Embed Size (px)
Citation preview
Camp. 3i#c~e~. Pbrsiol. Vol. 83.A. No. 3. pp. 421-4’5, 1986 Printed in Great B&in
U3OU-96~9jS6 $3.00 f 0.00
(‘ 1986 Pergamon Press Ltd
THE ROLE OF THE BLOOD-TESTIS BARRIER IN THE CHEMICAL COMPOSITION OF THE SEMINAL PLASMA OF THE FRESHWATER
TELEOST CLARIAS GARIEPINUS
G. J. STEYN and J. H. J. VAN VUREN
Research Unit for Fish Biology. Rand Afrikaans University, P.0. Box 524, Johannesburg 2000. Republic of South Africa. Telephone: 726-5000
Abstract-l. The chemical composition of the seminal and blood plasma of the sharptooth catfish C. gariepinus were analysed simultaneously. Concentration differences existed for various components in the seminal and blood plasma.
2. Differences found could mainly be attributed to the functioning of a blood-testis barrier. 3. The possible reason for the difference in concentration between specific components in the seminal
and blood plasma while others remained unchanged is further discussed.
INTRODUCTION
The seminal plasma of mammals is composed of various secretions contributed by the male accessory sex glands. By addition of the cell fraction to the plasma fraction during ejaculation, the spermatozoa become motile and presumably metabolically active (Nalbandov, 1976). The spermatozoa of external fertilizing teleosts, in contrast, are not being activated by the seminal plasma and only become motile after the addition of water. Generally, the seminal plasma of teleosts is a sole product of the testes and increases in volume, depending on the stage of maturation of the testes. The chemical composition of the seminal plasma of teleosts is of such a nature that it inhibits sperm motility and provides the necessary com- ponents which are needed for the maintenance of sperm metabolism. Significant chemical differences between the seminal and blood plasma of teleosts is probably due to the existence of a blood-testis bar- rier. The existence of a blood-testis barrier in teleosts were reported by Abraham ef c~f. (1979), as well as Marcaillou and Szollosi (1980). According to Abra- ham et al. (1980), the blood-testis barrier of the teleost Aphanius dispar is situated between the blood and spermatozoa in contrast to mammals where the barrier exists between the blood and spermatocytes. To date, no attempt has been made to determine the chemical effect of the blood-testis barrier. In the present study the chemical characteristics and con- centration were evaluated for both seminal and blood plasma of C, guriepintrs. Statistically significant differences in values. which may exist between these body fluids, could emphasize the importance of a particular component at a specific concentration.
MATERIALS AND METHODS
Twelve mature male sharptooth catfish (Chrius g~r- iepinus) were acclimatized for three weeks and injected with human chorionic gonadotrophins (Choragon) as weI1 as catfish pituitary extract at regular intervals and’s water
temperature of 23 ir: I C. Benzocaine hydrochloride (30 mg/l water) was used to anaesthetize the fish prior to sampling of milt and blood. Blood samples were taken from the dorsal aorta at the caudal penduncle. Heparin was used as anticoagulant. Large volumes of milt could not be stripped, therefore milt was collected by dissecting out and squeezing of the testes. Both blood and milt samples were centrifuged immediately after sampling at 3000 rpm. The milt of individuals l-4 (Table 1) were centrifuged at 5000 rpm in an attempt to obtain larger volumes of seminal plasma. The samples were prepared according to in- structions given for each test combination. A Shimadzu uv 200 double beam sp~trophotometer was employed for all calorimetric determinations.
CJsmolaIity and pW were determined with a Wescor 5100 B vapour pressure osmometer and Radiometer BMS 3 Mk 2 micro blood analyzer. respectively. Sodium and potassium concentrations were obtained on a Radiometer FLM 3 flame photometer while chloride values were read directly from a Buchler-Cotlove chloridometer. Calcium and mag- nesium concentration were determined with a Corning 940 calcium analyzer and a standard Merckotest-33X biochem- ical test combination respectively. The concentrations of glucose. total lipids, total protein. lactate. urea, pyruvate kinase, cholinesterase, alkaline phosphatase and cholesterol were determined with standard biochemical test combina- tion kits (Boehringer Mannheim). Fructose values were obtained according to the method described by Davison it ul. (1946). The data was statistically analysed with a BMDP 3D programme on a Sperry Univac II00 computer. Differences between parameter values of blood and seminal plasma were recorded as significant at the 5% level (P < 0.05).
