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Preliminary report of histological studies on the grey mullet gonad related to hormone treatment
Aquaculture, 5 (1975) 31-39 D Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
PRELIMINARY REPORT OF HISTOLOGICAL STUDIES ON THE GREY MULLET GONAD RELATED TO HORMONE TREATMENT
PO-CIkJNG PIEN* and I-CHIU LIAO** * Institute of Oceanography, National Taiwan Uniuersity, Taipei, Taiwan (Republic of China) ** Tungkang Marine Laboratory, Taiwan Fisheries Research Institute, Tungkang, Pingtung, Taiwan (Republic of China) Contribution
A No.26 from the Tungkang Marine Laboratory
(Received July 2nd, 1974)
ABSTRACT Pien, P.C. and Liao, I C., 1975. Preliminary report of histological studies on the grey mullet gonad related to hormone treatment. Aquaculture, 5: 31-39. Hormone treatment is an important and necessary step in the induced breeding of grey mullet. A histological study was made of the grey mullet gonad in relation to hormone treatment. It was found that hormone treatment usually had a favorable effect in inducing the maturation of females. No significant histological change was found in the males, however, after hormone treatment.
INTRODUCTION of the grey mullet (Mugil cephalus L.) has been carried Taiwan and preliminary success has been achieved in mass production of the fingerlings. However, little work has been done on basic studies such as the physiological, histological and biochemical effects induced by hormone treatment. There is only one report (Liao and Suzuki, 19’71) on this subject. More studies in this field are needed. During the season of mullet Artificial
propagation
out for 10 years
mass propagation
in
in 1973/1974,
of hormones
on the gonads
MATERIALS
AND METHODS
a preliminary
by investigation
study of tissue
was made
of the effect
sections.
The mullet used in this experiment were those captured near the coast of Tungkang during the spawning migration season. Control mullet for these experiments received no hormone treatment. Five female mullets and two males were used. The first hormone injections were usually given to the females within 1 h after being transferred into the stocking tank (2.5 m X 7 m X 1.5 m). Two of these females were dissected
32
13 h 33 min and 19 h 30 min respectively after the first injection. The other three females were given the second injection on the next day in addition to the first injection and were dissected at different times, 34 h 5 min, 30 h 50 min and 51 h 15 min respectively, after the first injection. The two males were given one injection and were dissected 24 h 10 min and 48 h 3 min later respectively. Measurements of body length and body weight were made just before dissection. After dissection, examination of gonad condition, measurement of gonad weight and fixation of part of the ovary or testis with Bouin solution were carried out. Histological sections of ‘7 pm were prepared by the paraffin technique and stained with Delafield’s hematoxylin and eosin for microscopic analysis. Sectioning and examination of the testis and ovaries of the control mullet were also performed to obtain information on the normal condition of gonads of fish receiving no hormone treatment. RESULTS
The gonadosomatic index (GSI) of female mullet migrating along the coast of Tungkang ranged from 11 to 21. The oocytes with a diameter of 0.6-0.7 mm were found mostly in the secondary or tertiary yolk stage and sometimes in the migratory nucleus stage (Table I). The classification of oocyte maturation stages in the present study, based on the general scheme used by Yamamoto et al. (1965) and Hayashi (1972), is as follows: (1) chromatin-nucleolus stage; (2) perinucleolus stage; (3) yolk vesicle stage; (4) primary yolk stage; (5) secondary yolk stage; (6) tertiary yolk stage; (7) migratory nucleus stage; and (8) mature stage. Different hormonal efficacy was obtained due to the variation in maturity among individuals. One female mullet had a GSI of 20.73 after the first injection (9 No.1 in Table II). The oocytes were found
TABLE
I
The gonad condition
of male and female grey mullet from Tungkang
coast
Date
Body length (cm)
Body weight (g)
Gonad weight (g)
Sex
GSI
Remarks
ll-1973 23l-1973 1-1973 2415-12-1973 15l-1974
40.1 41.0 38.7 40.4 42.1
1117.9 1 221.3 949.1 1178.3 1 250.1
169.9 231.4 189.9 132.4 256.5
0 0 9 9
15.20 18.95 20.01 11.24 20.52
Immature, Immature, Immature, Immature, Immature,
4242975-
36.9 41.9 46.0 39.3 32.8
838.5 1 284.7 2 002.5 859.6 536.7
90.6 151.2 395.2 80.8 23.1
d d d
10.81 11.77 19.74 9.40 4.30
Mature Mature Mature Mature Mature
