The induced ovulation technique using acute injections of hormones is important in the development of the mullet culture . Injected human chorionic gonadotropin (HCG) was successfully used to induce maturation and ovulation of female mullets, but high preoviposition mortality occurred and large numbers of fish did not oviposit when confined [2,3]. Mature broodstock of Liza ramada were stressed after ovarian maturation, acclimation to seawater, and induced spawning . Anesthesia induced by clove oil suppressed the cortisol stress response during routine handling procedures, transportation, and induction of spawning of L. ramada .
It is well established that stress is a serious threat to fish development and osmoregulation with significant effects on reproductive success [6–10]. In rainbow trout, repeated acute stress resulted in delayed ovulation and reduced egg size . Furthermore, female tilapia failed to spawn in crowded tanks where atresia was observed. A definite physiological function has not yet been attributed to somatolactin (SL) in fish. SL is involved in different physiological processes, such as regulation of some aspects of reproduction [11–14], and may modify the response to stress [15,16]. Other studies reported various effects of SL on acid–base balance , calcium regulation , phosphate and fat metabolism , and water background adaptation [20–22]. In addition, it was observed that the SL cells in the pars intermedia (PI) of the pituitary gland were activated during the reproductive phase in two species of the genus Oncorhynchus [23,24]. In addition, seasonal variations in the number, size, and intensity of the immunoreaction of SL cells concomitant with the development of the gonads and spawning were reported in Oreochromis niloticus and Mugil cephalus [7,13].
Understanding the physiological picture of fish during the reproductive cycle, seawater acclimation, and induced spawning is of essential value to know the possible reasons of preoviposition mortality of fish and to develop successful hatchery technology. Little data are available with regard to the physiology of mullets during the reproductive cycle and induced spawning, especially data related to possible changes in hydromineral balance and hypophyseal cell activity. It is well known that serum cortisol concentrations in L. ramada are markedly elevated in response to a variety of stress stimuli during the reproductive cycle and induction of spawning [4,25]. This study was planned to investigate the plasma ion levels and SL-immunoreactivity during handling, seawater acclimation, confinement, and induced spawning of mature L. ramada females kept in freshwater ponds. Such information may be critical for the development of reliable methods of spawning-induction in this and other commercially important species.
Materials and methods
These experiments were carried out during the natural spawning season of L. ramada. The fishes used in this study were caught, by draining water completely, from culture ponds of El-Serw Fish Research Station (freshwater habitat; Egypt). Mature broodstock of thin-lipped gray mullet, L. ramada, of at least 2 years old, with average weights ranging from 290 to 550 g and an average standard length ranging from 30 to 42 cm, were collected alive during the spawning season (November to January).
Fish transportation and anesthesia
Mature females of L. ramada were anesthetized in freshwater containing clove oil (Sigma-Aldrich Chemie Gmbh, Munich, Germany) before handling (20 mg/l) and transportation (5 mg/l) . After fish were caught, following the rapid induction of anesthesia during handling, and after 12 h test for transportation without and with clove oil-induced anesthesia, 10 fish per treatment were sampled for blood and pituitary glands.
Seawater acclimation and induction of spawning
Mature females of L. ramada were selected on the basis of the presence of a soft, swollen abdomen and protruding genital papillae. The maturity and the oocyte diameters of the females were staged by obtaining in-vivo biopsy of the ovary using a polyethelene cannula . For the measurement of oocyte diameter, the oocytes were preserved in a solution of 1% formalin in 0.9% NaCl. All of the females used possessed oocytes whose diameters were greater than 600 μm. Ripe males in which milt could be easily extruded, by gentle pressure on their bellies, were used.
Selected broodstock were acclimated in 2000–200l circular fiberglass tanks (10 fish/tank). The salinity and electrolyte composition of the water used for the different experimental groups are shown in (Table 1). In brief, fish were transferred into water with 10‰ salinity (for 12 h), which gradually increased to 35‰ (for another 12 h). After exposure to salinity, broodstock were transferred into 500 l fiberglass tanks equipped with constant running ozonated seawater (35‰) and aeration (one female+three male fishes/tank) for induction of spawning with HCG ‘pregnyl’ (Nile Co. for Pharmaceuticals, Cairo, Egypt). Water temperature ranged from 20 to 21°C. Female fish were injected by pregnyl into the dorsal musculature adjacent to the dorsal fin as a priming injection at a dose of 20 000 IU/kg body weight and after 24 h by a resolving injection of 40 000 IU HCG. All the male fishes received a single injection of HCG at a dosage of 1000 IU/kg body weight during the resolving injection of female. The used doses were calculated empirically depending on a series of preliminary experiments determining the optimal dose .