RESULTS
The results are presented in Table I. There were no statistically significant differences between the mean osmolality, sodium, urea and cholinesterase values of the seminal and blood plasma. Seminal plasma pH (7.73) was significantly (P < 0.009) higher compared to that of blood plasma (pH 7.27). Potassium values were higher (P <O.OOl), and calcium (P <O.OOl),
Tabl
e I.
Chem
ical
co
mpo
sitio
n of
th
e se
min
al
and
bloo
d pl
asm
a of
C.
g
ari
rpin
us
Fish
No
Sa
mpl
e
Osm
olah
ty
PH
Pota
ssiu
m
Sodi
um
Calc
ium
M
agne
sium
Ch
lorid
e G
luco
se
Fi?K
10sc
To
tal
prot
ein
Tota
l lip
ids
Lact
ate
Urea
Ch
oles
tero
l Py
ruva
te
kina
se
Chol
ines
tera
se
BP
179.
0 7.26
I.5
0 97
.0
10.5
8 0.
68
3.56
0.
19
3.75
0.
56
870.
97
32.2
6 87
0.97
48
.39
37.9
0 27
.40
40.2
0 56
.60
10.0
10
.0
8.0
12.0
17
2.50
17
.25
195.
50
5.75
50
.70
3.82
38
.24
0 75
.70
75.7
0 37
.85
37.8
5
I
SP
238.
0 21
8.0
7.20
6.
70
5.10
1.
60
127.
0 12
3.0
4.39
8.
87
0.14
0.
68
100.
0 88
.0
2 4
5 6
BP
SP
BP
SP
BP
215.
0 7.10
14
.10
104.
0 2.
29
0.29
240.
0 21
9.0
248.
0 6.
57
7.41
7.
061
2.80
7.
30
3.4
130.
0 12
7.0
135.
0 IO
.43
5.53
Il.
22
I.16
0.19
1.
30
99.0
95
.0
100.
0 13
3.33
22
.22
51.8
5
3.68
0.
58
4.45
2 11
71.4
3 57
.14
1028
.57
54.2
3 24
.20
56.2
12
.35
17.6
5 12
.35
500.
25
I I.5
0 40
8.25
75.6
9 37
.85
37.8
49
SP
BP
_ 22
8.0
209.
0 7.
33
7.82
10
.70
2.90
13
5.0
147.
0 2.
31
12.9
4 0.
43
I .93
93
.0
85.0
22
.22
32.4
3
0.39
4.
29
57.1
4 83
8.71
27
.60
24.7
0 19
.41
17.5
0 5.
75
333.
50
62.9
5 37
.85
34.0
6
SP
BP
SP
BP
251.
0 20
3.0
188.
0 22
9.0
245.
0 8.
15
7.61
8.
09
7.42
8.
21
19.9
0 2.
50
II.0
3.10
I I
.60
118.
0 III
.0
118.
0 15
4.0
145.
0 6.
47
il.85
9.
72
12.6
9 7.
12
I .06
I .
06
0.77
I .
25
0.77
70
.0
87.0
67
.0
99.0
85
.0
2.70
16
2.16
2.
70
97.3
1 8.
11
0.64
4.
71
64.5
2 64
5.16
30
.60
24.2
0 12
.50
15.0
0
241.
50
0 62
.95
44.2
4 38
.49
- 0.43
32
.26
23.2
0 15
.0
0
32.7
s
4.71
0.
43
677.
42
32.2
6 38
.50
25.1
0 15
.0
12.5
0 31
0.50
0
43.3
4 0
24.5
1 45
.42
7
SP
Alka
line
phos
phat
ase
33.0
16
.50
21.9
9 10
.99
33.0
21
.99
21.9
9 16
.50
21.9
9 16
.50
33.0
16
.50
33.0
21
.99
BP =
Bl
ood
plas
ma:
SP
=
Sem
inal
pl
asm
a;
.V =
M
ean;
SD
=
Stan
dard
de
viat
ion;
SE
=
Stan
dard
er
ror.
Tabl
e I
cont
inue
d:-
8 9
IO
II I2
R
SD
SE
Men
wrin
e
BP
198.
0
7.76
3.
60
SP
226.
0 8.1
I 16
.80
124.
0 8.
19
0.77
74
.0
0
BP
24X.
0 7.35
4.
40
12X.
0 9.
47
I.18
95.0
150.0
1.06
3.40
542.86
36.98
12.0
19x.37
SP
220.
0 BP
215.
