VALUES OF HAEMATOLOGICAL INDICES OF WELS (Silurus glanis L.) IN RELATIONSHIP TO THE LEVEL OF NUTRITION DURING THE PRE-SPAWNING PERIOD

Svobouod, Z., 1. Kolarova. H. Modra, V. Vajcova. J. Hamackova J. Koufil, P. Kozak: Values of HaemalOlogical Indices of Wels (SiluTlls glanis L.) in Relationship to the Level ofNllIrition during the Pre-spawning Period. Acta vet. Brno 1998,67: 235-242. The goal of this paper was to evaluate the effect of different nutrition levels on condition and health expressed by means of selected haematological indices of both males and females of wels (Silurus glanis L.) during the pre-spawning period. For 2 months of the pre-spawning period. brood wels were separated into groups reared similarly but with different level of nutrition. The ratio of fodder fish biomass: wels biomass in 1995 was 3.2: 1 in male group A; 1.1: 1 in male group B; 2.6: 1 in female group C and 0.9: I in female group D; while in 1997 this ratio was kept 2.3: 1. 0.7: I and 0.2: I in female groups A, Band C, respectively. Prior to the anticipated reproduction. haematological examination considered red blood count indices (red blood count RBC, haematocrit PCV, haemoglobin Hb. mean corpuscular volume MCV. mean corpuscular haemoglobin MCH. mean corpuscular haemoglobin concentration MCHC, erythrocyte sedimentation rate) and white blood count indices (leukocyte count Leuko, leukogramme), along with concentration of total protein in blood plasma (TP). Altogether. 15 males and females were checked in 1995 while 18 females were checked in 1997. Both males and females which had been reared during the 1995 and/or 1997 pre-spawning period with higher level of nutrition reached expressively higher relative weight gain and signiticantly higher total protein (TP) in blood plasma (P < 0.05: P < 0.0 I) compared to those with lower level of nutrition. Moreover. significantly higher pev. Hb and MCH (P < 0.05) were found in group of females with higher nutrition level in 1995. compared to those with a lower one. A significantly higher percentage of lymphocytes (P < 0.05 J. as well as a lower percentage of granulocytes and their developmental forms (P < 0.05) were found in a group of females with higher nutrition level in 1997 compared to those with lower nutrition levels. No significant differences were found in other haematological indices among the respective groups of brood wels. Results gained report for a more favourable condition of brood wels reared at higher nutrition level (ratio of fodder fish biomass: wels biomass 2.3: I 3.2: I) during the pre-spawning period and a higher non-specific disease resistance may be anticipated also. Relationships between nutrition level and values of production, haematological and reproductive characters of the fish under study were discussed. COlllplete red blood COllllt. cOlllplere white blood count. total plasllla protein. males. females Both the rearing and the nutrition level of fish during their pre-spawning period are important factors affecting quality of gametes and results of reproduction. Nevertheless, there are only sporadical references to the topic on individual fish species. The effect of nutrition level of reproductive traits were observed by V ach ta et al. (1992) in tench, by Springate et al. (1985) in rainbow trout. Reproduction is considered among important physiological stress of the fish organism (Jeney and Jeney 1992; Svobodova et al. Address for correspondence: Doc. MVDr. Zd~flka Svobodova. DrSc. Research Institute of Fi~h Culture and Hydrobiolog~ University of South Bohemia. Vodnany Zatisi nS/H.389 25 Vodnan). Czech Rt:publil: Phone: +.+20 3.+2 382.+02 El,'C +.+2.0342 382396 E-mail: tl:lJ:;hans@\"urh.cju.cz

The goal of this paper was to evaluate the effect of different nutrition levels on condition and health expressed by means of selected haematological indices of both males and females of wels (Silurus glanis L.) during the pre-spawning period.For 2 months of the pre-spawning period.brood wels were separated into groups reared similarly but with different level of nutrition.The ratio of fodder fish biomass: wels biomass in 1995 was 3.2: 1 in male group A; 1.1: 1 in male group B; 2.6: 1 in female group C and 0.9: I in female group D; while in 1997 this ratio was kept 2.3: 1. 0.7: I and 0.2: I in female groups A, Band C, respectively.
Prior to the anticipated reproduction.haematological examination considered red blood count indices (red blood count -RBC, haematocrit -PCV, haemoglobin -Hb.mean corpuscular volume -MCV.mean corpuscular haemoglobin -MCH.mean corpuscular haemoglobin concentration -MCHC, erythrocyte sedimentation rate) and white blood count indices (leukocyte count -Leuko, leukogramme), along with concentration of total protein in blood plasma (TP).Altogether.15 males and females were checked in 1995 while 18 females were checked in 1997.
Both males and females which had been reared during the 1995 and/or 1997 pre-spawning period with higher level of nutrition reached expressively higher relative weight gain and signiticantly higher total protein (TP) in blood plasma (P < 0.05: P < 0.0 I) compared to those with lower level of nutrition.
Moreover.significantly higher pev.Hb and MCH (P < 0.05) were found in group of females with higher nutrition level in 1995.compared to those with a lower one.A significantly higher percentage of lymphocytes (P < 0.05 J. as well as a lower percentage of granulocytes and their developmental forms (P < 0.05) were found in a group of females with higher nutrition level in 1997 compared to those with lower nutrition levels.No significant differences were found in other haematological indices among the respective groups of brood wels.Results gained report for a more favourable condition of brood wels reared at higher nutrition level (ratio of fodder fish biomass: wels biomass 2.3: I -3.2:I) during the pre-spawning period and a higher non-specific disease resistance may be anticipated also.Relationships between nutrition level and values of production, haematological and reproductive characters of the fish under study were discussed.

