Division of Physiology, University of Helsinki
Finnish Game and Fisheries Research Institute
The physiological effects of smolt transport were studied in connexion with comparative stockings of two-year-old Baltic salmon. Samples were taken just before transport from four hatcheries and after a five-day recovery at the stocking site. Physiological parameters describing the blood oxygen carrying capacity, energy reserves, and osmotic and ionic balance were determined.
Fish were transported from the fish farms under identical circumstances in regard to transport tanks, fish densities, and transport time. Continuous oxygenation was supplied in the transport tanks, and salt added to the transport water to give a salinity of circa 4 ‰. The experimental fish were allowed to recover for one week in small net pens in fresh water at the stocking site.
In all of the transport groups, the blood haematocrit value and haemoglobin concentration were lower following transport. The mean cellular haemoglobin content increased, however, with very high variation between individual fishes. The plasma glucose concentration, liver glycogen content, and muscle lipid content were reduced. The magnitude of this decrease differed considerably between the groups. The muscle water content decreased, but this decrease was statistically significant only in one of the groups. Plasma osmolality was not altered. However, a significant drop in plasma chloride and magnesium concentration was seen in one of the groups. The plasma protein concentration increased significantly in two of the groups.
The significance of these physiological changes is discussed, with special attention to the relation between smoltification and transport effects.
Cette étude porte sur les effets physiologiques du transport sur des saumons de la Baltique de deux ans. Des échantillons ont été prélevés dans quatre établissements piscicoles juste avant le transport et après cinq jours de récupération sur le site de repeuplement. On a déterminé les paramètres physiologiques relatifs à la capacité de transport de l'oxygène par le sand, aux réserves énergétiques et à l'équilibre osmotique et ionique.
Les conditions de transport sont restées identiques en ce qui concerne les bacs utilisés, la densité des tacons et la durée de l'opération. L'eau du bacs était oxygénée en permanence et avait une salinité de l'ordre de 5/1 000. Sur le site de repeuplement, on a laissé les poissons récupérer pendant une semaine dans de petits enclos en eau douce.
On a constaté, dans tous les groupes, une diminution de la valeur de l'hématocrite et de la concentration d'hémoglobine dans le sang. Toutefois, la teneur globulaire moyenne en hémoglobine avait augmenté, avec de très fortes variations d'un individu à l'autre. La concentration de glucose dans le plasma la teneur du foie en glycogène et la teneur des muscles en lipides avaient diminué. L'importance de cette diminution variait dans de très fortes proportions d'un groupe à l'autre. La teneur des muscles en eau avait elle aussi baissé mais cette baisse n'avait de valeur statistique que dans un seul groupe. L'osmolarité du plasma est restée inchangée. On a constaté une nette diminution de la teneur du plasma en chlorure et en magnésium dans l'un des groupes. La teneur du plasma en protéines s'était nettement accrue dans deux groupes.
Les auteurs examinent ces variations physiologiques et leurs effets sur le repeuplement.
In Finland the fish farms producing salmon smolts are mainly situated far away from the stocking sites on the coastline. During loading and transport (up to 500 km or 12 h) from the inland farms the fish are exposed to severe stress, the effects of which are not very thoroughly known.
The smolts seem to be very sensitive to handling. A medication treatment induces osmoregulatory imbalance (Bouck and Johnson, 1979) and even a slight descaling reduces the survival of coho salmon smolts in seawater (Bouck and Smith, 1979). During a routine transport the plasma cortisol, glucose and lactate concentrations increase and the liver glycogen content decreases (Wedemeyer, 1972; Specker and Schreck, 1980; Nikinmaa et al., 1981; ma) indicating a severe stress These disturbances are, at least in freshwater transport, associated with a lowered ionic concentration in plasma of coho salmon, rainbow trout and brown trout smolts (Aldrin, Mesager and Mevel, 1979; Soivio and Nikinmaa, 1981; Nikinmaa et al., 1982).
The physiology of juvenile salmon is greatly altered during parr-smolt transformation. Due to the differences between the physiological condition and stage of smoltification the stress responses and ability to recover from transport stress may greatly vary between separate fish groups during a short time interval.
Because the physiological status of the fish at the moment of stocking evidently affects the survival of the released smolts a project was started to clarify the optimal moment of parr-smolt transformation for transporting the smolts. In this study the physiological responses of fish with varying physiological background, owing to the evident differences in the smoltification stage, were investigated.