l-1973 l-1973 l-1973 2-1973 l-1974
Q
d
d
oocytes oocytes oocytes oocytes oocytes
at at at at at
secondary yolk stage tertiary yolk stage migratory nucleus stage secondary yolk stage tertiary yolk stage
33
at the secondary yolk stage, the same stage as in the control females. Another female (9 No.2 in Table II) which also received one injection had a GSI of 19.55 with an ovary opaque and yellow m appearance. The eggs were neither transparent nor separable. Microscopic examination of tissue sections showed that the oil droplets had a tendency to coalesce into a lesser number of droplets. The largest oil droplet was always in the center and up to 239 pm in diameter. The yolk globule measured 15 pm in diameter and the oocytes seemed to be in the very early migratory nucleus stage. The centering of oil droplets made the yolk globules gather peripherally in the cytoplasm. The nucleus started to shift toward the egg membrane because the oil droplets occupied the central portion of the oocyte. The zona radiata measured 10.6-14.5 pm and distinct radial striation was present. Among the three females that received two injections, one mullet had a GSI of 21.84 with an average oocyte diameter of 0.77 mm. A single oil droplet measured 290 ,um in diameter (0 No.3 in Table II). Eggs were not transparent. Examination of tissue sections showed that coalescence of the yolk globules occurred. Some coalesced yolk globules were global in shape and some massed together, both types being slightly eosinophilic. Distension of oocytes resulted in the zona radiata being as thin as 5.9-9.7 pm with indistinct radial striation. Another female mullet had a GSI of 37.07 (0 No.4 in Table II); and its ovary was fully distended in the body cavity and was translucent golden in appearance. Eggs were transparent and separable, and reached 0.93 mm in diameter. Yolk globules formed a homogeneous mass and were slightly eosinophilic. The single oil droplet measured 359 pm in diameter. A thin layer of strongly basophilic granules and vacuoles appeared between the yolk mass and egg membrane. The eggs were out of follicle cells and ready to ovulate. The mature stage of the eggs was thus reached by this female after two injections of hormone. Yet another female mullet had a GSI of 26.40 (0 No.5 in Table II); The eggs in the ovary did not seem to develop synchronously. Most of the eggs were transparent while the rest were opaque and yellow in appearance. Examination of the tissue section showed that the transparent eggs were the same as those in the mature stage and that the opaque eggs were degenerate. The GSI of male grey mullet captured along the coast of Tungkang ranged from 4 to 20. The testes filled the body cavity completely and were opaque, milky white, and both smooth and crumbly in texture. Milt could be easily extruded by pressure on the belly. In microscopic appearance, the testes were composed of numerous lobules fully packed with mature spermatozoa. A thin layer of connective tissue formed the wall of the lobules. Hormone treatment of the male mullets only made it easier to strip for milt but did not show any significant changes in the histological structure of the testes when compared with the control.
No.
3P+ 30RU + 50VE + 0.25BC 3P + 20RU + 1OOVE
3P + 20RU + 1OOVE
2P + 1ORU + 0.5BC
2Pt 1ORU + 0.5BC 2P+ 30RU + 0.5BC
l-1974
21-12-1973 16hlO
l- 2-1974 18h45
1323 h l18 h
l-1974 20 2-1974 07
18-12-1973 16h55
23h30
29-
-_..-
.
-
-
-
.
_ ._-
-
2P + ZORU + 0.5BC
25RU
22-12-1973 14 h 08
2- 2-1974 19 h 02
3P + 20RU
-
-
Dosage
19-12-1973 17 hO0
-
-
3P+ 30RU + 50VE + 0.5BC
28-
l-1974
Date
Dosage
Date
16h27
2nd injection
1st injection
* Measured after being stripped.
Sex
l-1974
l-1974
._-_
11.5
~-.
l-1974 30 2-1974 10
--
“_
-
37.2
3min
48 h
_- --
62.4*
_. _. _.
57.2*
335.4
862.6+
1270.4
40.8
901.5
398.6
292.7
376.8
Gonad weight (9)
1 004.6*
2 432.0
46.1
40.6
1825.5
1 497.2
1817.5
Body weight (9)
of glands);
46.2
45.0
46.0
Body length (cm)
24 h 10 min
51 h 15 min
3- 2-1974 22hOO
21.0
1423 b 318 h
30 h 50 min
22-12-1973 23 h 00
14.5
3.0
34 h
5min
19 h 30 min
13 h 33 min
Duration from 1st injection to dissection
of mullet (in number
20-12-1973 3 h 00
19 h 00
30-
6 h 00
29-
Dissection time
P: Pituitary
25.3
13.0
12.4
Total weight of pituitary injected
Various change found in gonad condition of male and female grey mullet after hormone treatment. RU: Synahorin (in Rabbit Unit); VE: Vitamin E (in mg); BC: Vitamin B complex (in ml).
TABLE II
7.23
5.69
26.40
37.07
21.84
19.55
20.73
GSI
__
.
-,
Testes were fully packed with mature sperms. Testes were fully packed with mature sperms. __;-____.
