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Evaluation of milt quality of Atlantic salmon
125
z4quaculture, 95 (1991)125-132 Elsevier Science Publishers B.V., Amsterdam
Evaluation of milt quality of Atlantic salmon Grete Hansen Aas, Terje Refstie and Bjarne Gjerde .4KVAFORSK, Institute ofAquaculture Research, P.O. Box IO, N-1432 .kNLH. (Accepted
Norway
10 October 1990)
ABSTRACT Aas, G.H., Refstie, T. and Gjerde, B., 1991. Evaluation of milt quality of Atlantic salmon. Aquaculture. 95: 125-132. Milt was collected individually from 27 Atlantic salmon, and sperm density, sperm motility, milt colour and osmolality, and content of sodium, potassium and glucose of seminal plasma recorded. Milt from each male fertilized samples of around 200 eggs taken from the same pool of eggs. Percentage of eyed-eggs was used as a measure of fertilization rate or milt quality. Large variation among males was observed for this trait. Variation among males for the recorded parameters was medium to high in magnitude. Correlation between sperm density registered by cell counting and spermatocrit was high (rc0.92). Repeatability waslowest for motility (0.72), and 0.92 or higher for other parameters. Of the parameters studied, sodium content in seminal plasma showed the highest correlation (r= 0.60) to fertilization rate. The remaining variation in fertilization rate could not be explained by any of the other parameters studied. However, all traits except for sperm motility and milt colour showed a significant correlation to fertilization rate. With repeated stripping within season, the sperm density gradually decreased and the sodium/ potassium ratio increased, resulting in decreasing fertilization rate.
INTRODUCTION
For farmed Atlantic salmon (Salvo saEar), hatching rate of eggs varies among different hatcheries (Ulgenes, 1983) and among batches within a hatchery (Rye et al., 1990). Milt quality is one of the factors which influence these results and can be delined as the ability to fertilize eggs. Several studies have described milt characteristics which can influence quality; for example, sperm density, sperm motility and the composition of the seminal plasma (Hwang and Idler, 1969; Mounib, 1978; Piironen and Hyvarinen, 1983; Stoss, 1983). However, few have correlated the milt characteristics to fertilizing ability. Some investigations of milt quality in connection with cryopreservation of sperm are reported (Hwang and Idler, 1969; Biiyiikhatipoglu and Holtz, 1978; Billard, 198 1; Stoss, 1983; Stoss and Holtz, 1983). 00448486/91/$03.50
0 199 1 -
Elsevier Science Publishers
B.V.
126
G.H. AAS ET AL.
Sperm density may influence fertilization rate. Sperm count and spermatocrit are two different measures of density. In rainbow trout Obraztsov ( 1985) found a correlation between sperm density and spermatocrit of 0.95-0.97. Results on rainbow trout sperm reported by Levanduski and Cloud ( 1988 ) suggested that the presence of non-motile sperm could reduce the fertility of normal motile sperm. However, this effect did not become significant until the concentration of motile sperm was less than 10%. Urine or*blood might follow the milt when the fish is stripped. This makes the milt slightly yellow or red, but further influence on fertilization is unknown. The aim of the present investigation were to ( 1) investigate variation in milt quality among males; (2) identify parameters correlated to fertilizing ability of sperm which can be used in evaluating milt quality before insemination; (3) investigate effects of repeated stripping within season on milt quality. MATERIALS
AND METHODS
Broodstock of Atlantic salmon were reared in seawater and transferred to brackish water three months before stripping at the Institute of Aquaculture’s Research, Sunndalsora. Males and females were selected at random and anaesthetized before stripping. Care was taken to avoid contamination of the milt and eggs with water or excreta. A pool of eggs from three to live females was used in each experiment. Milt was collected individually from 27 males and stored on ice in clean, dry plastic beakers until insemination. This always took place within 3 h after collection. Two different amounts of milt, corresponding to 1 ml and 0.5 ml milt per liter egg, were used to fertilize samples of about 200 eggs. After milt was added to the eggs, the eggs were stirred, and 15-30 s later water was added to complete the fertilization process. The eggs were then stirred once more. For each male and amount of milt, three parallels were used for fertilization. Altogether, there were six parallels per male. At the eyed-egg stage, eggs were shocked by shaking the tray, and the number of dead and live eggs were counted. The percentage of eyed-eggs was used as a measure of fertilization rate or milt quality. Mortality of embryo from fertilization to eyed-egg stage was treated as a constant, and, therefore, not recorded. Sperm density was evaluated using two different methods; first by measuring spermatocrit value using a microhematocrit centrifuge (Compur M 1100)) and secondly by counting sperm using Biirkers counting chamber after diluting the milt ( 1: 400 and 1: 800 depending on the spermatocrit value
