Camp. Biochem. Physiol. Vol. 90A, No. 1, pp. 49-51, 1988 Printed in Great Britain

0300-9629/88 $3.00 + 0.00 0 1988 Pergamon Press plc

CHEMICAL COMPOSITION OF BLOOD AND SEMINAL PLASMA OF BARBUS AENEUS (CYPRINIDAE)

W. VLOK and J. H. J. VAN VUREN Research Unit for Fish Biology, Rand Afrikaans University, PO Box 524, Johannesburg 2000,

South Africa

(Received 12 August 1987)

Abstract-l. The chemical composition of the seminal and blood plasma of Barbus aeneus (smallmouth yellowfish) were determined.

2. Various concentration differences between the components of the seminal and blood plasma were noted.

3. Differences found can be ascribed to the existence of a blood-testis barrier.

INTRODUCTION

The milt ejaculate is a suspension of spermatozoa in a fluid medium, the seminal plasma. The two com- ponents of milt differ in their origin, composition and function (Mann, 1964). Semen varies in composition, not only within different species but also between individuals of the same species (White and Macleod, 1963; Mann, 1964). Day-to-day variations in the concentration of some of the ,seminal plasma con- stituents from the same indivtdual do also occur (Mann, 1964).

In fish with external fertilization, the sperm is generally a testicular secretion1 with no additional discharges from the accessary: glands (Ginzburg, 1972). Suborder Gobioidei and Blennioidae (Ginz- burg, 1972) and Chrias guriephus (Steyn, 1984) are exceptional in having accessory glands. According to Ginzburg (1972) and Scott and Baynes (1980) the biochemical characteristics of fish milt could be deter- mined by evaluating the absence or presence of organic and inorganic components as well as the osmolality and pH of the seminal plasma. The pres- ence in seminal fluids of various chemical elements and components in fish is known to influence re- productive soundness (the same as in mammals) and it is therefore possible to propose morphological categories of spermatozoa to be used to evaluate the fertilizing capacity of the fish (Cruea, 1969). Abra- ham et al. (1979), as well as Marcaillou and Szollosi (1980) reported the existence of a blood-testis barrier in teleosts. The significant chemical differences exist- ing between the blood and seminal plasma are prob- ably due to the existence of a blood-testis barrier (Steyn and Van Vuren, 1986).

The blood-testis barrier (BTB) was previously described as an effective barrier within the tubule wall in the form of Sertoli cells connected to each other by specialized tight junctions between the Sertoli cells (Marcaillou and Sziilliisi, 1980). The BTB is a perme- ability barrier that exists between the circulatory system and the seminiferous tubules (Abraham et al., 1980). It is suggested that the primary barrier to penetration of the seminiferous tubules is the sur- rounding layer of contractile cells, but where this breached, specialized cell-to-cell junctions within the

epithelium constitute a secondary barrier to passage of materials into the testicular fluid (Dym and Faw- cett, 1970).

In the present study, an attempt was made to determine the effect of the blood-testis barrier on the chemical composition of the seminal plasma. This was done by studying the chemical characteristics as well as concentrations of constituents in both the seminal and blood plasma of Burbus aeneus. The results were statistically evaluated in an effort to determine whether differences in values were present which could indicate the existence of a blood-testis barrier.

MATERIALS AND METHODS

Adult, mature Barbus aeneus (smallmouth yellowfish) males were used in the experiments. Fish were caught during the breeding season while the annual upstream spawning migration was in progress.

Immediately after the fish were caught, blood was col- lected from the dorsal aorta in the caudal peduncle. Heparin was used as anticoagulant. The blood samples were immedi- ately centrifuged after sampling for 10 min at 3000 rpm and the plasma obtained poured into biofreeze vials and stored in liquid nitrogen and transferred to a laboratory freezer.

Glucose, total protein and total lipid concentrations were determined with standard biochemical test combinations (Boehringer Mannheim) while a Mercko, test 3338, test combination was used for magnesium concentrations. Fruc- tose concentrations were determined according to the method described by Davidson et al. (1949).