Slight distension of belly after 1st injection. Died from injury in ovary in early morning on Jan. 19. Ooeytes at secondary yolk stage. Poor activity resulted from injury. Oocytes with several oil droplets, yolk globule not coalesced. Floating swimming and poor activity found after 1st injection. Died at 3 h 00 on Dee. 20. Oocytes with single oil droplet and coalesced yolk globule. Obvious distension of belly found on Dec. 22. Eggs not separated when investigated at 13 h 55. 3-4 oil drop lets. Egg stripping and fertilization at 18 h 17. Poor fertilization result. Oocytes at mature stage. No response to 1st or 2nd injection, but release of many pieces of white substance at 23 h 00 on Feb. 2. Died at 22 h 00 on Feb. 3. Slight injury in ovary. Most of eggs were mature and the rest were atretic oocytes.
Remarks
35
DISCUSSION
Usually the female grey mullets migrating along the Tungkang coast are all sexually mature with well-developed gonads, but the eggs are not fully ripe for natural spawning. Most oocytes are in the secondary or tertiary yolk stage and sometimes in the migratory nucleus stage. There is a great increase in water quantity of the oocytes from the migratory nucleus stage to the fully mature stage. The changes are vigorous in the last stage when the diameter of the oocytes increases up to 0.9-0.95 mm. The oocytes in the final stages of yolk mass formation tend to crumble during sectioning. A series of morphological changes that might have occurred in the oocytes at this stage, such as the migration of the nucleus and the change of oocyte just before fertilization, were not completely studied due to the technical difficulties in sectioning by the paraffin method. Hayashi (1972) and Pollard (1972) also had similar problems. Because such problems were still present, the tracing of the migration of the nucleus and the final change of the egg just before fertilization were not perfected. An improved technique for section preparation is needed. The thin layer of basophilic granules found between the cytoplasm and membrane in the ripe eggs should be further studied in future in order to more clearly understand the substance of this layer which has never been described. At the fish market of Tungkang, one batch of mullet with distended bellies was obtained (Table III). Both the general features and histological structure of the ovaries seemed to be similar to those of the fish which responded well after two injections of hormone. The pituitary glands taken from both the male and female mullet were used in the present experiment and were found to be similar in efficiency. Liao and Suzuki (1971) also reported that the pituitary glands of male and female mullet were of equal importance in the function of induced breeding. This is convenient and advantageous since most mullet migrating along the coast of Tungkang are males. Ochiai and Umeda (1969) and Kuo et al. (1974) reported that the maturation of the ovary of MugiZ cephalus was of the “synchronism type”. Almost all the eggs reached the final maturation stage and were ovulated synchronously. The egg development in the early and middle stages was rather moderate and became rapid in the late stage. Among the three female mullets that received two injections of hormone, the mullet with a GSI of 26.40 had asynchronously developed ovarian eggs. A small number of the eggs were atretic. Whether this was caused by the poor state of health of the fish itself or by hormone treatment should be investigated. Concerning the degeneration, Shehadeh and Ellis (1970) reported that the refractoriness could be due to: (1) inadequate dosage; (2) the onset of oocyte degeneration prior to the initiation of injection; or (3) a combination of both. However, there is some difference between their experiment and the present experiment. The GSI of
36
37
TABLE III The gonad condition of ‘
Body length (cm)
Body weight (8)
Gonad weight (g)
GSI
Total number of eggs
Remarks
29-12-1973 29-12-1973 29-12-1973
42.6 49.5 46.1
1 609.7 2 743.6 2 310.4
503.1 961.1 854.3
31.25 35.03 36.98
1 348 000 2 423 000 2 214 000
Most part of eggs separated, single oil droplet.
their control female ranged from 5.60 to 12.30, much smaller than that in the present experiment. In addition, they used a different hormone dosage and injection interval. Degeneration of all the eggs in the ovary occurred in their experiment, while degeneration of only some of the eggs in the ovary occurred in our experiment. In general, the results we obtained indicate a close relationship between the physiological condition of the mullet spawner and the effect of hormone treatment. Mullet spawners that received injections of vitamin E were usually more active, lived longer and ovulated more smoothly (Liao, 1974). Further investigation on the causes of the degeneration of some of the eggs is necessary. It was generally expected that even the first injection might cause an increased GSI. However, the GSI of the two females treated with one injection were 20.73 and 19.55, lower than the GSI of the control. It is suggested by the authors that this may be caused by the poor state of health of fish or a shorter period between injection and dissection. In the present experiment, no comparison of the effects of different hormones was made. Liao and Suzuki (1971) reported that in the artificial prop‘agation of grey mullet, a combination of pituitary hormone and Synahorin was the most efficient way to induce breeding. Pituitary hormone alone might induce ovulation but with an inferior efficacy. HCG alone or Synahorin alone was found incapable of inducing ovulation. Further study in this field is needed. The optimum interval between the first and second injection, the suitable dosage, the effect of overdose or insufficient dose on ovary development, and the mechanism of hormone action on the mullet gonad should be studied in the future. _.._.-._
---__-
Fig.1. (1) Oocytes at secondary yolk stage; control female, x50; (2) Gocytes at tertiary yolk stage; control female, X 50; (3) Gocytes at secondary yolk stage; female mullet after one hormone injection (0 No.1, Table II), x50; (4) Oocytes with coalesced oil droplets; female mullet after one hormone injection (Q No.2, Table II), x 50; (5) Oocytes with fused yolk globules and single oil droplet; female mullet after two hormone injections (0 No.3, Table II), x50; (6) Atretic oocytes of female mullet after two hormone injections (Q No.5, Table II), x50; (7) Testicular lobules fully packed with spermatozoa; control male, x 10.0; (8) Testicular lobules fully packed with spermatozoa; male mullet after hormone treatment, X 100.