COlllplete red blood COllllt. cOlllplere white blood count. total plasllla protein. males. females
Both the rearing and the nutrition level of fish during their pre-spawning period are important factors affecting quality of gametes and results of reproduction.Nevertheless, there are only sporadical references to the topic on individual fish species.The effect of nutrition level of reproductive traits were observed by V ach ta et al. (1992) in tench, by Springate et al. (1985) in rainbow trout.Reproduction is considered among important physiological stress of the fish organism (Jeney and Jeney 1992;Svobodova et al. 1997).During artificial reproduction, the physiological stress is multiplied by stress of handling.Organisms in good conditions can adapt better to this stress.
The goal of experiments performed in 1995 and repeated in 1997 was to evaluate the effect of different nutrition le\'els on productive and reproductive traits, condition and health of wels (Silurus glanis L.) during the pre-spawning period.As the brood stock is very valuable and kept in limited number, altogether 15 fish in 1995 and 18 fish in 1997 were studied.We report in this study the results of evaluation of the effect of different nutrition levels on condition and health expressed by means of selected haematological indices of both males and females of wels during the pre-spav,:ning period.Relationships between values of haematological, production and reproducti ve traits of the fish under study are also discussed.
In the 1995 experiment, the brood wels were separated into the foJlowing 4 groups and stocked on April 24 into experimental ponds: group A -males with higher nutrition level (stocking biomass ratio of fodder fish: brood stock 3.2: I) group B -males with lower nutrition level (1.1:I ) group C -females with higher nutrition level (2.6: I) group D -females with lower nutrition level (0.9: I) Prior to the anticipated reproduction.i. e. June 26 and 29.fish were caught, identified.anaesthetized with Menocain.weighed and sampled for blood.Samples were taken from 15 specimen (5 males of groupA, 4 males of group B, 3 females of group C and 3 females of group D).
The 1997 experiment was carried out on females only and they were separated into 3 groups as followed: group A -females with higher nutrition level (stocking biomass ratio of fodder fish: broodstock 2.3:1) group B -females with lower nutrition level (0.7: I) group C -females with very low nutrition level (0.2: I) Fish were stocked on April 9. Prior to the anticipated reproduction, i. e. June 19, fish were caught.identified, anaesthetized with Menocain, weighed and the relative weight gain per day (%.d•! ) was calculated.Blood samples were taken from 18 specimen in total, i. e. 3 groups of 6 fish each.
Blood was sampled from a. er 1'.caudalis.Heparin in amount of 50 IV sodium heparin per I ml blood was used for stabilization.Samples were used for determination of red blood count (RBC), haematocrit (PCV), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), leukocyte count (Leuko), leukogramme and concentration of total protein in blood plasma (TP).Moreover, both male and female samples checked in 1995 were used to determine erythrocyte sedimentation rate.Methods foJlowed the Vnified Methods of Haematological Examination ofFish by S v 0 bod 0 v a et al. (1991 ).
The concentration of total protein in blood plasma was determined refractometricaJly.Results were evaluated using t-test in the STAT Plus software by VUVeL Bmo.

Results
A remarkably lower relative weight gain (0.050 and 0.079%•d-1 ) was recorded for males and females, respectively, kept during the 1995 pre-spawning period at a lower nutrition level compared to those kept at the higher one (0.182 and 0.253%•d-1 for males and females, respectively).Results ofhaematological indices of brood males and females of we Is kept at differing nutrition levels are given in Tables 1 and 2. For both males and females at the higher nutrition level, higher total protein concentrations in blood plasma (40.4 ± 6. IO and 38.5 ± 12.3 g'l-l, respectively) were recorded compared to those from the lower nutrition level (26.8 ± 5.28 and 27.6 ± 1.27 g'l-!, respectively; Fig. 1).This difference was found significant for males (P < 0.05).Moreover, significantly higher PCV, Hb and MCH (P < 0.05) were found in group of females with higher nutrition level compared to those with a lower one.No significant differences were found in other red and white blood indices among the compared groups of brood wels.The 1995 data were used as well to assess a sex-related difference in values of haematological indices.Within the fish kept at higher nutrition level.significantly higher both MCHC and MCH (P < 0.05)were found in females compared to males.Similarly, a significantly higher MCH of females (P < 0.0 I) compared to that of males was found in the fish kept at lower nutrition level.Values of erythrocyte sedimentation rate of males and females of all groups under study are given in Fig. 2. In all cases.higher sedimentation rate was found for females compared to males.This difference was significant for fish kept at lower nutrition level within the 20 min time limit (P < 0.05).reaching 22.1 ± 4.28 % for males and 32.7 ± 2.72 % for females.
In the 1997 pilot experiments, the highest relative weight gain (0.119 %'d-!) was recorded for female group A reared at higher nutrition level, followed by female group B at  Results of haematological investigation of wels females kept at three levels of nutrition are given in Table 3 and Fig with nutrition level were found for total protein concentration in blood plasma (TP).The highest TP was registered for female group A with higher nutrition level (33.1 ±4.92 g•l-I).the lowest one for female group C of the lowest nutrition level (24.1 ± 3.19g•I-I ).This difference was found significant between A and C groups, as well as between A and B group, the latter with lower nutrition level (27.0 ± 2.40 g'l-I) (P < 0.05).A significantly lower mean corpuscular volume (MCV) was found in female group A of higher nutrition level compared to Band C groups (P < 0.05).