0
7.80
7.24
II.70
5.70
129.0
142.0
3.04
8.03
0.83
0.87
X6.0
96.0
7.14
114.29
0
0
0.20
3.40
2x.57
514.29
1223
36.98
9.99
13.99
0
172.50
0
39.61
12.85
SP
BP
SP
BP
SP
BP
238.
0 26
1.0
213.
0 22
8.0
182.
0 22
3.0
7.52
7.
34
7.75
7.
12
8.07
7.
27
SP
222.
0 7.73
BP
22.8
9 0.
36
SP
20.0
7 0.
38
BP
6.62
0.
10
SP
5.80
0.
1 I
156.
0 12
.72
1.64
10
0.0
48.6
5
3.0
0.64
61
2.90
32
.26
37.1
0 28
IO
10
.0
10.0
31
0.50
0
36.1
2 0
37.4
3 36
.05
16.9
0 3.20
12.8
0 4.80
10.3
0 3.29
12.35
I.17
3.96
0.
34
1.15
18
.0
163.0
131.
0 164.0
127.
0 137.5
125.25
19.9
6 9.
80
5.77
2.
83
5.9
I IO.22
4.07
9.77
5.00
10.73
5.34
1.52
2.
21
0.44
0.
64
0.99
1.40
0.8
I 0.90
0.62
I.17
0.64
0.
36
0.29
0.
10
0.08
03
.0
93.0
79.0
102.0
96.0
94.90
86.2
0 5.
63
Il.78
1.
70
3.55
21
.43
192.86
21.4
3 85.71
21.4
3 106.86
12.9
4 50
.56
9.07
16
.0
2.87
0
1.06
0 2.13
0 1.06
0 0.
75
0 0.
38
0 0.
50
2.80
0.20
4.30
0.20
3.84
0.41
0.
62
0.17
0.
18
0.05
28
.57
457.14
28.5
7 628.57
28.5
7 738.25
39.2
1 20
9. I
I 12
.96
60.4
4 3.
74
15.4
3 3944
15.2
8 37.47
9.51
38.66
24.6
0 8.
98
II.70
2.
59
3.38
10
.99
7.99
9.99
12.99
9.99
12.26
12.5
0 2.
78
3.09
0.
80
0.89
0
138.0
0 270.25
0 270.97
3.35
10
3.02
5.
48
29.7
7 I .
59
0 71.21
0 10.32
0 46.98
0.42
18
.24
I.?
6.45
0.
40
38.2
7 26.21
‘6.4
1 27.36
34.5
8 36.97
40.5
5 19
.17
II.61
5.
54
3.36
Unit
mm
ol/k
g IO
?
1567
mm
& m
mol
/l m
g %
m
mol
ll m
eqil
mg
%
mm
ol/l
g %
m
g %
m
g f0
m
g %
m
g %
m
u/m
l VI
21.99
16.50
16.50
10.89
16.50
16.50
21.78
16.50
26.40
24.50
17.75
6.78
4.37
2.04
1.26
u I
Blood-testis barrier in fish 423
magnesium (P < 0.001) and chloride (P < 0.03) were significantly lower in comparison with the corre- sponding blood plasma values. Fructose was totally absent in the seminal plasma and glucose (P < O.OOl), total protein (P < O.OOl), total lipids (P < O.OOl), lactate (P < 0.005) and alkaline phosphatase (P < 0.01) values, were significantly lower in the seminal plasma. Cholesterol and pyruvate kinase is almost totally absent in the seminal plasma.
DISCUSSION
The values listed in Table 1, serve as an indication that the blood plasma of C. gariepinus is adjusted in order to establish a sperm suspending medium which contains optimum concentrations of specific chemical components. Conversion of the blood plasma into seminal plasma can be attributed to the blood-testis barrier. The composition of the seminal plasma should be sufficient to maintain the viability of the spermatozoa.
Blood and seminal plasma osmolahty values for C. gariepinus were the same and showed resemblence (Table 2) to that of Uncorh&rus keta (Morisawa et al., 1979). Although the osmolalities of these body fluids were the same, a significant difference was found in the occurrence and concentrations of vari- ous chemical components. Potassium values were much higher for instance in the seminal plasma of C. gariepinus, compared to blood plasma values. This tendency was also reported by Morisawa et al. (1979) for Carassius auratus, Cyprinus carpio and Salmo gairdnerii. According to Schlenk and Kahmann (1938) and Baynes et al. (1981) the in vioo inhibition of sperm motility can be attributed to potassium which is present in high concentrations in the seminal plasma of S. gairdnerii. Furthermore it was estab- lished that calcium and magnesium have a motility stimulating effect on S. gairdnerii spermatozoa. If the abovementioned hypothesis is correct, one could expect to find relatively high potassium and low calcium and magnesium concentrations (compared to blood) in the seminal plasma of teleosts. This ten- dancy can clearly be seen in the results obtained for C. gariepinus in the present study as well as those obtained by Morisawa et al. (1979) for 0. keta (Table 2).