38
Since all male mullet investigated had sexually mature testes fully packed with mature sperm, the hormone treatment only made it easier to strip the males, especially at the end of the mullet season. SUMMARY
During the season of grey mullet mass propagation in 1973/1974, histological studies were made of the effects of hormone treatment on mullet gonad. The results obtained are as follows: (1) The GSI of female mullet migrating near the coast of Tungkang ranged from 11 to 21 during the grey mullet spawning season. At that time, oocytes O-6-0.7 mm in diameter were found usually in the secondary or tertiary yolk stage, and sometimes in the migratory nucleus stage. (2) After one injection of hormone, the GSI values of two female mullets were 20.73 and 19.55 respectively; the oocytes of the former were in the secondary yolk stage, the same stage as those of the control females. Coalesced oil droplets (239 pm in diameter) were formed in the oocytes of the latter. (3) After the second injection, the oocytes of a female with a GSI of 21.84 were 0.77 mm in diameter and were transparent. The yolk globules coalesced together into a mass, with a maximum diameter of 150 pm. The oil droplets also coalesced into one large droplet 290 pm in diameter. Oocytes (0.93 mm in diameter) of another female with a GSI of 37.07 were found in the mature stage; the eggs were translucent in appearance and separable. A single oil droplet measuring about 359 pm was found. Most oocytes of the female with a GSI of 26.40 were found in the mature stage, but a small number of the oocytes were atretic. (4) The GSI of male fish captured during the spawning migration period along the coast of Tungkang ranged from 4 to 20. Testes were found fully packed with mature spermatozoa. (5) Hormone treatment of the male mullets only made it easier to strip them but did not cause any significant changes in histological structure. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the encouragements made by Professor T.Y. Chu, Director of the Institute of Oceanography, National Taiwan University, Dr H.C. Liu, associate professor of the same institute, and Dr C.C. Huang, Institute of Zoology, Academia Sinica. The authors are also deeply indebted to Mr T.P. Chen for reading the manuscript. The study was supported by a grant from the National Science Council under the administration of the Fisheries Division, JCRR.
39
REFERENCES Hayashi, I., 1972. On the ovarian maturation of the Japanese sea bass, Lateolabmx japonicus. Jap. J. Ichthyol., 19: 243-254 Kuo, C.M., Nash, C.E. and Shehadeh, Z.H., 1974. A procedural guide to induce spawning in grey mullet (Mugil cephalus L.). Aquaculture, 3: l-14 Liao, I.C. and Suzuki, K., 1971. Studies on the artificial propagation of grey mullet, Mugil cephalus Linnaeus i I. On the induced spawning. Proc. Spring Meeting Jap. Sot. Sci. Fish., 1971, 317 (in Japanese) Liao, I.C., 1974. Experiments on induced breeding of the grey mullet in Taiwan from 1963 to 1973. Presented at the IBP/PM Int. Symp. on the Grey Mullets and their Culture, Haifa, Israel 2-8, June, 1974 Ochiai, A. and Umeda, S., 1969. Spawning aspects of the grey mullet, Mugil cephalus L. living on the coastal region of Kochi Prefecture. Jap. J. Ichthyol., 16: 50-54 (in Japanese with English abstract) Pollard, D.A., 1972. The biology of a landlocked form of the normally catadromous salmoniform fish, Galaxias maculatus (Jenyns) - III. Structure of the gonads. Aust. J. Mar. Freshwater Res., 23: 17-38 Shehadeh, Z.H. and Ellis, J.N., 1970. Induced spawning of the striped mullet Mugil cephalus L. J. Fish. Biol., 2: 355-360 Yamamoto, K., Oota, I., Takano, K. and Ishikawa, T., 1965. Studies on the maturing process of the rainbow trout, Salmo gairdnerii - I. Maturation of the ovary of a oneyear old fish. Bull. Jap. Sot. Sci. Fish., 31: 123-132 (in Japanese with English abstract)
PRELIMINARY REPORT OF HISTOLOGICAL STUDIES ON THE GREY MULLET GONAD RELATED TO HORMONE TREATMENT
PO-CIkJNG PIEN* and I-CHIU LIAO** * Institute of Oceanography, National Taiwan Uniuersity, Taipei, Taiwan (Republic of China) ** Tungkang Marine Laboratory, Taiwan Fisheries Research Institute, Tungkang, Pingtung, Taiwan (Republic of China) Contribution
A No.26 from the Tungkang Marine Laboratory
(Received July 2nd, 1974)
ABSTRACT Pien, P.C. and Liao, I C., 1975. Preliminary report of histological studies on the grey mullet gonad related to hormone treatment. Aquaculture, 5: 31-39. Hormone treatment is an important and necessary step in the induced breeding of grey mullet. A histological study was made of the grey mullet gonad in relation to hormone treatment. It was found that hormone treatment usually had a favorable effect in inducing the maturation of females. No significant histological change was found in the males, however, after hormone treatment.
