3.5.1. Marine rotifers
3.5.2. Freshwater rotifers
3.5.3. Culture procedures
3.5.4. Harvesting/concentration of rotifers
3.5.1.1. Salinity
3.5.1.2. Temperature
3.5.1.3. Dissolved oxygen
3.5.1.4. pH
3.5.1.5. Ammonia (NH3)
3.5.1.6. Bacteria
3.5.1.7. Ciliates
Although Brachionus plicatilis can withstand a wide salinity range from 1 to 97 ppt, optimal reproduction can only take place at salinities below 35 ppt (Lubzens, 1987). However, if rotifers have to be fed to predators which are reared at a different salinity (± 5 ppt), it is safe to acclimatize them as abrupt salinity shocks might inhibit the rotifers’ swimming or even cause their death.
The choice of the optimal culture temperature for rearing rotifers depends on the rotifer-morphotype; L-strain rotifers being reared at lower temperatures than S-type rotifers. In general, increasing the temperature within the optimal range usually results in an increased reproductive activity. However, rearing rotifers at high temperature enhances the cost for food. Apart from the increased cost for food, particular care has also to be paid to more frequent and smaller feeding distributions. This is essential for the maintenance of good water quality, and to avoid periods of overfeeding or starvation which are not tolerated at suboptimal temperature levels. For example, at high temperatures starving animals consume their lipid and carbohydrate reserves very fast. Rearing rotifers below their optimal temperature slows down the population growth considerably. Table 3.1 shows the effect of temperature on the population dynamics of rotifers.
Table 3.1. Effect of temperature on the reproduction activity of Brachionus plicatilis. (After Ruttner-Kolisko, 1972).
|
Temperature (°C). |
15°C |
20°C |
25°C |
|
Time for embryonic development (days). |
1.3 |
1.0 |
0.6 |
|
Time for young female to spawn for the first time
(days). |
3.0 |
1.9 |
1.3 |
|
Interval between two spawnings (hours). |
7.0 |
5.3 |
4.0 |
|
Length of life (days). |
15 |
10 |
7 |
|
Number of eggs spawned by a female during her life. |
23 |
23 |
20 |
Rotifers can survive in water containing as low as 2 mg.l-1 of dissolved oxygen. The level of dissolved oxygen in the culture water depends on temperature, salinity, rotifer density, and the type of the food. The aeration should not be too strong as to avoid physical damage to the population.
Rotifers live at pH-levels above 6.6, although in their natural environment under culture conditions the best results are obtained at a pH above 7.5.
The NH3/NH4+ ratio is influenced by the temperature and the pH of the water. High levels of un-ionized ammonia are toxic for rotifers but rearing conditions with NH3-concentrations below 1 mg.l-1 appear to be safe.
Pseudomonas and Acinetobacter are common opportunistic bacteria which may be important additional food sources for rotifers. Some Pseudomonas species, for instance, synthesize vitamin B12 which can be a limiting factor under culture conditions (Yu et al., 1988).
Although most bacteria are not pathogenic for rotifers their proliferation should be avoided since the real risk of accumulation and transfer via the food chain can cause detrimental effects on the predator.
A sampling campaign performed in various hatcheries showed that the dominant bacterial flora in rotifer cultures was of Vibrio (Verdonck et al., 1994). The same study showed that the microflora of the live food was considerably different among hatcheries; especially after enrichment, high numbers of associated bacteria were found. The enrichment of the cultures generaly induces a shift in the bacterial composition from Cytophaga/Flavobacterium dominance to Pseudomonas/Alcaligenes dominance. This change is partly due to a bloom of fast growing opportunistic bacteria, favoured by high substrate levels (Skjermo and Vadstein, 1993).
The bacterial numbers after enrichment can be decreased to their initial levels by appropriate storage (6°C) and adjustment of the rotifer density (Skjermo and Vadstein, 1993). A more effective way to decrease the bacterial counts, especially the counts of the dominant Vibrionaceae in rotifers, consists of feeding the rotifers with Lactobacillus plantarum (Gatesoupe, 1991). The supplementation of these probiotic bacteria not only has a regulating effect on the microflora but also increases the production rate of the rotifers.
For stable rotifer cultures, the microflora as well as the physiological condition of the rotifers, has to be considered. For example, it has been demonstrated that the dietary condition of the rotifer Brachionus plicatilis can be measured by its physiological performance and reaction to a selected pathogenic bacterial strain (Vibrio anguillarum TR27); the V. anguillarum strain administered at 106-107 colony forming units (CFU).ml-1 causing a negative effect on rotifers cultured on a sub-optimal diet while the rotifers grown on an optimal diet were not affected by the bacterial strain. Comparable results were also reported by Yu et al. (1990) with a Vibrio alginolyticus strain Y5 supplied at a concentration of 2.5.104CFU.ml-1.