All samples were prepared according to specifications for the biochemical test combinations. Concentrations of the samples were determined with a Shimadzu UV 200 double beam spectrophotometer. Chloride concentrations were determined with a Buchler-Cotlove direct readout chlor- idometer while a Radiometer FLM 3 flame photometer was employed to determine sodium and potassium concen- trations. Calcium levels were determined with a Coming 940 calcium analyser while a radiometer BMS 3 Mk 2 micro blood analyser was used for pH and a Wescor 5100 B digital osmometer for osmolality determinations.

All data were statistically analysed with a BMDP 3 D program (t-test) on a Sperry Univac 1100 computer. Mean values, standard deviation and standard error were deter- mined and differences between blood and seminal plasma were recorded as significant at the 5% level (P > 0.05).

49

50 W. VLOK and J. H. J. VAN VUREN

Table 1. Chemical composition of blood and seminal plasma of Barbus aeneus (statistically significant differences: l P < 0.001; tP < 0.025)

Seminal plasma Blood plasma

Parameters x SE SD n K SE-.~.-SD- bin

____ Sodium *(mmol/l) 85.8333 1.603 6.802 I8 179.0500 5.247 23.467 20 Potassium Ymmolil) 56.9778 I.270 5.387 I8 2.2050 0.521 2.329 20

Chloride *(igil) Calcium *(mg %) Magnesium *(mmol/l) Glucose l (mg %) Fructose (mmol/l) Total lipid l (mg %) Total protein ‘(g “/a) Osmolality t(mmol/kg) nH

129.1579 2.292 9,990 4.0568 0.202 0.882 0. I229 0.006 0.027

14.2593 2.116 9.464 0.2122 0.037 0.164

80.2381 14.780 66.096 0.2166 0.048 0.214

309.421 I 4.064 17.727 X.6314 0.075 0.320

I9 108.5000 I9 II.1905 I9 0.6164 20 98.5891 20 0.4245 20 2629.6296 20 3.7321 19 324.4500 I8 7.1469

I.459 6.525 20 0.196 0.877 20 0.033 0.151 21

13.651 62.588 21 0.194 0.890 21

175.590 744.963 I8 0.156 0.714 21 4.298 19.220 20 0.075 0.338 20

RESULTS

The results are presented in Table 1. Statistically significant differences were evident in the glucose, total protein, total lipids, sodium, potassium, pH, magnesium, chloride and calcium concentrations (P > 0.001). The difference of significance in os- molality was slightly lower (P < 0.025). Fructose was the only parameter in which a non significant (NS) difference between the seminal and blood plasma occurred.

DISCUSSION

The concept of the blood-testis barrier was pro- posed at the beginning of the century (Ribbert, 1904; Bouffard, 1906) and was recently revised when it was shown that a permeability barrier exists between the circulatory system and the seminiferous tubules in species belonging to two important taxa:mammals (Brokelmann, 1963; Flickinger, 1967; Fawcett et el., 1970; Dym and Fawcett, 1970) and insects (SzBllosi en Marcaillon, 1977). Abraham et al. (1979) have recently shown this concept in the third important animal group, the teleosts.

According to Abraham et al. (1980) the blood-testis barrier is formed by tight junctions between Sertoli cells which provide a permeability barrier between the vascular spaces of the stroma and the lumina of ripe cysts. Components penetrate a short distance between the bases of the adjoining Sertoli cells before they are arrested by the tight junctional complexes (Abraham et al., 1980).

The higher chloride concentration in the seminal plasma of B. aeneus compared with that of the blood plasma, is the opposite of values found in C/arias gariepinus (Steyn and Van Vuren, 1986). Chloride and sodium play an important role in maintaining the osmotic balance in the sperm cells and it is important to evaluate these two components together. In the blood plasma of B. aeneus, sodium concentration is nearly double the value of sodium concentration in the seminal plasma. This is in contrast to results obtained in C/arias gariepinus (Steyn and Van Vuren, 1986). It is obvious that this difference in concen- tration will indicate that each species will have a specific concentration depending on the environ- mental and osmoregulatory stress on the organism.