INTRODUCTION of the grey mullet (Mugil cephalus L.) has been carried Taiwan and preliminary success has been achieved in mass production of the fingerlings. However, little work has been done on basic studies such as the physiological, histological and biochemical effects induced by hormone treatment. There is only one report (Liao and Suzuki, 19’71) on this subject. More studies in this field are needed. During the season of mullet Artificial
propagation
out for 10 years
mass propagation
in
in 1973/1974,
of hormones
on the gonads
MATERIALS
AND METHODS
a preliminary
by investigation
study of tissue
was made
of the effect
sections.
The mullet used in this experiment were those captured near the coast of Tungkang during the spawning migration season. Control mullet for these experiments received no hormone treatment. Five female mullets and two males were used. The first hormone injections were usually given to the females within 1 h after being transferred into the stocking tank (2.5 m X 7 m X 1.5 m). Two of these females were dissected
32
13 h 33 min and 19 h 30 min respectively after the first injection. The other three females were given the second injection on the next day in addition to the first injection and were dissected at different times, 34 h 5 min, 30 h 50 min and 51 h 15 min respectively, after the first injection. The two males were given one injection and were dissected 24 h 10 min and 48 h 3 min later respectively. Measurements of body length and body weight were made just before dissection. After dissection, examination of gonad condition, measurement of gonad weight and fixation of part of the ovary or testis with Bouin solution were carried out. Histological sections of ‘7 pm were prepared by the paraffin technique and stained with Delafield’s hematoxylin and eosin for microscopic analysis. Sectioning and examination of the testis and ovaries of the control mullet were also performed to obtain information on the normal condition of gonads of fish receiving no hormone treatment. RESULTS
The gonadosomatic index (GSI) of female mullet migrating along the coast of Tungkang ranged from 11 to 21. The oocytes with a diameter of 0.6-0.7 mm were found mostly in the secondary or tertiary yolk stage and sometimes in the migratory nucleus stage (Table I). The classification of oocyte maturation stages in the present study, based on the general scheme used by Yamamoto et al. (1965) and Hayashi (1972), is as follows: (1) chromatin-nucleolus stage; (2) perinucleolus stage; (3) yolk vesicle stage; (4) primary yolk stage; (5) secondary yolk stage; (6) tertiary yolk stage; (7) migratory nucleus stage; and (8) mature stage. Different hormonal efficacy was obtained due to the variation in maturity among individuals. One female mullet had a GSI of 20.73 after the first injection (9 No.1 in Table II). The oocytes were found
TABLE
I
The gonad condition
of male and female grey mullet from Tungkang
coast
Date
Body length (cm)
Body weight (g)
Gonad weight (g)
Sex
GSI
Remarks
ll-1973 23l-1973 1-1973 2415-12-1973 15l-1974
40.1 41.0 38.7 40.4 42.1
1117.9 1 221.3 949.1 1178.3 1 250.1
169.9 231.4 189.9 132.4 256.5
0 0 9 9
15.20 18.95 20.01 11.24 20.52
Immature, Immature, Immature, Immature, Immature,
4242975-
36.9 41.9 46.0 39.3 32.8
838.5 1 284.7 2 002.5 859.6 536.7
90.6 151.2 395.2 80.8 23.1
d d d
10.81 11.77 19.74 9.40 4.30
Mature Mature Mature Mature Mature
l-1973 l-1973 l-1973 2-1973 l-1974
Q
d
d
oocytes oocytes oocytes oocytes oocytes
at at at at at
secondary yolk stage tertiary yolk stage migratory nucleus stage secondary yolk stage tertiary yolk stage
33