Blood sampling and analytical procedures
During handling (transportation; anesthesia), seawater acclimation, and induction of spawning, 10 fish were sampled for blood by caudal severance. To obtain blood sample, fish were taken quickly out of the ponds and samples were obtained in less than 30 s for each fish. Blood was taken into microcentrifuge tubes and centrifuged. Plasma was separated and stored frozen at −20°C until required. Water chemistry and the different plasma ion levels were measured by using the autoanalyzer Synchron CX7 clinical system (Beckman Instruments, USA).
The fishes were anesthetized in a solution (20 mg/l) of clove oil (Sigma) before handling  and then perfused by the ascending aorta with 20 ml of normal saline, followed by 50 ml of Bouin's fluid at 4°C. The pituitary glands were immediately removed and postfixed in Bouin's fluid for 24 h at 4°C. The fixed pituitaries were thereafter dehydrated through graded ethanol solution, cleared, and embedded in paraplast (Melting point: 56–58°C). Consecutive median sagittal sections of the brain and the pituitary gland of 4-μm thickness were prepared.
Antiserum to chum salmon SL (SL. Lot No. 8906) was obtained from Dr. H. Kawauchi (School of Fisheries, Japan).
Immunocytochemical staining for the sections of the pituitary gland was performed with a vectastain avidin–biotin peroxidase complex (ABC) kit (Vector Laboratories, Inc. 30 Ingold Road, Burlingame, CA, USA) as described previously . In brief, sections were incubated with SL primary antibody diluted at 1 : 1500 overnight at 4°C. Thereafter, the sections were incubated with the biotinylated secondary antibody for 1 h and with avidin–biotin peroxidase complex for 45 min. Finally, the sections were washed and stained with 3, 3′-diaminobenzidine tetrahydrochloride for 3–5 min. After the enzyme reaction, the sections were counterstained with Thionin, washed in tap water, dehydrated in alcohol, cleared in xylene, and mounted in DPX.
To confirm the specificity of the immunoreactive procedures, adjacent sections were stained according to the above-described protocol, but incubation in the primary antiserum was omitted. In addition, normal bovine serum was used instead of primary antiserum. No positive structures or cells were found in these sections.
Cell number and size measurements
Quantification of SL immunoreactive cells in the PI was calculated from five sections of each individual animal. The number of SL immunoreactive cells for each animal was expressed as the mean±standard deviation. The sizes of SL immunoreactive cells were measured using computerized analysis (the Image-Pro Analysis package, Media Cybernetics) of digital images viewed by a microscope (Axioskop; Zeiss, Oberkochen, Germany). A 3CCD color video camera (Sony, Japan) was used for a minimum of 50 SL immunoreactive cells from each section. The cross-sectional area was measured for cells with the nucleus in the plane of section.
Results were analyzed with the statistical package for social sciences (SPSS). The paired-samples t-test was applied to compare means. The level for accepted statistical significance was P≤0.05.
Plasma ion levels
Table 2 shows that the initial levels of PO43−, Na+, K+, Ca2+, and Mg2+ for mature L. ramada females, kept in freshwater ponds, were 1.35±0.013 mmol/l, 162.5±3.56 mmol/l, 5.23±0.17 mmol/l, 17±0.4 mmol/l, and 1.35±0.008 mmol/l, respectively. These levels showed a slight increase during transportation without anesthesia. However, similar levels to the initial values were obtained by using clove oil (5 mg/l) as an anesthetic during transportation.
During seawater acclimation (10‰ salinity), the levels of the presented minerals were decreased to 1.37±0.013 mmol/l, 147±4.42 mmol/l, 2.9±0.24 mmol/l, 15.8±0.38 mmol/l, and 0.73±0.076 mmol/l for PO43−, Na+, K+, Ca2+, and Mg2+, respectively. Thereafter, they gradually increased with confinement to reach the initial values by 60 h.
Furthermore, the levels of PO43−, Na+, K+, Ca2+, and Mg2+ were significantly (P≤0.05) elevated during spawning (4.1±0.24 mmol/l, 228±4.06 mmol/l, 16.5±0.33 mmol/l, 17.75±0.38 mmol/l, and 4.8±0.28 mmol/l, respectively). Preoviposition mortality of ovulated females that died after 42 h from the beginning of injection was 2.27±0.016 mmol/l, 254±7.02 mmol/l, 4.32±0.1 mmol/l, 14.13±0.32 mmol/l, and 3.16±0.17 mmol/l, respectively.
