Camp. B&hem. Physiol. Vol. 9OA, NO. 3, pp. 387-389, 1988 0300-9629/88 $3.00 + 0.00 Printed in Great Britain 0 1988 Pergamon Press plc

PHYSICAL COMPOSITION OF THE SEMEN OF BARBUS AENEUS, THE SMALLMOUTH

YELLOWFISH (CYPRINIDAE)

W. VLOK* and J. H. J. VAN WREN

Research Unit for Fish Biology, Rand Afrikaans University, PO Box 524, Johannesburg 2000, Republic of South Africa

~Receive~ 4 November 1987)

Abstract-l. The physical composition of the semen of Barber ueneus (smallmouth yellowfish) was determined.

2. The mean values and standard deviations for some of the physical properties were as follows: sperm count = 8.41 x 106/mm3 + 2.55; percentage live cells = 92.5% f 9.92; spermatocrit = 61.9% & 5.74 and the total motility = 65 & 8.95 The colour of the sperm samples were white while the viscosity varied between 2 and 3 on the scale l-3.

INTRODUCTION

According to Ginzburg (1972) the sperm of fishes deposited in water is a suspension of the individual spermatozoa in a seminal fluid. The white of yellowfish sperm resembles milk. Information on the physical and chemical characteristics of the seminal plasma is used to determine the reproductive ability of fish (Crucea, 1969). The volume, colour, viscosity, percentage live cells, sperm con~ntration and sperm motility, to name but a few, are amongst the criteria which can successfully be used in the evaluation of sperm (Kruger et al., 1984). It is important to note that the chemical and physical changes in the en- vironment have an influence on the membrane poten- tial of bullsperm (Ibrahim and Gaal, 1979) and this phenomenon should not be ignored in the studying of fish sperm.

A correlation between spawning behaviour and percentage live sperm seems evident. A high per- centage of live sperm and sperm concentration is found for Cyprinus carpio (Kruger et al., 1984) which correlates with the environment (rapidly moving water) and other physical conditions (Le Roux and Steyn, 1968). The inactive sperm of C. carpio exhibit a rapid, progressive movement which will ensure adequate fertilization in the specific environment. Oreochromis mossambicus spawns in nests built in shallow slow-moving or standing water. After the female has released the ova, the male releases milt in close proximity of the nest (Axelrod and Burgess, 1978). According to Kruger et al. (1984) this might once again explain the lower percentage of live sperm and sperm concentrations as well as the less rapid, progressive movement of the 0. mossumbicus sperm. Colour and sperm concentration are also correlated. C. curpio has creamy-white semen and a sperm count of 6.52 x 109mm-3 while a lower count of

*To whom all correspondence should be addressed.

4 x 109mm-’ is evident in the clear, translucent sperm of 0. mossambicus (Kruger et al., 1984). According to Ginzburg (1972) the viscosity of the milt is influenced by the sperm concentration (den- sity). Sperm is activated with addition of water or medium in which it is deposited. After activation the sperm swim energetically (Ginzburg, 1972) and the movement is rarely rectilinear; usually it swims in a curved, often spiral direction (Schlenk and Kahman, 1938). The rate of movement declines rapidly (Ginz- burg, 1972; Kruger et al., 1984) and initially a high degree of motility is necessary to fertilize the eggs.

The present investigation was carried out in the field and the laboratory in order to establish the physical characteristics of the milt of But-bus ueneus and to compare it with values available for other teleosts.

MATERIALS AND METHODS

Mature Barbus ueneus males were caught during the annual breeding migration. The milt was collected in vials after the abdomen was carefully dried with paper towel to prevent contamination with water and mucus. By pressing lightly on the abdomen, milt was collected and immediately centrifuged at 3000 rpm in a crist centrifuge for 10 min. The seminal plasma was pippeted into 1 S-ml vials, chilled on ice in a cooler bag, and transferred to the laboratory where it was refrigerated.

Haymen’s diluting fluid was used to assess sperm counts with the aid of a Neubauer-Hawksley hemositomer (Dacie and Lewis, 1963). The percentage live sperm cells was determined according to the eosin-nigrosin differential staining technique described by Bfom (1950). The micro- hematocrit technique was employed to determine the sper- matocrit (Steyn and Van Vuren, 1987). The colour of the semen was noted directly after sampling and the degree of motility was measured according to the method described by Chao (1982). The total motility time was measured with a stopwatch, while the viscosity of the semen was measured on a scale of l-3, as described by Steyn and Van Vuren (1987).