The oxygen carrying capacity of the blood was evaluated from the haemoglobin concentration (Hb) and haematocrit value (Hct) of the blood. Plasma glucose and lactate concentration were analysed to detect the general stress responses and smoltification stage of the fish. To judge the energy storages the tissue samples were analysed for glycogen (liver) and total lipid (muscle) concentration. The diagnosis of the osmo-regulatory status of the fish was based on the water content of the muscle, the ionic and protein concentrations and the osmolality of the plasma.
Two-year old Baltic salmon (Salmo salar) from the stock of River Neva were used in the experiments. They were hatched from the eggs of a damfish population reared at Laukaa Fish Culture Research Station (LFCRS). The eggs were transferred to three private fish farms (Hanka Taimen Ltd., Nilakkalohi Ltd. and Saimaan Lohi Ltd.) at the eye spot stage. For the second year the fish were reared in outdoor earth-bottom ponds in all fish farms except LFCRS, where they were kept in 7 × 7 m2 concrete basins. They were reared in fresh water at natural temperatures fluctuating seasonally between near 0 and 20°C. All the groups were fed with pelletted dry salmon food with seasonally varying rations (Westman et al., 1982).
The fish were transported for stocking on 21 May, within the normal releasing time in southern Finland. Fish were starved for two days before transport. With a hand net 140 kg of fish were loaded into each transport tank (Ewos) with a volume of 2 400 1 (60 g fish/1 water). Salt (NaCl) was added to transport water in a concentration of 4 ppt (65–70 mmol/l NaCl). Continuous oxygenation was supplied to the tanks. The transport time was adjusted to 8 h for each group irrespectively of the transportation distance. Water temperature at the start of transport was 4.2–6.0°C and at unloading 12.0°C.
On the stocking site, in the estuary of the River Kymi (fresh water) 150 fish from each transport group were transferred in a 70-1 basin from the tank into a 1 m3 net cage, situated 20 m from the shore in 15 minutes. The fish were allowed to recover for five days in the net cages. During this time the fish were fed with pelletted salmon food twice a day. Because of the heavy spring flood the water salinity was 0 ‰.
Blood and tissue samples were taken from each stocking group before the transport (pre-transport samples) and after the five-day recovery period on the stocking site (post-transport samples). Water temperatures at samplings are shown in Table 1. A day before sampling, the fish were placed in individual restrainers made of black plastic tubing (cf. Soivio and Virtanen, 1980). For sampling, the fish were immobilized with a sharp blow on the head. Blood was drawn from the caudal vessels in heparinized tuberculine syringes and analysed immediately for haemoglobin concentration (Hb) and haematoctic value (Hct) with cyanmethaemoglobin method. The rest of the blood was centrifuged within 3 minutes and the separated plasma was analysed for glucose concentration with Boehringer test kit No. 124 842, plasma chloride (Cl) concentration with Radiometer CMT 10 chloride titrator, plasma magnesium (Mg2+) concentration with Wako test kit No. 273–32809, plasma total protein concentration with Biuret reaction and plasma osmolality with Wescor vapour osmometer.
For tissue determination small pieces of liver and white muscle were taken, white muscle always from the same place beneath the back fin. The glycogen content of liver was determined according to Harris et al. (1974). The total lipid of the muscle was determined with Boehringer test kit No. 124 303 from a 50-mg piece of muscle. To determine the water content a 100-mg piece of white muscle was dried to constant weight at 105°C.
The mean corpuscular haemoglobin concentration (MCHC) was calculated by dividing the Hb concentration with the Hct value. The condition factor (CF) was calculated from the formula
Body silvering was determined visually on a scale 0–4 (4 = completely silvered, 0 = typical parr colouring).
The hypo-osmoregulatory capacity of the fish was determined prior to transport by exposing ten specimens from each group to artificial sea water (salinity 28‰). The exposures were made in static conditions with continuous aeration in 200 1 water volume. After 48 h exposure, blood and tissue samples were collected for ion and water balance determinations (Table 2). Restrainers were not used in these experiments.
The physiological values determined from the pre-transport samples and the effects of seawater exposure are shown in Table 2. The values in Table 3 are for post-transport fish and differences from the pre-transport values are also given
The stage of smoltification in juvenile salmon can be determined from several physiological indices, e.g., the body silvering, hypo-osmotic regulatory capacity, ion and water balance in fresh water, and altered energy metabolism (Wedemeyer et al., 1980, Virtanen et al., 1981). In the stocking groups investigated, there were significant differences in these properties, indicating that the groups were at different stages of smoltification.