The slight decrease from physiological pH of the blood plasma of C. gariepinus (pH 7.27) is probably due to the use of the anaesthetic benzocaine hydro- chloride. Ferreira et al. (1981) indicated that 50 mg/l water of the abovementioned anaesthetic caused a pH decrease of 0.71 for C. carpio blood plasma and a progressive increase in pCOz values. The blood
plasma of C. gariepinus showed a decrease of 0. I3 (from physiological pH) when 30mg/l water benzo- Caine hydrochloride was used to anaesthetize the fish. The seminal plasma of C. gariepinus was distinctly alkaline (pH 7.73) and corresponded with that of many teleosts e.g. 0. keta (8.2-8.6) and S. gairdnerii (7.8) to name but a few (Morisawa et al., 1979; Baynes et al., 1981). A change in pH from 7.5 to 7.8 was recorded for the semen of S. gairdnerii within a few minutes after spawning (Baynes et al., 1981). The pH of S. gairdnerii semen increased further to 8, after a storage period of 24 hr in air. Sea-urchin sperm showed a 400% increase in oxygen consumption when the pH was increased from 7.8 to 8.0 (Roth- schild, 1956). Physical analysis of the semen of C. gariepinus (not reported on in this paper) showed that samples with the highest pH values had the highest percentage live cells. Together these results lead to the assumption that aerobic respiration and consequently maximal sperm survival occurs at a high pH.
The presence of glucose in the seminal plasma of C. gariepinus probably compensates for the absence of fructose. Fructose is the major reducing sugar in the semen of mammals (Mann, 1964). According to Gardiner (1978) the sperm of external fertilizing teleosts appears to be able to utilize internal energy sources (possibly lipids) via the Krebs cycle. In contrast, the sperm of internal fertilizing teleosts is capable of glycolytic metabolism. Terner and Korsh (1963) reported glycolytic metabolism up to a certain stage of spermatogenesis in an oviviparous Lepomis species. This termination of glycolytic metabolism during spermatogenesis is probably due to the changes in the sperm plasma membrane before ex- posure to a osmotically different fertilizing medium. Sperm of internal fertilizers, however, are released in an isosmotic environment and properties of the plasma membrane, such as glucose transport, need not be altered (Gardiner, 1978).
The variable nature of the blood glucose concen- trations of C. gariepinus could be attributed to as- phyxiation hyperglycemia which is according to Hattingh (1976) a major cause of experimental errors in blood glucose determinations in fish. Anaerobic incubation of fresh mammalian semen causes a progressive decline in fructose concentration and an accumulation of lactate simultaneously. Lactate is the final anaerobic product of glycolysis in mammalian sperm and cannot be oxidized any further. The addition of air to anaerobic samples causes a decline in sugar consumption due to the Pasteur effect and lactate is being oxidized at the same time (Mann. 1964). The high blood lactate levels of C. gariepinus could be due to the use of benzocaine hydrochloride
Table 2. Comparison of osmolality, pH and electrolyk values of Chrias gariepinus seminal plasma with those of other species (Mann. 1964; Gregory. 1970; Morisawa er al.. 1979)
Soecies oH Na K Ca Cl Me Osm -r--~-
Measuring unit r (mg %) (mg %) (mg %) (mg %) (mg Y0) (mOsm/l)
C. grrriepinus 7.73 288.05 48.3 5.34 308 1.55 222 0. kern 8.20 326.00 258.4 8.80 475 3.88 332 S. virreum - 383.3 97.0 3.20 467 1.60 s. ,scrlar ~ 237.0 X6.0 5.2 2.20 Human 7.4 281 89.0 25.0 155 14.0 - Bull 7.1 258 172.0 24.0 320 -
Cock 7.6 393 43 8 205 8.5 -
424 G. .I. STEYN and J. H. J. VAN VUREN
as an anaesthetic since it causes a rise in blood lactate levels (Ferreira rt ul., 1981). Lactate concentrations of mammals are higher in fresh semen than in peripheric blood or tissue (Mann, 1964). Although the seminal plasma lactate levels of C. gavirpir~s are low, in comparison to blood plasma, it corresponds to the concentration in mammals (35 mg% in humans and cattle, and 27% in boar semen). Lactate is supplied to mammalian semen by the seminal vesicles and is therefore present in a different fraction of the semen (Mann, 1964). In C. gariepinus, however. semen was collected in such a way that no con- tamination by seminal vesicular fluid could take place. Lactate presence in C. gariepinus milt is thus probably the product of anaerobic glycolysis during a specific stage of testes maturation. Pyruvic acid is the immediate precursor of lactate in the semen of animals where fructolysis (glycolysis) occurs and is therefore expected to be present in the seminal plasma (Mann, 1964). One of the enzymes involved in glycolysis, is pyruvate kinase. The present results indicated that this enzyme is located intracellularly. The only pyruvate kinase value which was recorded for C. gariepinus seminal plasma could have been the result of membrane injury caused by high speed centrifugation.