387

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

Table I. Physical characteristics of the semen of &rbus wwus

Parameter Mean value Maximum value Minimum value

Sperm cell colmt 8.41 * 2.55 x lob/mm3 13.75 x IO/mm’ 3.62 x IO/mm’ Percentage live cells 92.5 f 5.74% 91% 80% Spermatocrit 61.9 f 9.92% 82% 44% Colour The colour of the milt were white in all samples Viscosity The viscosity of the milt varied between 2 and 3

Means + SD; n = 10.

RESULTS

The results of the physical characteristics of the semen of B. ueneus are presented in Tables 1 and 2.

The sperm count varied between 3.62 x IO6 and 13.75 x lo6 cells/mm3 with an average of 8.41 + 2.55 x 106cells/mm3. The highest percentage of live cells was 97% with a lowest value of 80%. The mean value was 92.5 f 5.74%. The mean value for the spermatocrit was 61.9 f 9.92% with 44% as a minimum and 82% the maximum value. The semen was white in colour whilst the viscosity varied be- tween 2 and 3 on the scale used.

Sperm cells were non-motile after sampling and activation was induced with distilled water. Total motility time varied between 65 and 99 set (82.65 + 8.95). At the commencement of the experi- ment (time zero) the degree of motility was 5+ (+ + + + +). A progressive decline is noted in the degree of motility until motility ceased after 82.65 + 8.95 set (mean value).

DISCUSSION

The sperm counts of B. ueneus varied between 3.62 and 13.75 x 106/mm3 which compared favourably with the average sperm counts of 6.2 x 106/mm3 observed in Clarias gariepinus (Steyn, 1984). In com- parison with sperm counts in other teleosts these counts are relatively low. According to Ginzburg (1972) the total cell count varied from 2.5 x lo9 in the Acipenseridae to 73.2 x lo9 in the Clupeidae while sperm numbers in Cyprinus carpio were reported to be between 24.7 x lo9 (Clemens and Grant, 1965) and 2628 x lo9 (Musselius, 1951). Kruger et al. (1984) also presented sperm counts of 6.52 x lo6 for C. carpio and 4 x 104/mm’ in Oreochromis moss- ambicus. These values fluctuated extremely even within the same species. In view of the above- mentioned findings it seems necessary to sample milt from ripe running males only, which will ensure that the sample contains spermatozoa.

The sperm cell count for Barbus kimberleyensis (largemouth yellowfish) varied between 1.36 and 2.05 x lo9 cells/cm3 while an average value of 1.71 x lo9 cells/cm3 was found (Van Vliet, 1985). Even though the smallmouth and largemouth yellowfish are closely related, a definite difference in sperm cell counts is evident. The sperm count, how- ever, is in accordance with the breeding habits. Sperm with a high cell count is released in fast running water while less sperm are released in slow running or standing water as indicated by the results obtained for C. carpio and 0. mossambicus (Kruger et al., 1984). C. curpio spawns in fast running water while 0. mossambicus, a mouth breeder, picks up the

fertilized eggs just after fertilization in calm, well- ox! ;enated water.

The percentage of live sperm cells can be correlated with the breeding habits of the fish (Le Roux and Steyn, 1968; Axelrod and Burgess, 1978). The average value of 92.5% found in B. aeneus compares favour- ably with the high values determined for 0. mossum- bicus (71.8%) by Kruger et al. (1984). Kruger et al. (1984) are of the opinion that it can be ascribed to interspecific differences (P > 0.001). The resemblance in the percentage of live sperm between B. kimber- leyensis and B. aeneus can be expected from these two closely related species. The difference in percentage live cells found amongst teleosts is probably due to the breeding habits and time of sampling. At the commencement of the breeding season, a higher percentage of live sperm cells will be present than at the end of this period when lysomal activity occurs in the testis. Furthermore, sperm quality may also de- crease towards the end of the spawning season. The technique employed in the sampling of milt should also not be neglected.

