 17 B in primary hepatocyte culture in the rainbow trout Oncorhynchus mykiss General and Comparative Endocrinology, 105: 294-301.
• 20. Balbontin F, Espinosa X & Pang PKT (1978). Gonadal maturation and serum calcium level in two teleosts, the hake and the killifish. Comparative Biochemistry and Physiology, 61A: 617-621.
• 21. Trinder P (1960). Colorimetric microdetermination of calcium in serum. Analyst, 85: 889-894.
• 22. Fiske CH & Subbarow Y (1925). The colorimetric determination of phosphorus. Journal of Biological Chemistry, 66: 375-400.
• 23. Bailey RE (1957). The effect of estradiol on serum calcium, phosphorus and protein of goldfish. Journal of Experimental Zoology, 136: 455-469.
• 24. Pang PKT & Balbontin F (1978). Effects of sex steroids on plasma calcium levels in male killifish, Fundulus heteroclitus. General and Comparative Endocrinology, 36: 317-320.
• 25. Fontaine M, Chartier-Baraduc M, Deville J, Lopez E & Poncet M (1969). Sur les variations de la calcemie observées chez Salmo salar L. a diverses étapes de son cycle vital. Leur determinisme endocrinien et leur intervention eventuelle sur le comportement. Comptes Rendus Hebdomadaires des Séances de l'Académie des Sciences, 268: 1058-1061.
• 26. Bjornsson B Th & Haux C (1985). Distribution of calcium, magnesium and inorganic phosphate in plasma of estradiol-17B treated rainbow trout. Journal of Comparative Physiology, 155B: 347-352.
• 27. Nagler JJ, Ruby SM, Idler DR & So YP (1987). Serum phosphoprotein, phosphorus and calcium levels as reproductive indicators of vitellogenin in highly vitellogenic mature female and estradiol injected immature rainbow trout (Salmo gairdneri). Canadian Journal of Zoology, 65: 2421-2425.
• 28. Pang PKT (1973). Endocrine control of calcium metabolism in teleosts. American Zoologist, 13: 775-792.

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  21 Sept 1998
• Date of issue
  Aug 1998

History

• Accepted
  19 May 1998
• Received
  06 Aug 1997