In the group from Hanka-Taimen fish farm, a well developed body silvering, high oxygen carrying capacity, good capacity to regulate body water and ions in sea water and reduced glycogen and lipid storages indicate a good migratory readiness. On the other hand, the group from Saimaan Lohi fish farm was characterized by more distinctive parr marks, weaker hypo-osmoregulatory capacity and higher glycogen and lipid reserves. This, together with the decreased concentration of plasma protein and chloride, indicate that this group was at early stages in smoltification. The two other groups (LFCRS, Nilakkalohi) were, according to most of the parameters measured, intermediary in relation to the groups from Hanaka-Taimen and Saimaan Lohi.
It is important to notice that a high migratory readiness as expressed with well developed body silvering and hypo-osmoregulatory capacity is associated with reduced glycogen and lipid stores (cf. Farmer, Ritter and Ashfield, 1978; Woo, Bern and Nishioka, 1978). On the other hand, sufficient energy reserves are necessary for fish to sustain the stresses connected with transport and stocking. Farmer, Ritter and Ashfield (1978) pointed out that it is recommendable to release smolts at the beginning of their migratory period to ensure higher energy reserves at that time.
3.2.1 Blood oxygen carrying capacity
When comparing the pre- and post-transport situation, the most pronounced changes were a decrease (15–23 percent) in blood Hct. Due to a smaller reduction in blood haemoglobin (Hb) concentration the MCHC was increased which indicates changes in red cell volume (RCV). During stress and hypoxia the erythrocytes swell, which increases their oxygen affinity (Soivio and Nikinmaa, 1981a). On the other hand, we have evidence that changes in plasma osmolality of Baltic salmon cause changes in RCV. However, no significant changes in plasma osmolality were seen in this study. Nikinmaa et al. (1982) have shown a similar decrease in blood Hb concentration and MCHC in brown trout at the end of the week's recovery from a 14 h transport in salt water (6 ‰). The reason for this shrinking of erythrocytes remains an open question.
Nikinmaa, Soivio and Railo (1981) observed temperature dependence of Hb concentration in rainbow trout. So, part of the decrease seen in Hb concentration may be due to the temperature increase (9.5–8.3°C) during the transport and the recovery period.
In all the high variation in MCHC values indicates that no balance in RCV regulation had been achieved.
3.2.2 Energy metabolism
The great differences in plasma glucose levels between the groups prior to the transport had almost disappeared at the stocking site. In the group with the most pronounced hyperglycemia (Saimann Lohi) the plasma glucose concentration had reduced to 25 percent of its original level, and in two other groups the reduction was over 50 percent. However, in the group with originally lowest glucose level (Hanka-Taiman), there was no significant decrease and the post-transport values of this group were the highest.
The liver glycogen content decreased by 25–59 percent. The decrease was greatest in the group with originally highest glycogen reserves (Saimaan Lohi) so that the post-transport values did not significantly differ between the groups.
Plasma lactate concentrations were still clearly elevated in spite of the five days' recovery period in three of the groups but not in the group from Saimaan Lohi fish farm. The muscle lipid content on the other hand, was significantly affected (a decrease of 29 percent) only in the group from Saimaan Lohi, which had the highest lipid and glycogen reserves prior to transport.
The condition factor was not significantly changed during transport.
Reduction of energy reserves is a typical phenomenon in continuous stress, and it also takes place in transport (Hyvärinen et al., 1977; Nikinmaa, et al., 1982). Too low energy reserves may be a reason for post-transport mortality (Hyvärinen et al., 1977). In this study, the post-transport levels of blood glucose and liver glycogen were still on a lowered level in spite of five days' recovery with feeding. However, the energy balance of the transported fish cannot be considered to be extremely critical, because the glycogen reserves were not empty, and muscle lipid was only slightly reduced. The relatively moderate effect of transport on the energy reserves is possibly due to the following factors:
The fish were transported in salt water which decreases the energy costs of osmoregulation and stress symptoms associated with transport Wedemeyer, 1972; Long, McComas and Monk, 1977, Nikinmaa et al., 1982).
The fish were fed during recovery, and thus allowed to somewhat restore their energy reserves.
The changes in glucose, glycogen and lipid levels were clearly associated with the start situation. In the group from Saimaan Lohi, the most pronounced decrease in these values can be accounted partly for metabolic changes connected with delayed smoltification. Although the fish in this group lost their energy reserves more than those in the other groups, their plasma lactate concentration was not elevated. This indicates that fish transported at early stages of smoltification recover better from transport stress. However lactate was the only indication to support this hypothesis.