Endogenous respiration of spermatozoa probably depends mainly on the oxidation of intracellular lipids (Mann, 1964). Apart from the function as an energy source. external carbohydrates also contribute to the replenishment of intracellular lipid reserves (Terner and Korsch. 1962). Although lipids were found in the seminal plasma of C. gariepinus. it was found that lipids were almost entirely confined to the tails of salmonid spermatozoa and totally absent in the seminal plasma of 0. ketu (Mann, 1964; Mor- isawa et al.. 1979).
According to Mann (1964), bovine testes are an abundant source of cholesterol and various fatty acids. The extent to which the testes of C. gariepinus contributed to the cholesterol level in the seminal plasma is unknown. In the present survey the pres- ence of cholesterol in the seminal plasma of C. gariepinus could probably be the result of leaking after damage was done to the cell membrane during high speed centrifugation.
The vulnerability of sperm cell membranes and their high permeability for protein must carefully be considered before conclusions are made on the pres- ence of proteins and enzymes in the seminal plasma (Mann, 1964). Evidence exists that the secretory function of secondary sex glands involves changes in epithelial structure, for instance the burst and ex- folication of cells as well as the release of secretory granules. The high percentage of proteins and variety of oxidizing enzymes present in the seminal plasma, could probably be explained accordingly (Mann. 1964). The way in which milt was collected from C. gariepinus, has probably resulted in the leakage of enzymes and proteins from the testis tissue. Choli- nesterase presence in seminal plasma could be attrib- uted to degenerating germ cells in the testis (Ibrahim and Gaal, 1979). Histochemical studies on the testis of the rat showed cholineterase activity in the inter- stitial Leydig cells (Nachals and Scligmann. 1949). It was however. shown by Tibhs (1960) that seminal
cholinesterase is located in the sperm heads of Salmo trutta and Perca,flur!iatilis. A study on the distribu- tion of the enzyme in ram sperm cells showed that it was mainly confined to sperm cell tails (Mann. 1964). The extent to which testis tissue and sperm cells contributed to the presence of cholinesterase in the seminal plasma of C. garkpinrts, has not yet been established. Intracellular cholinesterase is involved in the control of the flagella wave and rotation actions in sperm cells (Tibbs. 1960). Sperm motility is being negatively affected when the seminal cholinesterase concentration is high and reduction in the surviving number of cryogenically preserved bull spermatozoa occurs accordingly (Ibrahim and Gaal, 1979). The mean seminal cholinesterase value for C. guricpinus
was low (40.55/~1) in comparison to that of bull semen (8331~1). No relationship was found between cholinesterase values, weak motility and low percen- tage live spermatozoa in the semen of C. guriepinus. It is therefore believed that cholinesterase has an unimportant function in the cryogenic preservation of C. gariepinus spermatozoa. It was further shown by Steyn et al. (1985) that C. gariepinus semen can successfully be preserved at - 196 C.
Finally. the results obtained in the present study could be used to identify the most important com- ponents in the seminal plasma of C. guriepinus and thus to develop effective extenders for the cryo- preservation of spermatozoa. The findings on the presence of enzymes lead us to conclude that until more research is done on the effect of sedimentation and centrifugation on the concentrations of enzymes in the seminal plasma, it is needless to determine the concentration of the enzymes concerned. Further- more it is important to determine the concentration of proteins since the cryogenic properties and buffering capacity thereof will help to increase the effectivity of cryodiluents.