EVALUATION OF MILT QUALITY OF ATLANTIC SALMON
127
of each sample). Counting chambers were always kept in a moist atmosphere for at least 15 min before cell counting. Motility of sperm in each sample was evaluated within two hours after collection. A droplet of milt was placed under a microscope and a droplet of freshwater was added before the percentage of motile sperm was calculated. Only forward moving sperm were judged to be motile, while vibrating sperm were judged to be immotile. A scale from 0 to 10 was used, where 0 and 10 respectively corresponded to 0% and 100% motile sperm. The colour of each sample of milt was judged visually. The three colours observed were white, pale red and pale yellow. Milt from each sample was centrifuged and the seminal plasma removed and frozen for further analysis. Osmotic pressure of seminal plasma was measured with an osmometer (Knauer, Semi-micro-osmometer ), the content of sodium and potassium with an atomabsorption spectrophotometer ( AOAC Methods ( 1980) Nr. 3.020-3.022), and the content of glucose with a spectrophotometer using a Mercotest 14335 Glucose-reagentkit. Repeatabilities of the recorded parameters were studied by analysing two milt samples from each male for cell counting, motility, osmolality and glucose, and three samples for spermatocrit. For each parameter, repeatability was calculated as the ratio between the variance component among fish and the total variance. A total of seven to nine males was stripped repeatedly every second week from 24 October to 4 December 1988 and altogether four times during the breeding season. The same operation as described previously was carried out. The effect of milt volume on fertilization was studied using a two-way nested analysis of variance including the effect of individual male, and the effect of milt volume within male as random effects in addition to a random error. Simple and multiple linear regressions were used to analyze the effect of each parameter on fertilization rate. In the simple analysis, a second degree polynomial was included in the model. RESULTS
Means and variation between males for the traits studied are given in Table 1. The fertilization results among males showed large variation ranging from 0.5% to 97.3%. The coefficient of variation (CV) was 19 or higher for all traits except for the sodium/potassium ratio which showed a medium CVvalue. Average number of sperm per egg was 6.2 x 10’ (s.d. = 3.1 x 10’) when 0.5 ml of milt per liter egg was used, and 12.6~ lo5 (sd. =6.4x 105) using 1.O ml of milt per liter egg. The calculated repeatabilities were 0.94 and 0.97 for sperm density registered by cell counting and spermatocrit, respectively; 0.72 for motility; 0.99 for osmolality and 0.92 for glucose. The correlation between the two mea-
G.H. AA.9 ET AL.
128 TABLE
1
Mean (X), standard deviation (s.d. ), coefficient of variation (CV), minimum (min) and maximum (max) values of traits. Computed statistics are based on mean values of from one to six samples per male Traits
N
Eyed eggs (O/O )
21
64.1
21 27
Sperm density counting ( X 109) spermatocrit (O/o) Sperm motility (score l-10) Seminal plasma Osmolality (mOsm/kg) Sodium (mmol/l ) Potassium (mmol/l) Sodium/Potassium Glucose (mmol/l)
5
15
Spermafocrlf
Fig. 1. Sperm density measured
s.d.
X
cv
min
max
33.6
_
0.5
91.3
9.54 23.4
3.2 8.0
34 -
3.5 9.1
11.9 45.0
21
6.8
1.6
24
3.5
9.5
25 27 21 21 27
245 92.8 21.5 1.97 0.45
55 23.9 5.3 0.28 0.15
22 26 19 14 33
111 33.0 14.6 1.33 0.23
320 133.9 31.0 2.51 0.90
25
35
value,
45
%
by cell count plotted against spermatocrit
value (n = 27).
sures of sperm density was high (r= 0.92 ). Observed values for each method are illustrated in Fig. 1. In the two-way nested analysis of variance, the effect of male was highly significant and explained 89.8% of the total variation in fertilization rate. The effect of milt volume within male was not significant (P> 0.05 ) and explained only 2.7% of the variation. The regression of fertilization rate on motility was not significant (P> 0.05 ) , nor the effect of milt colour on fertilization rate. The regression of fertilization rate on each parameter, except for sperm motility and milt colour, was significant and linear (P
129
EVALUATION OF MILT QUALITY OF ATLANTIC SALMON
non-significant second-degree polynomial. The correlation of fertilization rate with sperm density registered by cell counting and spermatocrit was 0.52 and 0.40 respectively, with osmolality 0.57; with sodium 0.60; with potassium 0.48; with sodium/potassium 0.52, and with glucose 0.42. The plots between fertilization rate and spermatocrit, and between fertilization rate and sodium, are illustrated in Figs. 2 and 3. The linear regression line is drawn in each figure. A similar plot was observed between fertilization rate and osmolality. Correlations among some of the parameters registered in seminal plasma are presented in Table 2. Applying a stepwise forward regression analysis to all parameters studied, sodium was the first parameter included in a model with fertilization rate as
30
20
Spermatocrrt
value,
%
Fig. 2. Spermatocrit value plotted against fertilization rate (n = 27 ).