3.2.3 Ionic and osmotic balance
The changes in osmotic and ionic balance were insignificant or quite small in all of the groups. No indices of impaired osmotic balance, often connected with transport in fresh water (Wedemeyer, 1972; Nikinmaa et al., 1982), were seen in the physiological state of transported fish. This again can partly be accounted for by saltwater transport, which decreases osmotic stress, and partly by feeding during the recovery period, which increases the energy available for osmotic work. It is evident that the changes in the concentrations of plasma protein and ions are due to the proceeding of smoltification, rather than the effects of transport. Virtanen (unpubl.) has found a temporary decrease in plasma concentrations of protein and certain ions during smoltification.
The results indicate that the physiological status of smolts is still somewhat unbalanced five days after transport. This may be a significant reason for high post-release mortality of the reared smolts compared with the natural ones (Toivonen, 1977). In order to reduce delayed mortality, attention should be paid to the following factors:
Fish to be released must have an optimal physiological condition and stage of smoltification.
Transport stress must be minimized, e.g., by decreasing the handling of fish, using sufficiently low fish densities and adding salt to transport water.
Fish must be allowed to recover from transport at the stocking site. A recovery time of more than a week is recommendable, and fish must be fed during this time.
Aldrin, J.F., J.L. Mesager and M. Mevel, 1979 Essai sur le stress de transport chez le salmon coho juvenile (Oncorhynchus kisutch). Aquaculture, 17(4):279–89
Bouck, G.R., and D.A. Johnson, 1979 Medication inhibits tolerance to seawater in coho salmon smolts. Trans.Am.Fish.Soc., 1–8:63–6
Bouck, G.R., and S.D. Smith, 1979 Mortality of experimentally descaled smolts of coho salmon (Oncorhynchus kisutch) in fresh and salt water. Trans.Am.Fish.Soc., 108:67–9
Coetzee, N. and J. Hattingh. 1976 Effects of sodium chloride on the freshwater fish Labeo capensis during and after transport. Zool.Afr., 12:244–7
Farmer, G.L., J.A. Ritter and D. Ashfield, 1978 Seawater adaptation and parr-smolt transformation of juvenile Atlantic salmon, Salmo salar. J.Fish.Res.Board Can., 35(1):93–100
Harris, R.C. et al., 1974 Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris in man at rest. Methods and variance of values. Scand.J.Clin.Lab.Invest., 33:109–20
Hyvarinen, H. et al., 1977 Jarvilohen is jarvitaimenen istutusstressi ja sen merkitys istutuksen onnistumiselle. Suomen Kalastuslehti, 84:116–20
Long, C.W., J.R. McComas and B.H. Monk, 1977 Use of salt (NaCl) water to reduce mortality of chinook salmon (Oncorhynchus tschawytscha) during handling and hauling. Mar.Fish.Rev., 39(7):6–9
Nikinmaa, M., A. Soivio and E. Railo, 1981 Blood volume of Salmo gairdneri: influence of ambient temperature. Comp.Biochem.Physiol.(A Comp.Physiol.), 69:767–9
Nikinmaa, M. et al., 1982 Hauling stress in brown trout (Salmo trutta). Physiological responses to transport in fresh or salt water, and recovery in brackish water. MS
Soivio, A. and M. Nikinmaa, 1981 Kirjolohen fysiologisesta tilasto kuljetuksen ja sita seuraavan toipumisen aikana. (The physiological condition of rainbow trout during transport and the subsequent recovery period). Suomen Kalatalous, 49:49–56
Soivio, A., 1981a The swelling of erythrocytes in relation to the oxygen affinity of the blood of the rainbow trout, Salmo gairdneri Richardson. In Stress and fish, edited by A.D. Pickering. London, Academic Press, pp. 103–19
Soivio, A. and Virtanen, E., 1980 Methods for physiological experiments on fish. Ekotoxikologiska metoder for akvatisk miljo. Nordforsk, Raport (16):1–15 p.