Ackno~ledRrmmrs~The senior author is indebted to the CSIR and RAU for financial assistance.
REFERENCES
Abraham M., Rahamim E. and Tibika H. (1979) The blood-testis barrier in teleosts. .Ih.\. / Con,qr. Eur. wmp. Endocrinol. Sot. (Sorrcn/o ).
Abraham M.. Rahamim E.. Tibika H., Golenscr E. and Kieselstein M. (1980) The blood testis barrier in Aphuniu.r &spar (Teloestei). Cell Thuc Rex. 21 I, 207 2 14.
Baynes S. M., Scott A. P. and Dawson A. P. (19x1) Rainbow trout .Saln~~~ ,qhdncri Richardson. sper- matozoa: effects of cations and pH on motility. J. Fish Bid. 19, 259 767.
Davison C. S.. Lewis J. H.. Tagnon H. J.. Adam M. A. and Taylor F. H. L. (1946) Method for fructose analysis in semen. New EtzgI. J. Md. 234. 279.
Ferreira J. T.. Smit G. L. and Schoonbee H. .I (1981) Haematological evaluation of the anaesthetic benzocainc hydrochloride in the freshwater fish Cx/winu.\ wrpkj L. J. Fish Bid. 18, 291 297.
Gardincr 0. M. (197X) Utilization of extracellular glucose by spermatozoa of two viviparous fishes. C’wq~. Biockvt~. Ph~~.rird 59. I65 16X.
Gregory R. W. (1970) Physical and chemical properties of walleye sperm and seminal plasma. Trtnr.c. Am. Fixh. Sock. 99, 51x 525.
Blood- testis barrier in fish
Hattingh J. (1976) The influence of carbon dioxide on the blood sugar concentration in the fresh-water fish Laheo cupmsis (Smith). Camp. Biochenr. PkJ~iol. 53. 2355236.
lbrahim M. A. R. and Gaal J. (1979) EtTects of seminal plasma cholinesterase on the viability and freezing prop- erties of bull spermatozoa. Ac,to wt. Aud. Sci. Hunt. 27, 403 407.
hlann T. ( 1964) The Biochervistrj~ of Smcv7 md of tlw Mnk Reproductiw Trwt. p. 493. Methuen & Co. Ltd. London.
Marcaillou C. and Szollosi A. (1980) The blood testis barrier in a nematode and a fish: A generalizable concept. J. ultrttstr. Rex. 70, 128136.
Morisawa M.. Hirano T. and Suzuki K. (1979) Changes in blood and seminal plasma composition of the mature salmon Or7~or/~wc/1u.s ketu during adaptation to fresh- water. Camp. Bioc~hm~. Physiol. 64, 3255329.
Nachals M. M. and Seligman A. M. (1949) Evidence for the specificity of esterase and lipase by use of three chromo- genic substrates. J. hiol. Chew. 181, 343-347.
425
Nalbandov A. V. (1976) Reproductiw Physiology of‘ Mum- muls und Birds. p. 334. Freeman & Co.. San Francisco.
Rothschild L. (1956) Fertilixtion. Methuen & Co., London. Schlenk W. and Kahmann H. (1938) Die chemische zusam-
mensetzung des spermliquors und ihre physiologische bedentungy Biochek. Z. i95, 283-301.
Stevn G. J.. van Vuren J. H. J., Schoonbee H. J. and Chao Nai-Hsien (1985) Preliminary investigations on the cryo- preservation of Clurius guric~pinus (Clariidae:Pisces) sperm. Water S.A. 11, 15-18.
Terner C. and Korsh G. (1962) The biosynthesis of CI4-labelled lipids by isolated bull spermatozoa. Bio- chemistry 1, 3677372.
Terner C. and Korsh G. (1963) The oxidative metabolism of pyruvate. acetate and glucose in isolated fish sper- matozoa. J. cell. cornp. Physiol. 62, 243-250.
Tibbs J. (1960) Acetylcholinesterase in flagellated systems. Biochim. hiophn. Actu 41, 115-122.
![An Appraisal of Introduced African Catfish Clarias ... · of introduced African catfish is poorly understood [2,4]. African sharptooth catfish Clarias gariepinus is arguably the most](https://img.pdfslide.us/doc/110x75/5eaeac3e62773f09dd34694f/an-appraisal-of-introduced-african-catfish-clarias-of-introduced-african-catfish.jpg)