~ &
100
-
T
d 80% UJ x
60-
L-3 91
-
‘;i<
40-
g -i: :: > Y
zo-
.
n
,
40gIt 30
50
Sodurn
,
I 70
In seminal
90
130
110
pfasma (mmol/l
)
Fig. 3. Sodium content in seminal plasma plotted against fertilization rate (n = 27 ).
G.H. AAS ET AL.
130 TABLE 2 Correlation coefficients among parameters
measured in seminal plasma
Trait
Potassium
Sodium/Potassium
Glucose
Osmolality
Sodium Potassium Sodium/Potassium Glucose
0.87
0.72 0.29
0.32 0.09 0.50
0.69 0.67 0.44 0.27
TABLE 3 Mean (with standard error) for parameters measured on seven to nine fish with four repeated strippings within season Traits
Stripping no. 1
Fertilization rate (%) Density registered by cell counting ( 109) Spermatocrit % Motility (scale from 0 to 10) Osmolality (mOsm/kg) Sodium (mmol/l) Potassium (mmol/l) Sodium/Potassium Glucose (mmol/l)
81.7k5.6 10.9f0.9 24.3 * 3.3 8.1 f0.4 266f 14 98.2 f 9.6 27.lk2.3 2.12kO.06 0.53+0.06
2
4
3 63.9* 12.9 7.81t0.6 17.9f 1.9 7.OkO.8 197+20 73.3* 10.7 19.2k2.0 2.23f0.12 0.26 f 0.04
4l.Of 12.5 4.5kO.7 18.9f2.4 5.1 f0.8 218? 18 82.0f 11.7 20.0f2.8 2.43f0.17 0.26 ? 0.02
33.24 12.2 13.14 1.1 253+21 96.Ok6.8 19.7+ 1.6 2.89kO.09 0.51+0.04
dependent variable. None of the other parameters contributed significantly to explain the remaining variation in fertilization rate. The fertilization rate and sperm density gradually decreased and the sodium/potassium ratio increased with repeated stripping during the spawning season (Table 3 ) . DISCUSSION
The average sperm density found by cell counting corresponds well with results reported by Scott and Baynes ( 1980) and Piironen and HyvHrinen ( 1983 ). Piironen and Hyvarinen ( 1983 ) found twice as much glucose in the seminal plasma in landlocked Atlantic salmon as found in this experiment (177.8 mg/l=0.99 mmol/l). For sperm density, osmolality, and glucose the repeatability was found to be 0.92 or higher. Therefore, it is sufficient to take one measurement per male. Sperm motility achieved a lower repeatability (r=0.72), indicating that the methodology for recording motility may be improved. The high correlation found between sperm density measured by spermato-
EVALUATION OF MILT QUALITY OF ATLANTIC SALMON
131
crit and by cell counting (r=0.92) is in agreement with Obraztsov ( 1985) who reported an even higher correlation (r=0.95-0.97) in rainbow trout. Therefore, little is to be gained by measuring both parameters. Spermatocrit is the less time-consuming method of the two, and also the easiest to use under practical conditions. Refstie ( 1986) recommended the use of at least 1 ml milt per liter eggs under practical conditions. From the observed average density this corresponds to 1.9 x 1O6sperm per egg (assuming 5000 eggs/liter). In these experiments, a high fertilization rate was observed with fewer sperm per egg. Therefore, the recommended 1 ml of milt per liter egg might be lowered. Of the parameters studied, sodium showed the highest correlation to fertilization rate. However, sodium content in seminal plasma explained a rather small proportion of the total variation. The remaining variation could not be explained by the other parameters measured in this experiment. This is possibly due to the rather high correlations between sodium and other parameters registered. In order to discriminate between milt of different qualities with higher accuracy, additional information.about milt quality is therefore required. The plot between fertilization rate and sodium (Fig. 3 ) might indicate a threshold value of around 87 mmol/l of sodium in seminal plasma for an adequate fertilization rate, and that a certain level of sodium in the seminal plasma is required for a high fertilization rate. In existing literature (Morisawa et al., 1983; Stoss, 1983; Cosson et al., 198 5 ), a relationship between fertilization ability and motility is often mentioned, but no figures are given. Stoss ( 1983) claimed that sperm motility is a valuable parameter when sperm cell viability is estimated, and he maintains that motility and fertilization ability are, in general, well correlated. Levanduski and Cloud ( 1988) found fertilization ability was reduced when the proportion of motile sperm was less than 10%. The non-significant relationship between sperm motility and fertilization rate observed in this experiment might be caused by the higher proportion of motile sperm, which was never less than 35%. Cosson et al. ( 1985 ) describe stroboscopy as a more objective way to measure motility. However, they did not correlate sperm motility to fertilization ability. Morisawa et al. ( 1983) examined the effect of potassium and osmolality on the motility of sperm cells of salmonids. Their results suggested that osmolality was not an important determinant of sperm motility in salmonids, and that sperm motility was suppressed by the seminal potassium in the sperm duct. Of the parameters measured, spermatocrit is the quickest and cheapest to carry out and is recommended for use under practical conditions. Sodium is
132
G.H. AAS ET AL.