The average spermatocrit for the semen of B. ueneus (61.9%) compares favourably with that of Salmo salar (60%) and the 55% of Stizostedion uitreum (Hwang and Idler, 1969; Gregory, 1970). These values differ markably from the value of 27% (9% minimum, 57% maximum) recorded for C. gariepinus (Steyn, 1984). It was also established that high spermatocrits were not accompanied by high numbers and quality of sperm in C. guriepinus (Steyn and Van Vuren, 1987). This is in contrast to the findings of Clemens and Grant (1965) which showed a linear relation between increasing seminal hy- dration and decreasing cell counts for Cyprinus carpio and Parasalmo mykiss milt. In C. gariepinus a sperm- atocrit of 19% gave a sperm count of 9.88 million cells/mm3, while a spermatocrit of 20% gave a sperm count of only 2.99 million cells/mm3. This same

Table 2. Total sperm motility (set) of the sperm after activation

Motility grade Sample _ number 4(++++) 3(+++) 2(+ +) I(+) 0

1 17.0 33.0 46.0 63.0 99.0 2 14.0 32.0 49.0 65.0 85.0 3 16.0 34.5 49.5 68.0 87.0 4 17.0 33.0 48.5 64.0 81.5 5 17.5 33.5 50.0 64.0 90.0 6 17.0 33.5 48.5 62.0 75.0 7 20.0 36.0 50.5 67.5 88.0 8 18.0 32.5 47.5 63.5 80.5 9 16.0 27.0 40.5 57.5 65.0

10 16.0 28.0 42.0 59.5 75.5 Mean 16.85 32.3 47.2 63.4 82.65

SD 1.48 2.63 3.23 3.06 8.95

Means + SD.

Fish sperm 389

phenomenon was noted in the sperm of B. aeneus

where a sperm count of 8 million/mm3 and more had a spermatocrit between 62% and 70%. On the other hand, a spermatocrit of 82% gave a sperm cell count of only 3.62 x 106. According to Steyn (1984) late stage spermatids do aggregate in milt after sampling and these cells cannot be counted separately during the counting process in the hemositometer. The vol- ume of the spermatids is much higher than that of the mature sperm cells which results in a high sper- matocrit (volume) with a low cell count. It is evident that a high cell count will not always ensure a high spermatocrit and therefore it is not advisable to use spermatocrit as a criterion in determining the quality of sperm.

The colour of the milt of B. aeneus (white) compares favourably with that of other teleosts. According to Le Roux and Steyn (1968) and Axelrod and Burgess (1978) there is a definite similarity between the colour of the semen and the breeding habits of C. carpio

(substrate breeder) and 0. mossambicus (mouth breeder). According to these authors the semen of fish breeding in a stream is white, while the semen of mouth and nestbreeders in still water is clear. Kruger et al. (1984) concluded that a correlation existed between the colour and sperm concentration in C. Carpio (cream-white with a cell count of 6.52 x 106/mm3) and 0. mossambicus (clear semen with a cell count of 4 x 104/mm3). These species are examples of stream and mouth breeders respectively. Barbus aeneus can therefore be classified as a stream breeder if milt colour is regarded as a major criterion.

The sperm cell of B. aeneus is non-motile after sampling and this is in accordance with the findings of Ginzburg (1972) and Scott and Baynes (1980) for other teleosts. Kruger et al. (1984) have found active sperm in the seminal plasma of 0. mossambicus after sampling. This could be the result of contamination with urine. The sperm cell of B. aeneus was not active after sampling and had to be activated by the addi- tion of distilled water to the milt. Van Vliet (1985) states that the sperm of B. kimberleyensis is activated when the protective seminal plasma is diluted by the medium directly after ejaculation. The sperm samples of B. aeneus which were evaluated had a high degree of motility (+ + + + +) which steadily declined to inactivity over a period of 65-99 sec. The sperm of B.

aeneus thus have a relatively short motility in which the eggs must be fertilized.

According to Ginzburg (1972), the sperm of fish breeding in cold water have a shorter motility time than the fish breeding in warmer water. Furthermore, the motility of sperm of stream breeders terminates much quicker than the fish breeding in calmer water. It is very difficult to compare the sperm motility of fish because a standard activator and a standard activating temperature are not used (Steyn, 1984). Steyn (1984) suggests the use of distilled water as an activator is the most appropriate medium since it cannot be affected by ion concentration fluctuations.

The high viscosity of the semen (2-3) can be ascribed to the high cell counts because there is a correlation between the viscosity and sperm cell counts (Kruger et al., 1984; Van Vliet, 1985). Since

milt viscosity is determined on an arbitrary scale the results are directly influenced by the judgement of the observer. This could lead to confusion when the scales of determination are divided in a number of groups according to colour variation.

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

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