Specker, J.L. and C.B. Schreck, 1980 Stress responses to transportation and fitness for marine survival in coho salmon (Oncorhynchus kisutch) smolts. Can.J.Fish.Aquat.Sci. 37(5):765–9
Toivonen, J., 1977 Differences in recaptures of wild and hatchery reared salmon smolts. ICES, C.M 1977/M:7 (mimeo)
Wedemeyer, G., 1972 Some physiological consequences of handling stress in juvenile coho salmon (Oncorhynchus kisutch) and steelhead trout (Salmo gairdneri). J.Fish.Res.Board Can., 29(12)1780–3
Wedemeyer G.A., 1980 R.L. Saunders and W.C. Clarke, Environmental factors affecting smoltification and early marine survival of anadromous salmonids. Mar.Fish.Rev., 1–14
Virtanen, E., 1981 et al, Physiological condition and smoltification of one-year old Baltic salmon (Salmo salar) in heated brackishwater effluents and fresh water. Schr. Bundesforschungsanst.Fish.Hamb., (16/17)vol.2:121–30
Woo, N.Y.S., 1978 H.A. Bern and R.S. Nishioka, Changes in body composition associated with smoltification and premature transfer to seawater in coho salmon (Oncorhynchus kisutch) and king salmon (O. tsawytscha). J.Fish Biol., 13(4):421–8
Table 1 Sampling dates and water temperatures at sampling
|Pre-transport samples||Post-transport samples|
|Sampling date||Temperature °C||Sampling date||Temperature°C|
Table 2 Physiological parameters for the stocking groups prior to transport in fresh water and the change caused by 48 hours' seawater exposure at a salinity of 28%. Means ± SKM's and n are given
|body silvering index (0–4)||2.7±0.10||(18)||2.9±0.23||(20)||2.4±0.14||(20)||2.2±0.16||(20)|
|total protein (g/l)||33.6±0.9||(13)||29.9±1.4||(18)||22.7±2.6||(17)||20.1±2.9||(12)|
|total lipids (%)||0.69±0.06||(18)||0.55±0.05||(20)||0.58±0.04||(20)||0.75±0.05||(20)|
|CHANGE CAUSED BY SEAWATER EXPOSURE IN:|
|Blood MCHC (g/l) Plasma||35.9±6.3||(9)||19.3±9.7||(10)||1.7±2.1||(9)||12.2±6.6||(5)|
|Muscle water (%)||-1.48±0.19||(8)||-1.20±0.22||(10)||-1.60±0.37||(10)||-3.38±0.32||(8)|
Table 3 Physiological parameters for the stocking groups after transport and subsequent recovery in fresh water.
Means ± SEM's and n are given on the upper line, below the absolute and relative changes from pre-transport
values (Table 2). The statistical significance of the change is given with the following symbols:
NS = not significant;
○ = P<0.1;
** = P<0.01;
*** = P<0.001
|Parameter:||The stocking group originating from:|
|-0.075***||(23 %)||-0.053***||(15 %)||-0.072***||(21 %)||-0.087***||(22 %)|
|-9.6**||(12 %)||-2.2NS||(2.3 %)||-15.0***||(17 %)||-5.9NS||(6.3 %)|
|+83.8○||(33 %)||+44.1NS||(16 %)||+15.6○||(6 %)||+60.5**||(25 %)|
|-1.05***||(54 %)||-0.08NS||(6 %)||-1.17**||(57 %)||-2.86***||(75 %)|
|+110***||(196 %)||+116***||(264 %)||+202***||(388 %)||-7NS||(7 %)|
|0NS||(0 %)||-0.034NS||(4.0 %)||+0.007NS||(0.9 %)||-0.022NS||(2.6 %)|
|-0.40○||(25 %)||-0.47NS||(29 %)||-1.12***||(50 %)||-1.60***||(59 %)|
|total lipids (%)||0.65±0.08||(8)||0.54±0.05||(10)||0.49±0.05||(9)||0.54±0.01||(10)|
|-0.04NS||(6 %)||-0.01NS||(1.8 %)||-0.09NS||(16 %)||-0.22***||(29 %)|
|-0.79*||(1.0 %)||-0.49NS||(0.6 %)||-0.51NS||(0.7 %)||-0.19NS||(0.2 %)|
|-3.1NS||(2.4 %)||-6.0*||(4.5 %)||+1.2NS||(0.9 %)||+6.9○||(5.8 %)|
|-0.17NS||(10 %)||-0.55***||(26 %)||-0.08NS||(5 %)||+0.08NS||(5 %)|
|total protein (g/l)||30.5±2.5||(5)||35.3±1.6||(10)||28.2±1.5||(8)||31.9±3.8||(3)|
|-3.1NS||(9 %)||+5.4*||(18 %)||+5.6○||(25 %)||+11.9*||(59 %)|
|+6.9NS||(2.3 %)||-4.5NS||(1.5 %)||+0.8NS||(0.3 %)||+0.9NS||(0.3 %)|