Halotricha and Hypotricha ciliates, such as Uronema sp. and Euplotes sp., are not desired in intensive cultures since they compete for feed with the rotifers. The appearance of these ciliates is generally due sub-optimal rearing conditions, leading to less performing rotifers and increased chances for competition. Ciliates produce metabolic wastes which increase the NO2 - N level in the water and cause a decrease in pH. However, they have a positive effect in clearing the culture tank from bacteria and detritus. The addition of a low formalin concentration of 20 mg.l-1 to the algal culture tank, 24 h before rotifer inoculation can significantly reduce protozoan contamination. Screening and cleaning of the rotifers through the use of phytoplankton filters (< 50 µm) so as to reduce the number of ciliates or other small contaminants is an easy precaution which can be taken when setting up starter cultures.
Brachionus calyciflorus and Brachionus rubens are the most commonly cultured rotifers in freshwater mass cultures. They tolerate temperatures between 15 to 31°C. In their natural environment they thrive in waters of various ionic composition. Brachionus calyciflorus can be cultured in a synthetic medium consisting of 96 mg NaHCO3, 60 mg CaSO4.2H2O, 60 mg MgSO4 and 4 mg KCl in 1 1 of deionized water. The optimal pH is 6-8 at 25°C, minimum oxygen levels are 1.2 mg.l-1. Free ammonia levels of 3 to 5 mg.l-1 inhibit reproduction.
Brachionus calyciflorus and Brachionus rubens have been successfully reared on the microalgae Scenedesmus costato-granulatus, Kirchneriella contorta, Phacus pyrum, Ankistrodesmus convoluus and Chlorella, as well as yeast and the artificial diets Culture Selco® (Inve Aquaculture, Belgium) and Roti-Rich (Florida Aqua Farms Inc., USA). The feeding scheme for Brachionus rubens needs to be adjusted as its feeding rate is somewhat higher than that of B. plicatilis.
3.5.3.1. Stock culture of rotifers
3.5.3.2. Upscaling of stock cultures to starter cultures
3.5.3.3. Mass production on algae
3.5.3.4. Mass production on algae and yeast
3.5.3.5. Mass culture on yeast
3.5.3.6. Mass culture on formulated diets
3.5.3.7. High density rearing
Intensive production of rotifers is usually performed in batch culture within indoor facilities; the latter being more reliable than outdoor extensive production in countries where climatological constraints do not allow the outdoor production of microalgae. Basically, the production strategy is the same for indoor or outdoor facilities, but higher starting and harvesting densities enable the use of smaller production tanks (generally 1 to 2 m3) within intensive indoor facilities. In some cases, the algal food can be completely substituted by formulated diets (see 3.5.3.6.).
Culturing large volumes of rotifers on algae, baker’s yeast or artificial diets always involves some risks for sudden mortality of the population. Technical or human failures but also contamination with pathogens or competitive filter feeders are the main causes for lower reproduction which can eventually result in a complete crash of the population. Relying only on mass cultures of rotifers for reinoculating new tanks is too risky an approach. In order to minimize this risk, small stock cultures are generally kept in closed vials in an isolated room to prevent contamination with bacteria and/or ciliates. These stock cultures which need to generate large populations of rotifers as fast as possible are generally maintained on algae.
The rotifers for stock cultures can be obtained from the wild, or from research institutes or commercial hatcheries. However, before being used in the production cycle the inoculum should first be disinfected. The most drastic disinfection consists of killing the free-swimming rotifers but not the eggs with a cocktail of antibiotics (e.g. erythromycin 10 mg.l-1, chloramphenicol 10 mg.l-1, sodium oxolinate 10 mg.l-1, penicillin 100 mg.l-1, streptomycin 20 mg.l-1) or a disinfectant. The eggs are then separated from the dead bodies on a 50 µm sieve and incubated for hatching and the offspring used for starting the stock cultures. However, if the rotifers do not contain many eggs (as can be the case after a long shipment) the risk of loosing the complete initial stock is too big and in these instances the rotifer should be disinfected at sublethal doses; the water of the rotifers being completely renewed and the rotifers treated with either antibiotics or disinfectants. The treatment is repeated after 24 h in order to be sure that any pathogens which might have survived the passage of the intestinal tract of the rotifers are killed as well. The concentration of the disinfection products differs according to their toxicity and the initial condition of the rotifers. Orientating concentrations for this type of disinfection are 7.5 mg.l-1 furazolidone, 10 mg.l-1 oxytetracycline, 30 mg.l-1 sarafloxacin, or 30 mg.l-1 linco-spectin.