a better measure of milt quality, but measurement requires analytical equipment. With repeated stripping, fertilization rate and sperm density decreased and sodium/potassium ratio increased. This would be expected, taking into account observed relationships between these parameters. However, no systematic decrease in osmolality was observed. It is worth noticing that sperm motility decreased with repeated stripping. Milt quality of males stripped late in the spawning season for the first time should therefore be investigated.
REFERENCES
AOAC Methods, 1980. Official Methods of Analysis of the Association of Official Analytic Chemists, 13th edn. W. Horwitz, (Editor), Association of Official Analytic Chemists, Washington, DC, 10 18 pp. Billard, R., 198 1. Short-term preservation of sperm under oxygen atmosphere in rainbow trout (Salmogairdneri). Aquaculture, 23: 287-293. Bilytikhatipoglu, S. and Holtz, W., 1978. Preservation of trout sperm in liquid or frozen state. Aquaculture, 14: 49-56. Cosson, M.P., Billard, R., Gatti, J.L. and Christen, R., 1985. Rapid and quantitative assessment of trout spermatozoa motility using stroboscopy. Aquaculture, 46: 7 l-75. Hwang, P.C. and Idler, D.R., 1969. A study of major cations, osmotic pressure, and pH in seminal components of Atlantic salmon. J. Fish. Res. Board Can., 26: 4 13-419. Levanduski, M.J. and Cloud, J.G., 1988. Rainbow trout (Salmo gairdneri) semen: effect of non-motile sperm on fertility. Aquaculture, 75: 17 1- 179. Morisawa, M., Suzuki, K. and Morisawa, S., 1983. Effects of potassium and osmolality on spermatozoan motility of salmonid fishes. J. Exp. Biol., 107: 105-l 13. Mounib, MS., 1978. Cryogenic preservation of fish and mammalian spermatozoa. J. Reprod. Fertil., 53: 13-18. Obraztsov, A.N., 1985. Estimation of sperm concentration in rainbow trout (Salmo gairdneri Rich.). Genetic and ecological implications in fish culture. pp. 11 l-l 16 (in Russian, with English summary). Piironen, J. and Hyvlrinen, H., 1983. Composition of the milt of some teleost fishes. J. Fish Biol., 22: 351-361. Refstie, T., 1986. Reproduksjon - livssyklus. In: T. Gjedrem (Editor), Fiskeoppdrett med Fremtid. Landbruksforlaget, Oslo, pp. 46-58. Rye, M., Lillevik, KM. and Gjerde, B., 1990. Survival in early life of Atlantic salmon and rainbow trout; estimates of heritabilities and of genetic correlations among survival traits and of genetic correlations between survival and growth. Aquaculture, 89: 209-2 16. Scott, A.P. and Baynes, S.M., 1980. A review of the biology, handling and storage of salmonid spermatozoa. J. Fish Biol., 17: 707-739. Stoss, J., 1983. Fish gamete preservation and spermatozoan physiology. In: W.S. Hoar, D.J. Randall and E.M. Donaldson (Editors), Fish Physiology, Vol. IXB., Academic Press, London, pp. 305-350. Stoss, J. and Holtz, W., 1983. Successful storage of chilled rainbow trout (Salmo gairdneri) spermatozoa for up to 34 days. Aquaculture, 3 1: 269-274. Ulgenes, Y., 1983. Miljra, oppbevaring, hartdtering og emaering av stamtisk. 24 August 1983 Fiskeoppdrett 83, 14 pp.
