Camp. Biochem. Physiol. Vol. 86A, No. 2, pp. 315-317, 1987 0300-9629187 $3.00 + 0.00 Printed in Great Britain ,Q 1987 Pergamon Journals Ltd

SOME PHYSICAL PROPERTIES OF THE SEMEN FROM ARTIFICIALLY INDUCED SHARPTOOTH

CATFISH (CLARIAS GARIEPINUS)

G. J. STEYN and J. H. J. VAN WREN

Research Unit for Fish Biology, Rand Afrikaans University, P.O. Box 524, Johannesburg 2000, Republic of South Africa. Telephone: 726-5000

(Receiced 6 May 1986)

Abstract-l. A programme for the artificial induction of spermatogenesis and spermiation in Clurius gariepinus is presented.

2. The physical properties of the semen were determined. 3. Semen colour was white in all cases and mean values for the other physical properties were as

follows: semen volume = 9.91 ml; spermatocrit = 27%; sperm count = 6.2 x 106/mm’; percentage live spermatozoa = 87% and total motility time = 118 sec.

INTRODUCTION

Reproduction mode and strategy of animals are revealed to a certain extent by the physical properties of the semen. Mammalian spermatozoa for instance are activated by addition of the seminal plasma whereas the sperm cells of external fertilizing teleosts become motile upon contact with the medium in which it is released under natural conditions e.g. water. Fertilization in mammals involves a single to a few ova, therefore a relative low sperm count is sufficient to achieve fertilization. In teleosts in gen- eral, where thousands of eggs need to be fertilized, a much higher sperm count could thus be expected. Sperm counts in salmonids, are very much higher in comparison to that of mammals (Ginzburg, 1972). The low sperm count of the cichlid Oreochromis mossambicus as determined by Kruger et al. (1984), could serve as an indication of the relatively small number of eggs to be fertilized. According to Ginzberg (1972), fishes spawning in running water tend to have high sperm counts with short motility times, while still-water spawners appear to have lower sperm counts with longer motility times. Finally semen colour could serve as an indication of high or low cell counts (Salisbury et al., 1978) and the presence of high concentrations riboflavin (White and MacLeod, 1953).

The aim of the present investigation was to artificially induce spermatogenesis and spermiation in the sharptooth catfish Clarias gariepinus under labo- ratory conditions in order to determine the physical properties of the semen.

MATERIALS AND METHODS

Mature male catfish (C/arias gariepinus) were netted in the Roodeplaat dam near Pretoria in the Transvaal. They were acclimated for a period of 1 month in commercially available porta pools. Spermatogenesis and spermiation was artificially induced through the application of human cho- rionic gonadotrophins (HCG) and alcohol preserved catfish pituitary extract (CPE) (Schoonbee et al., 1980). Individuals were injected in groups of five at a time. The injection programme started before commencement of the natural

breeding season and was continued until the peak of the breeding season. Administration of hormones was main- tained in order to obtain 5 ml semen from each individual. Some of the injected fish were occasionally killed in order to monitor the stage of spermatogenesis and seminal hydration. If insufficient seminal hydration had occurred during these investigations, the administration of hormones was continued on the remaining fish in that specific group. Information on the injection procedures which resulted in successful spermiation is presented in Table 1.

Semen was collected in 50 ml glass beakers by dissecting out and squeezing of the testes. The biochemical and physical properties were then determined for each semen sample. The biochemical properties of C. gariepinus has already been published in a separate paper (Steyn and van Vuren, 1986).

The various physical properties of the semen of 14 individuals were determined by applying previously de- scribed methods. Semen volume was measured with a 10 ml measuring cylinder and the colour was noted. Semen vis- cosity was estimated according to a scale of l-3, where, 1 = watery, 2 = in between and 3 = thick (not dripping easily). Spermatocrit values were determined according to the microhematocrit technique which is in general use in hematology. Assessment of the sperm count was made with the aid of a Neubauer-Hawksley hemocytometer (Dacie and Lewis, 1963). The percentage live spermatozoa was determined according to the nigrosin+osin staining tech- nique (Blom, 1950). Spermatozoa were activated by mixing a minute volume of semen with the tip of a toothpick into a drop of distilled water on a glass slide. Motility was then evaluated with a light microscope ( x 400). A stop watch was used to determine motility time while motility degree was estimated according to the following scale: + = very weak, + + = weak, + + + = moderate, + + + + = strong, + + + + + = very strong (Steyn et al., 1985). Motility grade and motility time are plotted in the same figure to illustrate the decreasing type of movement of C. gariepinus sperm (Fig. I).

RESULTS

A decrease in the number of hormone dosages injected to achieve spermiation, occurred from August to November (Table 1). The individual (9) which yielded the largest volume (25 ml) semen, was one of the group which was injected during October.

315

316 G. J. STEYN and J. H. J. VAN WREN

wwww g rL&n.ELy,

~vvvvvvvvvvvv

+++++= very strong ++++ - strong +++ = Moderate ++ - Weak

+ - Very weak

Seconds

Fig. I.