At the Laboratory of Aquaculture & Artemia Reference Center the stock cultures for rotifers are kept in a thermo-climatised room (28°C ± 1°C). The vials (50 ml conical centrifuge tubes) are previously autoclaved and disposed on a rotating shaft (4 rpm). At each rotation the water is mixed with the enclosed air (± 8 ml), providing enough oxygen for the rotifers (Fig. 3.4.). The vials on the rotor are exposed to the light of two fluorescent light tubes at a distance of 20 cm (light intensity of 3000 lux on the tubes).
The culture water (seawater diluted with tap water to a salinity of 25 ppt) is aerated, prefiltrated over a 1 µm filter bag and disinfected overnight with 5 mg.l-1 NaOCl. The next day the excess of NaOCl is neutralized with Na2S2O3 (for neutralization and color reaction see worksheet 3.1.) and the water is filtered over a 0.45 µm filter.
Inoculation of the tubes is carried out with an initial density of 2 rotifers.ml-1. The food consists of marine Chlorella cultured according to the procedure described in 2.3. The algae are centrifuged and concentrated to 1-2.108 cells.ml-1. The algal concentrate is stored at 4°C in a refrigerator for a maximum period of 7 days, coinciding with one rotifer rearing cycle. Every day the algal concentrate is homogenized by shaking and 200 µl is given to each of the tubes. If fresh algae are given instead of the algal concentrate 4 ml of a good culture is added daily.
After one week the rotifer density should have increased from 2 to 200 individuals.ml-1 (Fig. 3.5.). The rotifers are rinsed, a small part is used for maintenance of the stock, and the remaining rotifers can be used for upscaling. Furthermore, after some months of regular culture the stock cultures will be disinfected as described earlier in order to keep healthy and clean stock material. However, the continuous maintenance of live stock cultures of Brachionus does not eliminate the risk of bacterial contamination.
Treatment with anti-biotics might lower the bacterial load, but also implies the risk for selection of antibiotic-resistant bacteria. However, the commercial availability of resting eggs could be an alternative to maintaining stock cultures and reducing the chances for contamination with ciliates or pathogenetic bacteria (see Fig. 3.7.).
The upscaling of rotifers is carried out in static systems consisting of erlenmeyers of 500 ml placed 2 cm from fluorescent light tubes (5000 lux). The temperature in the erlenmeyers should not be more than 30°C. The rotifers are stocked at a density of 50 individuals.ml-1 and fed 400 ml freshly-harvested algae (Chlorella 1.6.106 cells.ml-1); approximately 50 ml of algae being added every day to supply enough food. Within 3 days the rotifer concentration can increase to 200 rotifers.ml-1 (Fig. 3.5.). During this short rearing period no aeration is applied.
Once the rotifers have reached a density of 200-300 individuals.ml-1 they are rinsed on a submerged filter consisting of 2 filter screens. The upper mesh size (200 µm) retains large waste particles, while the lower sieve (50 µm) collects the rotifers. If only single strainers are available this handling can be carried out with two separate filters. Moreover, if rinsing is performed under water the rotifers will not clog and losses will be limited to less than 1%.
The concentrated rotifers are then distributed in several 15 l bottles filled with 2 l water at a density of 50 individuals.ml-1 and a mild tube aeration provided. In order to avoid contamination with ciliates the air should be filtered by a cartridge or activated carbon filters. Fresh algae (Chlorella 1.6 × 106 cells.ml-1) are supplied daily. Every other day the cultures are cleaned (double-screen filtration) and restocked at densities of 200 rotifers.ml-1. After adding algae for approximately one week the 15 l bottles are completely full and the cultures can be used for inoculation of mass cultures.
Undoubtedly, marine microalgae are the best diet for rotifers and very high yields can be obtained if sufficient algae are available and an appropriate management is followed. Unfortunately in most places it is not possible to cope with the fast filtration capacity of the rotifers which require continuous algal blooms. If the infrastructure and labor is not limiting, a procedure of continuous (daily) harvest and transfer to algal tanks can be considered. In most places, however, pure algae are only given for starting up rotifer cultures or to enrich rotifers (see 3.5.3.1. and 3.6.1.1.).