z4quaculture, 95 (1991)125-132 Elsevier Science Publishers B.V., Amsterdam
Evaluation of milt quality of Atlantic salmon Grete Hansen Aas, Terje Refstie and Bjarne Gjerde .4KVAFORSK, Institute ofAquaculture Research, P.O. Box IO, N-1432 .kNLH. (Accepted
Norway
10 October 1990)
ABSTRACT Aas, G.H., Refstie, T. and Gjerde, B., 1991. Evaluation of milt quality of Atlantic salmon. Aquaculture. 95: 125-132. Milt was collected individually from 27 Atlantic salmon, and sperm density, sperm motility, milt colour and osmolality, and content of sodium, potassium and glucose of seminal plasma recorded. Milt from each male fertilized samples of around 200 eggs taken from the same pool of eggs. Percentage of eyed-eggs was used as a measure of fertilization rate or milt quality. Large variation among males was observed for this trait. Variation among males for the recorded parameters was medium to high in magnitude. Correlation between sperm density registered by cell counting and spermatocrit was high (rc0.92). Repeatability waslowest for motility (0.72), and 0.92 or higher for other parameters. Of the parameters studied, sodium content in seminal plasma showed the highest correlation (r= 0.60) to fertilization rate. The remaining variation in fertilization rate could not be explained by any of the other parameters studied. However, all traits except for sperm motility and milt colour showed a significant correlation to fertilization rate. With repeated stripping within season, the sperm density gradually decreased and the sodium/ potassium ratio increased, resulting in decreasing fertilization rate.
INTRODUCTION
For farmed Atlantic salmon (Salvo saEar), hatching rate of eggs varies among different hatcheries (Ulgenes, 1983) and among batches within a hatchery (Rye et al., 1990). Milt quality is one of the factors which influence these results and can be delined as the ability to fertilize eggs. Several studies have described milt characteristics which can influence quality; for example, sperm density, sperm motility and the composition of the seminal plasma (Hwang and Idler, 1969; Mounib, 1978; Piironen and Hyvarinen, 1983; Stoss, 1983). However, few have correlated the milt characteristics to fertilizing ability. Some investigations of milt quality in connection with cryopreservation of sperm are reported (Hwang and Idler, 1969; Biiyiikhatipoglu and Holtz, 1978; Billard, 198 1; Stoss, 1983; Stoss and Holtz, 1983). 00448486/91/$03.50
0 199 1 -
Elsevier Science Publishers
B.V.
126
G.H. AAS ET AL.
Sperm density may influence fertilization rate. Sperm count and spermatocrit are two different measures of density. In rainbow trout Obraztsov ( 1985) found a correlation between sperm density and spermatocrit of 0.95-0.97. Results on rainbow trout sperm reported by Levanduski and Cloud ( 1988 ) suggested that the presence of non-motile sperm could reduce the fertility of normal motile sperm. However, this effect did not become significant until the concentration of motile sperm was less than 10%. Urine or*blood might follow the milt when the fish is stripped. This makes the milt slightly yellow or red, but further influence on fertilization is unknown. The aim of the present investigation were to ( 1) investigate variation in milt quality among males; (2) identify parameters correlated to fertilizing ability of sperm which can be used in evaluating milt quality before insemination; (3) investigate effects of repeated stripping within season on milt quality. MATERIALS
AND METHODS
Broodstock of Atlantic salmon were reared in seawater and transferred to brackish water three months before stripping at the Institute of Aquaculture’s Research, Sunndalsora. Males and females were selected at random and anaesthetized before stripping. Care was taken to avoid contamination of the milt and eggs with water or excreta. A pool of eggs from three to live females was used in each experiment. Milt was collected individually from 27 males and stored on ice in clean, dry plastic beakers until insemination. This always took place within 3 h after collection. Two different amounts of milt, corresponding to 1 ml and 0.5 ml milt per liter egg, were used to fertilize samples of about 200 eggs. After milt was added to the eggs, the eggs were stirred, and 15-30 s later water was added to complete the fertilization process. The eggs were then stirred once more. For each male and amount of milt, three parallels were used for fertilization. Altogether, there were six parallels per male. At the eyed-egg stage, eggs were shocked by shaking the tray, and the number of dead and live eggs were counted. The percentage of eyed-eggs was used as a measure of fertilization rate or milt quality. Mortality of embryo from fertilization to eyed-egg stage was treated as a constant, and, therefore, not recorded. Sperm density was evaluated using two different methods; first by measuring spermatocrit value using a microhematocrit centrifuge (Compur M 1100)) and secondly by counting sperm using Biirkers counting chamber after diluting the milt ( 1: 400 and 1: 800 depending on the spermatocrit value