at the secondary yolk stage, the same stage as in the control females. Another female (9 No.2 in Table II) which also received one injection had a GSI of 19.55 with an ovary opaque and yellow m appearance. The eggs were neither transparent nor separable. Microscopic examination of tissue sections showed that the oil droplets had a tendency to coalesce into a lesser number of droplets. The largest oil droplet was always in the center and up to 239 pm in diameter. The yolk globule measured 15 pm in diameter and the oocytes seemed to be in the very early migratory nucleus stage. The centering of oil droplets made the yolk globules gather peripherally in the cytoplasm. The nucleus started to shift toward the egg membrane because the oil droplets occupied the central portion of the oocyte. The zona radiata measured 10.6-14.5 pm and distinct radial striation was present. Among the three females that received two injections, one mullet had a GSI of 21.84 with an average oocyte diameter of 0.77 mm. A single oil droplet measured 290 ,um in diameter (0 No.3 in Table II). Eggs were not transparent. Examination of tissue sections showed that coalescence of the yolk globules occurred. Some coalesced yolk globules were global in shape and some massed together, both types being slightly eosinophilic. Distension of oocytes resulted in the zona radiata being as thin as 5.9-9.7 pm with indistinct radial striation. Another female mullet had a GSI of 37.07 (0 No.4 in Table II); and its ovary was fully distended in the body cavity and was translucent golden in appearance. Eggs were transparent and separable, and reached 0.93 mm in diameter. Yolk globules formed a homogeneous mass and were slightly eosinophilic. The single oil droplet measured 359 pm in diameter. A thin layer of strongly basophilic granules and vacuoles appeared between the yolk mass and egg membrane. The eggs were out of follicle cells and ready to ovulate. The mature stage of the eggs was thus reached by this female after two injections of hormone. Yet another female mullet had a GSI of 26.40 (0 No.5 in Table II); The eggs in the ovary did not seem to develop synchronously. Most of the eggs were transparent while the rest were opaque and yellow in appearance. Examination of the tissue section showed that the transparent eggs were the same as those in the mature stage and that the opaque eggs were degenerate. The GSI of male grey mullet captured along the coast of Tungkang ranged from 4 to 20. The testes filled the body cavity completely and were opaque, milky white, and both smooth and crumbly in texture. Milt could be easily extruded by pressure on the belly. In microscopic appearance, the testes were composed of numerous lobules fully packed with mature spermatozoa. A thin layer of connective tissue formed the wall of the lobules. Hormone treatment of the male mullets only made it easier to strip for milt but did not show any significant changes in the histological structure of the testes when compared with the control.
No.
3P+ 30RU + 50VE + 0.25BC 3P + 20RU + 1OOVE
3P + 20RU + 1OOVE
2P + 1ORU + 0.5BC
2Pt 1ORU + 0.5BC 2P+ 30RU + 0.5BC
l-1974
21-12-1973 16hlO
l- 2-1974 18h45
1323 h l18 h
l-1974 20 2-1974 07
18-12-1973 16h55
23h30
29-
-_..-
.
-
-
-
.
_ ._-
-
2P + ZORU + 0.5BC
25RU
22-12-1973 14 h 08
2- 2-1974 19 h 02
3P + 20RU
-
-
Dosage
19-12-1973 17 hO0
-
-
3P+ 30RU + 50VE + 0.5BC
28-
l-1974
Date
Dosage
Date
16h27
2nd injection
1st injection
* Measured after being stripped.
Sex
l-1974
l-1974
._-_
11.5
~-.
l-1974 30 2-1974 10
--
“_
-
37.2
3min
48 h
_- --
62.4*
_. _. _.
57.2*
335.4
862.6+
1270.4
40.8
901.5
398.6
292.7
376.8
Gonad weight (9)
1 004.6*
2 432.0
46.1
40.6
1825.5
1 497.2
1817.5
Body weight (9)
of glands);
46.2
45.0
46.0
Body length (cm)
24 h 10 min
51 h 15 min
3- 2-1974 22hOO
21.0
1423 b 318 h
30 h 50 min
22-12-1973 23 h 00
14.5
3.0
34 h
5min
19 h 30 min
13 h 33 min
Duration from 1st injection to dissection
of mullet (in number
20-12-1973 3 h 00
19 h 00
30-
6 h 00
29-
Dissection time
P: Pituitary
25.3
13.0
12.4
Total weight of pituitary injected
Various change found in gonad condition of male and female grey mullet after hormone treatment. RU: Synahorin (in Rabbit Unit); VE: Vitamin E (in mg); BC: Vitamin B complex (in ml).
TABLE II
7.23
5.69
26.40
37.07
21.84
19.55
20.73
GSI
__
.
-,
Testes were fully packed with mature sperms. Testes were fully packed with mature sperms. __;-____.