Semen colour was white in all cases. Viscosity varied from 1 to 2 on the viscosity scale. A viscosity of 3 was observed once (Sample 4). Spermatocrit values ranged from 9 to 57%. The lowest sperm count was 3.0 x lO’/mm’ while the highest value was 9.9 x 106/mm3. The mean sperm count was 6.2 x 106/mm3. Most of the samples yielded 80% or more live cells. Sample 3 had the lowest percentage live cells, namely 63%. Sample 9 had the highest percentage live cells, namely 97% (Table 2). Sperm motility decreased rapidly on average, form grade + + + + + to + within 62 set and activity totally ceased after 118 set (Fig. 1).

DISCUSSION

Fouchk (1986) showed that blood plasma LH and testosterone levels of C. gariepinus reach a peak in October-November during the natural reproduction cycle in the Transvaal. The results obtained in the present survey confirmed the peak of the reproductive cycle of the above-mentioned species in the Transvaal and therefore the most suitable time to artificially induce spermiation. Seminal hydration is dependent on gonadotrophin levels (Clemens and Grant, 1965). The variation which existed between the semen vol- umes of C. gariepinus could be attributed to the extent to which seminal hydration has taken place. According to Clemens and Grant (1965) a linear relation existed between increasing seminal hydration and decreasing cell counts for Cyprinus carpio and Salmo gairdneri semen. This means that the higher the viscosity of the semen, the higher the sperm counts and spermatocrit values. This was, however, not evident with C. gariepinus semen. Sample 4 which showed the highest spermatocrit value also had the lowest cell count and the highest viscosity. Sample 5 has a low spermatocrit value (19%) with a high cell count (9.9 x 10/mm3). Sample 6 which had a com- parable spermatocrit value (20%) revealed a low cell count. In general, these confusing correlations be- tween spermatocrit values and cell counts could be attributed to the method of semen sampling. Squeez- ing of the testis sometimes lead to contamination of the sample with the much larger spermatid cells. These cells tended to aggregate and could not be

Fish semen 317

Table 2. Phvsical orooerties of C. zarieoinus semen

Fish number

Physical properties

PH Volume Colour Viscosity Spermatocrit Sperm colmt Live sperm Motility time measured against motility grade +++++ ++++ +t+ ++ +

Total motility time

Measuring

1 2 3 4 5 6 7 8 9 IO II 12 13 14 X unit

1.2 7.1 1.4 7.3 a.2 8.1 8.2 8.1 7.8 7.5 7.8 8.0 7.7 -log,, LB+1 4 12.3 8.0 1.5 10.5 8.6 7.7 10.2 25.0 5.0 127 13.7 II.6 8.0 9.91 ml

White

I I 2 3 I I 2 I I 2 I 2 I I Scale I9 9 21 57 I9 20 42 22 I9 51 28 34 I9 22 27 %

6.7 3.6 6,4 0.8 9.9 3,0 6.2 7,3 5,8 9.4 6.6 4.8 932 734 632 x IO”/mm’ 80 91 63 70 95 91 94 87 91 93 91 89 91 93 87 %

000x00000000000 seconds

I8 I5 25 X 25 20 27 29 I8 23 I9 22 24 20 22 seconds 25 28 41 0 33 28 41 39 30 33 34 35 35 31 33 seconds

35 43 52 29 44 45 56 55 46 51 52 50 50 43 47 seconds 57 54 62 42 64 63 72 74 64 66 69 67 61 59 62 seconds

129 95 I25 68 120 I27 149 130 124 139 123 II4 II0 100 II8 seconds

counted individually under the microscope, sub- sequently resulting in cell counts which do not coin- cide with the spermatocrit values and semen viscosity. It would thus seem that dissecting out and squeezing of the testis is not always the recommended method of semen sampling for the determination of values for the parameters concerned. This could be avoided by gently squeezing the testis as well as making sure that spermiation has taken place to such an extent that enough spermatozoa have been liberated. Never- theless, spermatocrit values of 19% and lower are considered as accurate determinations since no sper- matid contamination appeared to have taken place. Subsequently, viscosity values and cell counts of such samples could also be regarded as accurate.

The variation which existed in the percentage live cells, is probably due to various physiological vari- ables of the individual. The semen sample (3) which had the lowest percentage live spermatozoa, also had the lowest pH value. Good quality semen (i.e. high cell counts with high percentage live spermatozoa) tended to have high pH values. Baynes ef al., (1981) showed that the pH value of fresh Sulmo gairdneri semen increased from 7.5 to 7.8 within a few minutes after sampling. When S. gairdneri semen was further stored under oxygen, the pH value increased to 8 after 24 hr. According to Mann (1964) a 400% increase in oxygen uptake of sea urchin spermatozoa occurred when the seminal pH was elevated from 7.8 to 8.0. Together these results lead to the assumption that maximal sperm survival occurs at a high pH. It is well known that teleost sperm motility time could be experimentally prolonged by diluting the semen with saline (Drabkina, 1961). A combination of an increased seminal pH and salinity could effectively be utilized in artificial spawning programmes. When sperm motility of this species is compared with that of other species, it should be kept in mind that distilled water was used as activator in the present study. A recent study on the semen of C. gariepinus, however, showed borehole water and distilled water to be equally effective activating agents.

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

REFERENCES

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