EVALUATION OF MILT QUALITY OF ATLANTIC SALMON
127
of each sample). Counting chambers were always kept in a moist atmosphere for at least 15 min before cell counting. Motility of sperm in each sample was evaluated within two hours after collection. A droplet of milt was placed under a microscope and a droplet of freshwater was added before the percentage of motile sperm was calculated. Only forward moving sperm were judged to be motile, while vibrating sperm were judged to be immotile. A scale from 0 to 10 was used, where 0 and 10 respectively corresponded to 0% and 100% motile sperm. The colour of each sample of milt was judged visually. The three colours observed were white, pale red and pale yellow. Milt from each sample was centrifuged and the seminal plasma removed and frozen for further analysis. Osmotic pressure of seminal plasma was measured with an osmometer (Knauer, Semi-micro-osmometer ), the content of sodium and potassium with an atomabsorption spectrophotometer ( AOAC Methods ( 1980) Nr. 3.020-3.022), and the content of glucose with a spectrophotometer using a Mercotest 14335 Glucose-reagentkit. Repeatabilities of the recorded parameters were studied by analysing two milt samples from each male for cell counting, motility, osmolality and glucose, and three samples for spermatocrit. For each parameter, repeatability was calculated as the ratio between the variance component among fish and the total variance. A total of seven to nine males was stripped repeatedly every second week from 24 October to 4 December 1988 and altogether four times during the breeding season. The same operation as described previously was carried out. The effect of milt volume on fertilization was studied using a two-way nested analysis of variance including the effect of individual male, and the effect of milt volume within male as random effects in addition to a random error. Simple and multiple linear regressions were used to analyze the effect of each parameter on fertilization rate. In the simple analysis, a second degree polynomial was included in the model. RESULTS
Means and variation between males for the traits studied are given in Table 1. The fertilization results among males showed large variation ranging from 0.5% to 97.3%. The coefficient of variation (CV) was 19 or higher for all traits except for the sodium/potassium ratio which showed a medium CVvalue. Average number of sperm per egg was 6.2 x 10’ (s.d. = 3.1 x 10’) when 0.5 ml of milt per liter egg was used, and 12.6~ lo5 (sd. =6.4x 105) using 1.O ml of milt per liter egg. The calculated repeatabilities were 0.94 and 0.97 for sperm density registered by cell counting and spermatocrit, respectively; 0.72 for motility; 0.99 for osmolality and 0.92 for glucose. The correlation between the two mea-
G.H. AA.9 ET AL.
128 TABLE
1
Mean (X), standard deviation (s.d. ), coefficient of variation (CV), minimum (min) and maximum (max) values of traits. Computed statistics are based on mean values of from one to six samples per male Traits
N
Eyed eggs (O/O )
21
64.1
21 27
Sperm density counting ( X 109) spermatocrit (O/o) Sperm motility (score l-10) Seminal plasma Osmolality (mOsm/kg) Sodium (mmol/l ) Potassium (mmol/l) Sodium/Potassium Glucose (mmol/l)
5
15
Spermafocrlf
Fig. 1. Sperm density measured
s.d.
X
cv
min
max
33.6
_
0.5
91.3
9.54 23.4
3.2 8.0
34 -
3.5 9.1
11.9 45.0
21
6.8
1.6
24
3.5
9.5
25 27 21 21 27
245 92.8 21.5 1.97 0.45
55 23.9 5.3 0.28 0.15
22 26 19 14 33
111 33.0 14.6 1.33 0.23
320 133.9 31.0 2.51 0.90
25
35
value,
45
%
by cell count plotted against spermatocrit
value (n = 27).
sures of sperm density was high (r= 0.92 ). Observed values for each method are illustrated in Fig. 1. In the two-way nested analysis of variance, the effect of male was highly significant and explained 89.8% of the total variation in fertilization rate. The effect of milt volume within male was not significant (P> 0.05 ) and explained only 2.7% of the variation. The regression of fertilization rate on motility was not significant (P> 0.05 ) , nor the effect of milt colour on fertilization rate. The regression of fertilization rate on each parameter, except for sperm motility and milt colour, was significant and linear (P
129
EVALUATION OF MILT QUALITY OF ATLANTIC SALMON
non-significant second-degree polynomial. The correlation of fertilization rate with sperm density registered by cell counting and spermatocrit was 0.52 and 0.40 respectively, with osmolality 0.57; with sodium 0.60; with potassium 0.48; with sodium/potassium 0.52, and with glucose 0.42. The plots between fertilization rate and spermatocrit, and between fertilization rate and sodium, are illustrated in Figs. 2 and 3. The linear regression line is drawn in each figure. A similar plot was observed between fertilization rate and osmolality. Correlations among some of the parameters registered in seminal plasma are presented in Table 2. Applying a stepwise forward regression analysis to all parameters studied, sodium was the first parameter included in a model with fertilization rate as
30
20
Spermatocrrt
value,
%
Fig. 2. Spermatocrit value plotted against fertilization rate (n = 27 ).