Slight distension of belly after 1st injection. Died from injury in ovary in early morning on Jan. 19. Ooeytes at secondary yolk stage. Poor activity resulted from injury. Oocytes with several oil droplets, yolk globule not coalesced. Floating swimming and poor activity found after 1st injection. Died at 3 h 00 on Dee. 20. Oocytes with single oil droplet and coalesced yolk globule. Obvious distension of belly found on Dec. 22. Eggs not separated when investigated at 13 h 55. 3-4 oil drop lets. Egg stripping and fertilization at 18 h 17. Poor fertilization result. Oocytes at mature stage. No response to 1st or 2nd injection, but release of many pieces of white substance at 23 h 00 on Feb. 2. Died at 22 h 00 on Feb. 3. Slight injury in ovary. Most of eggs were mature and the rest were atretic oocytes.
Remarks
35
DISCUSSION
Usually the female grey mullets migrating along the Tungkang coast are all sexually mature with well-developed gonads, but the eggs are not fully ripe for natural spawning. Most oocytes are in the secondary or tertiary yolk stage and sometimes in the migratory nucleus stage. There is a great increase in water quantity of the oocytes from the migratory nucleus stage to the fully mature stage. The changes are vigorous in the last stage when the diameter of the oocytes increases up to 0.9-0.95 mm. The oocytes in the final stages of yolk mass formation tend to crumble during sectioning. A series of morphological changes that might have occurred in the oocytes at this stage, such as the migration of the nucleus and the change of oocyte just before fertilization, were not completely studied due to the technical difficulties in sectioning by the paraffin method. Hayashi (1972) and Pollard (1972) also had similar problems. Because such problems were still present, the tracing of the migration of the nucleus and the final change of the egg just before fertilization were not perfected. An improved technique for section preparation is needed. The thin layer of basophilic granules found between the cytoplasm and membrane in the ripe eggs should be further studied in future in order to more clearly understand the substance of this layer which has never been described. At the fish market of Tungkang, one batch of mullet with distended bellies was obtained (Table III). Both the general features and histological structure of the ovaries seemed to be similar to those of the fish which responded well after two injections of hormone. The pituitary glands taken from both the male and female mullet were used in the present experiment and were found to be similar in efficiency. Liao and Suzuki (1971) also reported that the pituitary glands of male and female mullet were of equal importance in the function of induced breeding. This is convenient and advantageous since most mullet migrating along the coast of Tungkang are males. Ochiai and Umeda (1969) and Kuo et al. (1974) reported that the maturation of the ovary of MugiZ cephalus was of the “synchronism type”. Almost all the eggs reached the final maturation stage and were ovulated synchronously. The egg development in the early and middle stages was rather moderate and became rapid in the late stage. Among the three female mullets that received two injections of hormone, the mullet with a GSI of 26.40 had asynchronously developed ovarian eggs. A small number of the eggs were atretic. Whether this was caused by the poor state of health of the fish itself or by hormone treatment should be investigated. Concerning the degeneration, Shehadeh and Ellis (1970) reported that the refractoriness could be due to: (1) inadequate dosage; (2) the onset of oocyte degeneration prior to the initiation of injection; or (3) a combination of both. However, there is some difference between their experiment and the present experiment. The GSI of
36
37
TABLE III The gonad condition of ‘
Body length (cm)
Body weight (8)
Gonad weight (g)
GSI
Total number of eggs
Remarks
29-12-1973 29-12-1973 29-12-1973
42.6 49.5 46.1
1 609.7 2 743.6 2 310.4
503.1 961.1 854.3
31.25 35.03 36.98
1 348 000 2 423 000 2 214 000
Most part of eggs separated, single oil droplet.
their control female ranged from 5.60 to 12.30, much smaller than that in the present experiment. In addition, they used a different hormone dosage and injection interval. Degeneration of all the eggs in the ovary occurred in their experiment, while degeneration of only some of the eggs in the ovary occurred in our experiment. In general, the results we obtained indicate a close relationship between the physiological condition of the mullet spawner and the effect of hormone treatment. Mullet spawners that received injections of vitamin E were usually more active, lived longer and ovulated more smoothly (Liao, 1974). Further investigation on the causes of the degeneration of some of the eggs is necessary. It was generally expected that even the first injection might cause an increased GSI. However, the GSI of the two females treated with one injection were 20.73 and 19.55, lower than the GSI of the control. It is suggested by the authors that this may be caused by the poor state of health of fish or a shorter period between injection and dissection. In the present experiment, no comparison of the effects of different hormones was made. Liao and Suzuki (1971) reported that in the artificial prop‘agation of grey mullet, a combination of pituitary hormone and Synahorin was the most efficient way to induce breeding. Pituitary hormone alone might induce ovulation but with an inferior efficacy. HCG alone or Synahorin alone was found incapable of inducing ovulation. Further study in this field is needed. The optimum interval between the first and second injection, the suitable dosage, the effect of overdose or insufficient dose on ovary development, and the mechanism of hormone action on the mullet gonad should be studied in the future. _.._.-._
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Fig.1. (1) Oocytes at secondary yolk stage; control female, x50; (2) Gocytes at tertiary yolk stage; control female, X 50; (3) Gocytes at secondary yolk stage; female mullet after one hormone injection (0 No.1, Table II), x50; (4) Oocytes with coalesced oil droplets; female mullet after one hormone injection (Q No.2, Table II), x 50; (5) Oocytes with fused yolk globules and single oil droplet; female mullet after two hormone injections (0 No.3, Table II), x50; (6) Atretic oocytes of female mullet after two hormone injections (Q No.5, Table II), x50; (7) Testicular lobules fully packed with spermatozoa; control male, x 10.0; (8) Testicular lobules fully packed with spermatozoa; male mullet after hormone treatment, X 100.