Significantly
higher percentage of lymphocytes (P < 0.05) was registered for A female group compared to B female group of lower nutrition level.Similarly, the female A group was found to have a lower percentage of granulocytes and their developmental forms (myelocytes, metamyelocytes, neutrophile granulocytes -band forms and segmented forms) compared to B (P < 0.01) and C (P < 0.05) groups.No significant differences were found in other indices of red and white blood count among the respective groups of female wels.

Discussion
An important period of brood fish preparation for reproduction starts after overwintering.In case of brood wels kept in ponds for artificial reproduction, the pre-spawning period is considered extremely important for reproductive results, and above all from the point of view of fish recovering after reproduction.The cause is mainly in the character of changes of gonadosomatic index.In wels females, it has a sheer-growing course in the last one or one-and-half month prior to spawning (Kouril et aL 1981).Rearing of the respective groups of \liels males and females in ponds was performed and evaluated in spring 1995 and repeatedly in spring 1997.All groups were kept under the same conditions with the only difference being in nutrition leveL This was reflected in the daily relative weight gain, i.e. the higher nutrition level (higher ratio of fodder fish biomass: broodstock biomass), the higher was the daily relative weight gain.With a group of females kept at a very low nutrition level (0.2: I), there was even a negative relative weight gain.
Apart from these production traits.the broodfish condition and health \vas checked by means of haematological indices at the end of the pre-spawning period, i. e. prior to the anticipated reproduction.The concentration of total protein in blood plasma is considered one of the most important indices for assessment of condition and health of fish in feeding experiments (Svobodova and Parova 1977: Jirasek et al. 1993: Kolarova et al. 1998).Our evaluation of wels males and females kept at different nutrition levels also showed significant differences in total protein concentration in blood plasma.with the benefit for groups kept at higher nutrition level.It was proved repeatedly in 1995 for males and in 1997 for females.Values of other haematological indices also evidenced a better condition and health of brood wels kept at higher nutrition level during the pre-spawning period.This can be exemplified on significantly higher values of haematocrit.haemoglobin concentration and mean corpuscular haemoglobin in a group of females with higher nutrition level compared to those with lower nutrition level at the end of 1995 pre-spawning period.As a significantly higher percentage of lymphocytes and a lower percentage of granulocytes and their developmental forms were found in a group of females with higher nutrition level in 1997 compared to those with lower nutrition levels, this group may be considered having a higher non-specific resistance.
The data reported and discussed in this study evidence unambiguously for more favourable condition and health of wels reared at higher nutrition level compared to those kept at lower nutrition level.Despite of these facts, no differences in female reproductive traits of the respective groups were proven repeatedly, e. g. relative fecundity and mean weight of I egg did not differ significantly within the two (1995) or three (1997) different nutrition levels (Kouril eta!. 1996;HamaCkova etaI.1998). Hamackova etal. (1998) stated that the gonad weight of wels females kept at three various nutrition levels during the pre-spawning period did not increase in consequence of food intake only but also due to utilization of supply substances.In contrary, it was proven for male group with the higher nutrition level in 1995 that there was a highly significant (P < 0.01) difference in relative count of stripped spermatozoa (K 0 u i 'i I et al. 1996).
Though the number offish analyzed in 1995 was very low, we tried to compare the values of haematological indices of wels males and females.Higher erythrocyte sedimentation rate in all groups of females compared to males was in accordance with data in the literature (Sabioncello et al. 1969;Svobodova et al. 1981;Habekovic 1987).A significant difference in erythrocyte sedimentation rate between males and females of groups at lower nutrition level is also worth of mention.This difference was significant (P < 0.01) within the 20 min limit (males 22.1 ± 4.28%, females 32.7 ± 2.72%).
Fig. 3. Total protein concentration in blood plasma of Silurus giallis L. females reared at different nutrition levels during the pre•spawning period (June 19, 1997).A -higher nutrition level, B -lower nutrition level, C -very low nutrition level.

Table I
Haematological indices of Silurus glallis L. males reared at different nutrition levels during the pre-spawning period(June 26 and 29, 1995) Haematological indices of Silurlts glallis L. females reared at different nutrition levels during the pre-spawning period(June 26 and 29,1995)