~ &
100
-
T
d 80% UJ x
60-
L-3 91
-
‘;i<
40-
g -i: :: > Y
zo-
.
n
,
40gIt 30
50
Sodurn
,
I 70
In seminal
90
130
110
pfasma (mmol/l
)
Fig. 3. Sodium content in seminal plasma plotted against fertilization rate (n = 27 ).
G.H. AAS ET AL.
130 TABLE 2 Correlation coefficients among parameters
measured in seminal plasma
Trait
Potassium
Sodium/Potassium
Glucose
Osmolality
Sodium Potassium Sodium/Potassium Glucose
0.87
0.72 0.29
0.32 0.09 0.50
0.69 0.67 0.44 0.27
TABLE 3 Mean (with standard error) for parameters measured on seven to nine fish with four repeated strippings within season Traits
Stripping no. 1
Fertilization rate (%) Density registered by cell counting ( 109) Spermatocrit % Motility (scale from 0 to 10) Osmolality (mOsm/kg) Sodium (mmol/l) Potassium (mmol/l) Sodium/Potassium Glucose (mmol/l)
81.7k5.6 10.9f0.9 24.3 * 3.3 8.1 f0.4 266f 14 98.2 f 9.6 27.lk2.3 2.12kO.06 0.53+0.06
2
4
3 63.9* 12.9 7.81t0.6 17.9f 1.9 7.OkO.8 197+20 73.3* 10.7 19.2k2.0 2.23f0.12 0.26 f 0.04
4l.Of 12.5 4.5kO.7 18.9f2.4 5.1 f0.8 218? 18 82.0f 11.7 20.0f2.8 2.43f0.17 0.26 ? 0.02
33.24 12.2 13.14 1.1 253+21 96.Ok6.8 19.7+ 1.6 2.89kO.09 0.51+0.04
dependent variable. None of the other parameters contributed significantly to explain the remaining variation in fertilization rate. The fertilization rate and sperm density gradually decreased and the sodium/potassium ratio increased with repeated stripping during the spawning season (Table 3 ) . DISCUSSION
The average sperm density found by cell counting corresponds well with results reported by Scott and Baynes ( 1980) and Piironen and HyvHrinen ( 1983 ). Piironen and Hyvarinen ( 1983 ) found twice as much glucose in the seminal plasma in landlocked Atlantic salmon as found in this experiment (177.8 mg/l=0.99 mmol/l). For sperm density, osmolality, and glucose the repeatability was found to be 0.92 or higher. Therefore, it is sufficient to take one measurement per male. Sperm motility achieved a lower repeatability (r=0.72), indicating that the methodology for recording motility may be improved. The high correlation found between sperm density measured by spermato-
EVALUATION OF MILT QUALITY OF ATLANTIC SALMON
131
crit and by cell counting (r=0.92) is in agreement with Obraztsov ( 1985) who reported an even higher correlation (r=0.95-0.97) in rainbow trout. Therefore, little is to be gained by measuring both parameters. Spermatocrit is the less time-consuming method of the two, and also the easiest to use under practical conditions. Refstie ( 1986) recommended the use of at least 1 ml milt per liter eggs under practical conditions. From the observed average density this corresponds to 1.9 x 1O6sperm per egg (assuming 5000 eggs/liter). In these experiments, a high fertilization rate was observed with fewer sperm per egg. Therefore, the recommended 1 ml of milt per liter egg might be lowered. Of the parameters studied, sodium showed the highest correlation to fertilization rate. However, sodium content in seminal plasma explained a rather small proportion of the total variation. The remaining variation could not be explained by the other parameters measured in this experiment. This is possibly due to the rather high correlations between sodium and other parameters registered. In order to discriminate between milt of different qualities with higher accuracy, additional information.about milt quality is therefore required. The plot between fertilization rate and sodium (Fig. 3 ) might indicate a threshold value of around 87 mmol/l of sodium in seminal plasma for an adequate fertilization rate, and that a certain level of sodium in the seminal plasma is required for a high fertilization rate. In existing literature (Morisawa et al., 1983; Stoss, 1983; Cosson et al., 198 5 ), a relationship between fertilization ability and motility is often mentioned, but no figures are given. Stoss ( 1983) claimed that sperm motility is a valuable parameter when sperm cell viability is estimated, and he maintains that motility and fertilization ability are, in general, well