A high potassium concentration inhibits the mo- tility of sperm (Schlenk, 1933; Schlenk and Kahman, 1938; Baynes et al., 1981) and the high potassium

concentration in the seminal plasma of the small- mouth yellowfish (B. aeneus) can thus ensure that the sperm cells will not be activated prior to release during the breeding season.

The potassium concentration in the seminal plasma of B. aeneus (56.98 mmol/l) compares favourably with the value of 53.43 mmol/l found for B. kimber- ieyensis (Van Vliet, 1985) while Steyn and Van Vuren (1986) recorded a value of 12.35 mmol/l in the sharp- tooth catfish. The difference is probably also an indication of species specificity.

Calcium (Mann, 1964) and magnesium (Cruea, 1969) are co-factors in enzyme reactions. The occur- rence of these components in the seminal plasma could therefore be ascribed to the fact that sperm cell membranes were damaged through centrifugation during analysis. These ions are possibly involved in the sperm contra-cellular metabolism during activa- tion when energy is needed for motility (Baynes et al., 1981). All the above-mentioned ions play a role in maintaining osmolality in the sperm cells. Morisawa et al. (1979) mentioned that the mean osmolality in the blood and seminal plasma is nearly identical in Oncorhynchus keta. This was also evident in C. gariepinus (Steyn, 1984).

The pH of the seminal plasma (8.63) of the small- mouth yellowfish is higher than that of the blood plasma (7.15). This corresponds with the results recorded by Van Vliet (1985) for B. kimberleyensis (largemouth yellowfish) and Steyn and Van Vuren (1986), where the pH in the seminal plasma is some- what higher than in the blood plasma of C. garie- pinus. Baynes et al. (1981) mentioned that the pH in the fresh semen of Salmo gairdnerii rose from 7.5-7.8 after collection. When oxygen is sufficiently supplied, the pH can rise to 8 within 24 hr. With an increase of 400% in oxygen concentration, pH in the sperm of the sea urchin rose from 7.848.0 (Rothschild, 1956). Teleost sperm have an optimum aerobic respiration at a pH of 8 (Steyn and Van Vuren, 1986) and the pH recorded for the seminal plasma (8.63) of B. aeneus leads to the conclusion that the sperm are kept unde aerobic conditions prior to release. This is probably a mechanism to ensure that good quality sperm with a sufficient supply of oxygen are available for fertil- ization

The concentration of glucose in the blood plasma (98.55 mg %) is higher than in the seminal plasma (14.26 mg %) of B. aeneus while fructose is found in low concentrations in the semen of the smallmouth yellowfish. This is in contrast to what was found in

Fish blood-testis barrier 51

the seminal plasma of C. gariepinus where fructose was absent (Steyn and Van Vuren, 1986). The glucose concentration (12.94 mg %) in the seminal plasma of C. gariepinus (Steyn and Van Vuren, 1986) corre- sponds to that of B. ueneus. Van Vliet (1985) found fructose (mean, 1.26 mg %) in the seminal plasma of B. kimberleyensis while these values varied between 0 and 3.97 mg %. It therefore seems as if glucose is the primary external source of energy for the sperm cell.

The function of protein in the seminal plasma is not known, but it is possible that it may have a protective function (White and Macleod, 1963; Mann, 1964; Cruea, 1969). Small amounts of protein were evident in the seminal plasma of B. aeneus and it is also possible that some may have been released after mechanical damage to the cell membranes oc- curred during centrifugation. The concentration of lipids (80.24 mg %) in the seminal plasma is higher than that of protreins and it is possible that the lipids have a greater function in protecting the sperm cells than proteins.

Finally, it can be concluded that a blood-testis barrier is in existence in the testis of B. aeneus. This is evident in the specific regulation of ion and organic component concentration levels.

Acknowledgements-The senior author is indebted to the CSIR and RAU for financial assistance.

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