Immunostaining of somatolactin cells in the pituitary gland
The SL-producing cells were located in the PI bordering the neurohypophysis. The stress during handling (anesthesia; transportation), seawater acclimation, and induction of spawning affected the activity of SL immunoreactive cells. As illustrated in Fig. 1a and b, Fig. 2a and b, there was no change in both the secretory activity and the synthetic activity of SL immunoreactive cells during anesthesia; both granulated and degranulated cells are present. There was only a slight increase in cell number during anesthesia (Table 3). A dramatic increase in SL immunoreactive cell secretory activity was observed during transportation as reflected by increased cell number (Table 3), hypotrophy, and decrease in immunoreactivity (Fig. 3a and b, Fig. 4a and b) as well as no change in the synthetic activity during transportation without anesthesia (Fig. 3a and 3b).
There was a gradual increase in SL immunoreactive cell secretory activity during seawater acclimation (10‰ salinity) as reflected by a decrease in immunoreactivity (Fig. 5a and b, Fig. 6a and b) as well as an increase in cell number and size (Table 3). However, a dramatic increase in SL immunoreactive cell synthetic activity was observed during confinement as reflected by strong immunoreactivity, increase in size, and hyperplasia of cells, but no change was observed in the secretory activity (Fig. 7a and b, Fig. 8a and b and Table 3).
During preoviposition mortality (Fig. 9a and b) and spawning (Fig. 10a and b), there was a dramatic increase in SL immunoreactive cell secretory activity as reflected by hypotrophy and a decrease in immunoreactivity as well as a decrease in cell number in comparison with controls (Fig. 11a and b, Fig. 12a and b and Table 3).
Handling, transportation, and seawater acclimation are common stressors in fish culture, resulting in poor overall performance, diseases, and even mortality. Thus, for farmers, fish stress can result in a substantial economic loss, and for researchers, stress can confound experimental results. Anesthetics provide a useful means of reducing physical damage and preventing the exacerbation of handling stress. Clove oil at a dose of 20 mg/l effectively suppressed the electrolytes stress response, and may be used as a useful anesthetic for reducing the adverse effects of stress during handling. Our results support previous findings that clove oil is effective in suppressing the cortisol stress response and is a reasonable alternative to MS-222 in steelhead trout Oncorhynchus mykiss  and L. ramada . In addition, clove oil can be used on O. mykiss and Acipenser transmontanus fishes before gamete harvesting without significant adverse impacts on gametes [28,29]. In contrast, exposure to anesthetic agents such as benzocaine, MS-222, metomidate, and isoeugenol elicited a stress response in Salmo salar, Hippoglossus hippoglossus, and Gadus morhua .
The transportation procedure induced stress response, as represented by an increase in the levels of Na+, Ca2+, and Mg2+ in mature broodstock of L. ramada and a decrease in serum phosphorus and K+ levels. In contrast, anesthesia with clove oil at a dose of 5 mg/l, during transportation, was effective in suppressing blood electrolytes, stress response, and maintaining baseline concentrations. Similarly, a concentration of 2.5 mg/l clove oil appeared sufficient for complete loss of reactivity to fright stimuli and sedate mature L. ramada for 7–10 h for the purpose of transportation . In addition, a similar study for the investigation of clove oil efficacy in transportation of juvenile rainbow trout demonstrated that levels of clove oil between 2 and 5 ppm may be useful in fish transport for 6–8h . This immunocytochemical investigation indicated that a dramatic increase in SL immunoreactive cell secretory activity was observed during transportation. This suggests that SL has an important role in the adaptive response of fishes to stress. In support of this view, environmental stress was shown to cause rapid activation of SL-secreting cells in the PI associated with an increase in plasma SL levels in O. mykiss, suggesting that this hormone has an important role in the adaptive response of fish to stress .
In this study, during seawater acclimation, plasma ion levels of L. ramada were decreased. In contrast, a gradual increase in SL immunoreactive cell secretory activity was observed during seawater acclimation. After 48 h of confinement in seawater, final values of electrolytes returned to initial values. However, a dramatic increase in SL immunoreactive cell synthetic activity was observed during confinement, without change in the secretory activity. This may be due to acclimation to captivity and/or tank confinement, which was probably less stressful. Our results support previous findings, whereas the opposite effect of confinement on plasma cortisol concentration was reported in Chelidonichthys kumu , Acanthopagrus butcheri , and L. ramada . In addition, plasma SL levels in H. hippoglossus tended to increase in response to acute stress, in parallel with plasma cortisol levels . As cortisol treatment has been shown to increase tolerance to seawater in fishes [34,35], SL as cortisol is considered as a seawater-adapting hormone.