correlated. Levanduski and Cloud ( 1988) found fertilization ability was reduced when the proportion of motile sperm was less than 10%. The non-significant relationship between sperm motility and fertilization rate observed in this experiment might be caused by the higher proportion of motile sperm, which was never less than 35%. Cosson et al. ( 1985 ) describe stroboscopy as a more objective way to measure motility. However, they did not correlate sperm motility to fertilization ability. Morisawa et al. ( 1983) examined the effect of potassium and osmolality on the motility of sperm cells of salmonids. Their results suggested that osmolality was not an important determinant of sperm motility in salmonids, and that sperm motility was suppressed by the seminal potassium in the sperm duct. Of the parameters measured, spermatocrit is the quickest and cheapest to carry out and is recommended for use under practical conditions. Sodium is
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a better measure of milt quality, but measurement requires analytical equipment. With repeated stripping, fertilization rate and sperm density decreased and sodium/potassium ratio increased. This would be expected, taking into account observed relationships between these parameters. However, no systematic decrease in osmolality was observed. It is worth noticing that sperm motility decreased with repeated stripping. Milt quality of males stripped late in the spawning season for the first time should therefore be investigated.
REFERENCES
AOAC Methods, 1980. Official Methods of Analysis of the Association of Official Analytic Chemists, 13th edn. W. Horwitz, (Editor), Association of Official Analytic Chemists, Washington, DC, 10 18 pp. Billard, R., 198 1. Short-term preservation of sperm under oxygen atmosphere in rainbow trout (Salmogairdneri). Aquaculture, 23: 287-293. Bilytikhatipoglu, S. and Holtz, W., 1978. Preservation of trout sperm in liquid or frozen state. Aquaculture, 14: 49-56. Cosson, M.P., Billard, R., Gatti, J.L. and Christen, R., 1985. Rapid and quantitative assessment of trout spermatozoa motility using stroboscopy. Aquaculture, 46: 7 l-75. Hwang, P.C. and Idler, D.R., 1969. A study of major cations, osmotic pressure, and pH in seminal components of Atlantic salmon. J. Fish. Res. Board Can., 26: 4 13-419. Levanduski, M.J. and Cloud, J.G., 1988. Rainbow trout (Salmo gairdneri) semen: effect of non-motile sperm on fertility. Aquaculture, 75: 17 1- 179. Morisawa, M., Suzuki, K. and Morisawa, S., 1983. Effects of potassium and osmolality on spermatozoan motility of salmonid fishes. J. Exp. Biol., 107: 105-l 13. Mounib, MS., 1978. Cryogenic preservation of fish and mammalian spermatozoa. J. Reprod. Fertil., 53: 13-18. Obraztsov, A.N., 1985. Estimation of sperm concentration in rainbow trout (Salmo gairdneri Rich.). Genetic and ecological implications in fish culture. pp. 11 l-l 16 (in Russian, with English summary). Piironen, J. and Hyvlrinen, H., 1983. Composition of the milt of some teleost fishes. J. Fish Biol., 22: 351-361. Refstie, T., 1986. Reproduksjon - livssyklus. In: T. Gjedrem (Editor), Fiskeoppdrett med Fremtid. Landbruksforlaget, Oslo, pp. 46-58. Rye, M., Lillevik, KM. and Gjerde, B., 1990. Survival in early life of Atlantic salmon and rainbow trout; estimates of heritabilities and of genetic correlations among survival traits and of genetic correlations between survival and growth. Aquaculture, 89: 209-2 16. Scott, A.P. and Baynes, S.M., 1980. A review of the biology, handling and storage of salmonid spermatozoa. J. Fish Biol., 17: 707-739. Stoss, J., 1983. Fish gamete preservation and spermatozoan physiology. In: W.S. Hoar, D.J. Randall and E.M. Donaldson (Editors), Fish Physiology, Vol. IXB., Academic Press, London, pp. 305-350. Stoss, J. and Holtz, W., 1983. Successful storage of chilled rainbow trout (Salmo gairdneri) spermatozoa for up to 34 days. Aquaculture, 3 1: 269-274. Ulgenes, Y., 1983. Miljra, oppbevaring, hartdtering og emaering av stamtisk. 24 August 1983 Fiskeoppdrett 83, 14 pp.