38
Since all male mullet investigated had sexually mature testes fully packed with mature sperm, the hormone treatment only made it easier to strip the males, especially at the end of the mullet season. SUMMARY
During the season of grey mullet mass propagation in 1973/1974, histological studies were made of the effects of hormone treatment on mullet gonad. The results obtained are as follows: (1) The GSI of female mullet migrating near the coast of Tungkang ranged from 11 to 21 during the grey mullet spawning season. At that time, oocytes O-6-0.7 mm in diameter were found usually in the secondary or tertiary yolk stage, and sometimes in the migratory nucleus stage. (2) After one injection of hormone, the GSI values of two female mullets were 20.73 and 19.55 respectively; the oocytes of the former were in the secondary yolk stage, the same stage as those of the control females. Coalesced oil droplets (239 pm in diameter) were formed in the oocytes of the latter. (3) After the second injection, the oocytes of a female with a GSI of 21.84 were 0.77 mm in diameter and were transparent. The yolk globules coalesced together into a mass, with a maximum diameter of 150 pm. The oil droplets also coalesced into one large droplet 290 pm in diameter. Oocytes (0.93 mm in diameter) of another female with a GSI of 37.07 were found in the mature stage; the eggs were translucent in appearance and separable. A single oil droplet measuring about 359 pm was found. Most oocytes of the female with a GSI of 26.40 were found in the mature stage, but a small number of the oocytes were atretic. (4) The GSI of male fish captured during the spawning migration period along the coast of Tungkang ranged from 4 to 20. Testes were found fully packed with mature spermatozoa. (5) Hormone treatment of the male mullets only made it easier to strip them but did not cause any significant changes in histological structure. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the encouragements made by Professor T.Y. Chu, Director of the Institute of Oceanography, National Taiwan University, Dr H.C. Liu, associate professor of the same institute, and Dr C.C. Huang, Institute of Zoology, Academia Sinica. The authors are also deeply indebted to Mr T.P. Chen for reading the manuscript. The study was supported by a grant from the National Science Council under the administration of the Fisheries Division, JCRR.
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REFERENCES Hayashi, I., 1972. On the ovarian maturation of the Japanese sea bass, Lateolabmx japonicus. Jap. J. Ichthyol., 19: 243-254 Kuo, C.M., Nash, C.E. and Shehadeh, Z.H., 1974. A procedural guide to induce spawning in grey mullet (Mugil cephalus L.). Aquaculture, 3: l-14 Liao, I.C. and Suzuki, K., 1971. Studies on the artificial propagation of grey mullet, Mugil cephalus Linnaeus i I. On the induced spawning. Proc. Spring Meeting Jap. Sot. Sci. Fish., 1971, 317 (in Japanese) Liao, I.C., 1974. Experiments on induced breeding of the grey mullet in Taiwan from 1963 to 1973. Presented at the IBP/PM Int. Symp. on the Grey Mullets and their Culture, Haifa, Israel 2-8, June, 1974 Ochiai, A. and Umeda, S., 1969. Spawning aspects of the grey mullet, Mugil cephalus L. living on the coastal region of Kochi Prefecture. Jap. J. Ichthyol., 16: 50-54 (in Japanese with English abstract) Pollard, D.A., 1972. The biology of a landlocked form of the normally catadromous salmoniform fish, Galaxias maculatus (Jenyns) - III. Structure of the gonads. Aust. J. Mar. Freshwater Res., 23: 17-38 Shehadeh, Z.H. and Ellis, J.N., 1970. Induced spawning of the striped mullet Mugil cephalus L. J. Fish. Biol., 2: 355-360 Yamamoto, K., Oota, I., Takano, K. and Ishikawa, T., 1965. Studies on the maturing process of the rainbow trout, Salmo gairdnerii - I. Maturation of the ovary of a oneyear old fish. Bull. Jap. Sot. Sci. Fish., 31: 123-132 (in Japanese with English abstract)