The levels of PO43−, Na+, K+, and Mg2+ were significantly elevated during spawning, but Ca2+ level was decreased. Moreover, preoviposition mortality was accompanied by a physiological disturbance similar to that obtained at ovulation. In addition, a dramatic increase in SL immunoreactive cell secretory activity was observed during spawning and preoviposition mortality. During ovulation of L. ramada, gonadosomatic index increased to the maximum values within 33–64 h (38.4–46.5%), which is two times higher than those values of prespawning females . This may cause a high degree of stress in ovulated females. In L. ramada, ovulation was accompanied not only with rapid elevation in electrolytes and high secretory activity of SL immunoreactive cells but also with high cortisol levels . In the stressed state, elevated cortisol is important for central nervous system activation, increasing blood glucose concentration and elevating mean blood pressure, all of which are important for coping with stress . Cortisol is also thought to curtail the stress-induced inflammatory/immune reaction that might otherwise lead to tissue damage . This may explain the hydro–mineral imbalance observed, in this study, during ovulation and before the death of the fishes. In addition, sustained elevated levels of cortisol seem to be the cause of the immunosuppression that renders the fish vulnerable to the pathogens .
In summary, plasma ion concentrations and SL immunoreactive cell secretion in L. ramada are markedly elevated in response to a variety of stress stimuli. Furthermore, serum cortisol concentrations in L. ramada are markedly elevated in response to a variety of stress stimuli . Therefore, caution must be exercised not only in sampling the fish but also in relating temporal changes in cortisol and plasma ion levels to physiological functions.
The investigators are extremely grateful to Dr. H. Kawauchi (School of Fisheries Science, Kitasato University, Iwate, Japan) for kindly donating the antiserum of chum salmon somatolactin.
1. Mousa MA. Induced spawning
and embryonic development of Liza ramada
reared in freshwater ponds. Anim Reprod Sci. 2010;119:115–122
2. Mousa MA Biological studies on the reproduction of mullet (Mugil cephalus
L.) in Egypt [thesis]. 1994 Egypt Ain Shams University
3. Mousa MA. Hormonal induction of oocyte final maturation and ovulation in thin-lipped grey mullet, Liza ramada
(Risso). Bull Nat Inst Oceanogr Fish A R E. 1999;25:331–355
4. Mousa SA, Mousa MA. Involvement of corticotropin-releasing factor and adrenocorticotropic hormone in the ovarian maturation, seawater acclimation
and induced spawning
of Liza ramada
. Gen Comp Endocrinol. 2006;146:167–179
5. Mousa MA. The efficacy of clove oil as an anaesthetic during the induction of spawning
of thin-lipped grey mullet, Liza ramada
(Risso). J Egypt Ger Soc Zool. 2004;45:515–535
6. Flik G, Wendelaar Bonga SE. Stress
in very young and adult fish. Vie Milieu. 2001;51:229–236
7. Mousa MA, Mousa SA. Immunocytochemical study on the localization and distribution of the somatolactin
cells in the pituitary gland and the brain of oreochromis niloticus (teleostei, cichlidae). Gen Comp Endocrinol. 1999;113:197–211
8. Wendelaar Bonga SE. The stress
response in fish. Physiol Rev. 1997;77:591–625
9. Campbell PM, Pottinger TG, Sumpter JP. Stress
reduces the quality of gametes produced by rainbow trout. Biol Reprod. 1992;47:1140–1150
10. Schreck CB, Contreras Sanchez W, Fitzpatrick MS. Effects of stress
on fish reproduction, gamete quality and progeny. Aquaculture. 2001;197:3–24
11. Planas JV, Swanson P, Rand Weaver M, Dickhoff WW. Somatolactin
stimulates in vitro gonadal steroidogenesis in coho salmon, Oncorhynchus kisutch
. Gen Comp Endocrinol. 1992;87:1–5
12. Rand Weaver M, Swanson P. Plasma somatolactin
levels in coho salmon (Oncorhynchus kisutch
) during smoltification and sexual maturation. Fish Physiol Biochem. 1993;11:175–182
13. Mousa MA, Mousa SA. Implication of somatolactin
in the regulation of sexual maturation and spawning
of Mugil cephalus
. J Exp Zool. 2000;287:62–73
14. Vissio PG, Andreone L, Paz DA, Maggese MC, Somoza GM, Strussmann CA. Relation between the reproductive status and somatolactin
cell activity in the pituitary of pejerrey, Odontesthes bonariensis
(atheriniformes). J Exp Zool. 2002;293:492–499
15. Rand Weaver M, Pottinger TG, Sumpter JP. Plasma somatolactin
concentrations in salmonid fish are elevated by stress
. J Endocrinol. 1993;138:509–515
16. Johnson LL, Norberg B, Willis ML, Zebroski H, Swanson P. Isolation, characterization and radioimmunoassay of Atlantic halibut somatolactin
and plasma levels during stress
and reproduction in flatfish. Gen Comp Endocrinol. 1997;105:194–209
17. Kakizawa S, Kaneko T, Hirano T. Elevation of plasma somatolactin
concentrations during acidosis in rainbow trout (Oncorhynchus mykiss
). J Exp Biol. 1996;199:1043–1051
18. Kaneko T, Hirano T. Role of prolactin and somatolactin
in calcium regulation in fish. J Exp Biol. 1993;184:31–45
19. Lu M, Swanson P, Renfro JL. Effect of somatolactin
and related hormones on phosphate transport by flounder renal tubule primary cultures. Am J Physiol. 1995;268(3 Pt 2):R577–R582
20. Zhu Y, Thomas P. Effects of light on plasma somatolactin
levels in red drum (Sciaenops ocellatus
). Gen Comp Endocrinol. 1998;111:76–82
21. Zhu Y, Yoshiura Y, Kikuchi K, Aida K, Thomas P. Cloning and phylogenetic relationship of red drum somatolactin
cDNA and effects of light on pituitary somatolactin
mRNA expression. Gen Comp Endocrinol. 1999;113:69–79
22. Canepa MM, Pandolfi M, Maggese MC, Vissio PG. Involvement of somatolactin
in background adaptation of the cichlid fish, Cichlasoma dimerus
. J Exp Zoolog A Comp Exp Biol. 2006;305:410–419
23. Olivereau M, Rand Weaver M. Immunocytochemical study of the somatolactin
cells in the pituitary of Pacific salmon, Oncorhynchus nerka
and O. keta
at some stages of the reproductive cycle. Gen Comp Endocrinol. 1994;93:28–35
24. Olivereau M, Rand Weaver M. Immunoreactive somatolactin
cells in the pituitary of young, migrating, spawning
and spent chinook salmon, Oncorhynchus tshawytscha
. Fish Physiol Biochem. 1994;13:141–151
25. Mousa MA. Seasonal changes in gonads of the nile perch, Lates niloticus
(teleostei, Centropomidae) during the reproductive cycle in the nile river. J Egypt Ger Soc Zool. 2004;43:23–44
26. Shehadeh ZH, Kuo CM, Milisen KK. Validation of an in vivo method for monitoring ovarian development in the grey mullet (Mugil cephalus
L). J Fish Biol. 1973;5:489–496
27. Pirhonen J, Schreck CB. Effects of anaesthesia with MS-222, clove oil and CO2 on feed intake and plasma cortisol in steelhead trout (Oncorhynchus mykiss
). Aquaculture. 2003;220:507–514
28. Holcomb M, Woolsey J, Cloud JG, Ingermann RL. Effects of clove oil, tricaine and CO2
on gamete quality in steelhead and white sturgeon. N Am J Aquacul. 2004;66:228–233
29. Velíšek J, Svobodová Z, Piačková V. Effects of clove oil anaesthesia on rainbow trout (Oncorhynchus mykiss
). Acta Vet Brno. 2005;74:139–146
30. Zahl IH, Kiessling A, Samuelsen OB, Olsen RE. Anesthesia
in Atlantic salmon (Salmo salar
), atlantic cod (Gadus morhua
) and atlantic halibut (Hippoglossus hippoglossus
). Fish Physiol Biochem. 2010;36:719–730
31. Keene JL, Noakes DLG, Moccia RD, Soto CG. The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss
(Walbaum). Aquacul Res. 1998;29:89–101
32. Clearwater SJ, Pankhurst NW. The response to capture and confinement stress
of plasma cortisol, plasma sex steroids and vitellogenic oocytes in the marine teleost, red gurnard. J Fish Biol. 1997;50:429–441
33. Haddy JA, Pankhurst NW. Stress
-induced changes in concentrations of plasma sex steroids in black bream. J Fish Biol. 1999;55:1304–1316
34. Bern HA, Madsen SS. A selective survey of the endocrine system of the rainbow trout (Oncorhynchus mykiss
) with emphasis on the hormonal regulation of ion balance. Aquaculture. 1992;100:237–262
35. Mommsen TP, Vijayan MM, Moon TW. Cortisol in teleosts: dynamics, mechanisms of action and metabolic regulation. Rev Fish Biol Fish. 1999;9:211–268
36. Bamberger CM, Schulte HM, Chrousos GP. Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocr Rev. 1996;17:245–261