Batch cultivation is probably the most common method of rotifer production in marine fish hatcheries. The culture strategy consists of either the maintenance of a constant culture volume with an increasing rotifer density or the maintenance of a constant rotifer density by increasing the culture volume (see 3.5.3.4.). Extensive culture techniques (using large tanks of more than 50 m3) as well as intensive methods (using tanks with a volume of 200-2000 l) are applied. In both cases large amounts of cultured microalgae, usually the marine alga Nannochloropsis, are usually inoculated in the tanks together with a starter population containing 50 to 150 rotifers.ml-1.
Depending on the strategy and the quality of the algal blooms baker’s yeast may be supplemented. The amount of yeast fed on a daily basis is about 1 g.million-1 of rotifers, although this figure varies depending on the rotifer type (S,L) and culture conditions. Since algae have a high nutritional value, an excellent buoyancy and do not pollute the water, they are used as much as possible, not only as a rotifer food, but also as water conditioners and bacteriostatic agents.
In contrast to most European rearing systems, Japanese developed large culture systems of 10 to 200 metric tons. The initial stocking density is relatively high (80-200 rotifers.ml-1) and large amounts of rotifers (2-6 × 109) are produced daily with algae (4-40 m3) supplemented with yeast (1-6 kg).
The mass production on algae and yeast is performed in a batch or semi-continuous culture system. Several alterations to both systems have been developed, and as an example the rearing models used at The Oceanic Institute in Hawaii are described here:
· Batch culture systemThe tanks (1 200 l capacity) are half filled with algae at a density of 13-14 × 106 cells.ml-1 and inoculated with rotifers at a density of 100 individuals.ml-1. The salinity of the water is 23 ppt and the temperature maintained at 30°C. The first day active baker’s yeast is administered two times a day at a quantity of 0.25 g/10-6 rotifers. The next day the tanks are completely filled with algae at the same algal density and 0.375 g baker’s yeast per million rotifers is added twice a day. The next day the rotifers are harvested and new tanks are inoculated (i.e. two-day batch culture system).
· Semi-continuous culture
In this culture technique the rotifers are kept in the same tank for five days. During the first two days the culture volume is doubled each day to dilute the rotifer density in half. During the next following days, half the tank volume is harvested and refilled again to decrease the density by half. On the fifth day the tank is harvested and the procedure started all over again (i.e. five-day semi-continuous culture system).
The nutritional composition of algae-fed rotifers does not automatically meet the requirements of many predator fish and sometimes implies an extra enrichment step to boost the rotifers with additional nutritional components such as fatty acids, vitamins or proteins (see 3.6.). Also, the addition of vitamins, and in particular vitamin B12, has been reported as being essential for the culture of rotifers (Yu et al., 1989).
Baker’s yeast has a small particle size (5-7 µm) and a high protein content and is an acceptable diet for Brachionus. The first trials to replace the complete natural rotifer diet by baker’s yeast were characterized by varying success and the occurrence of sudden collapses of the cultures (Hirayama, 1987). Most probably the reason for these crashes was explained by the poor digestibility of the yeast, which requires the presence of bacteria for digestion. Moreover, the yeast usually needs to be supplemented with essential fatty acids and vitamins to suit the larval requirements of the predator organisms. Commercial boosters, but also home-made emulsions (fish oils emulgated with commercial emulgators or with egg-yolk lecithin), may be added to the yeast or administered directly to the rotifer tank (see 3.6.1.3.). Better success was obtained with so called w-yeast-fed rotifers (rotifers fed on a yeast preparation produced by adding cuttlefish liver oil at a 15% level to the culture medium of baker’s yeast) which ensured a high level of (n-3) essential fatty acids in the rotifers (Watanabe et al., 1983). The necessity of adding the component in the food of the rotifer or to the rotifers’ culture medium was later confirmed by using microparticulate and emulsified formulations (Watanabe et al., 1983; Léger et al., 1989). Apart from fresh baker’s yeast, instant baker’s yeast, marine yeast (Candida) or caked yeast (Rhodotorula) may also be used.
The most frequently used formulated diet in rotifer culture in Europe is Culture Selco® (CS) available under a dry form. It has been formulated as a complete substitute for live microalgae and at the same time guarantees the incorporation of high levels of EFA and vitamins in the rotifers. The biochemical composition of the artificial diet Culture Selco® consists of 45% proteins, 30% carbohydrates, 15% lipids (33% of which are (n-3) HUFA), and 7% ash. Its physical characteristics are optimal for uptake by rotifers: the particle, having a 7 µm particle size, remaining in suspension in the water column with a relatively strong aeration, and not leaching. However, the diet needs to be suspended in water prior to feeding, which facilitates on one hand the possibilities for automatic feeding but on the other hand requires the use of aeration and cold storage. The following standard culture procedure has been developed and tested on several rotifer strains in 100 l tanks.
Cylindro-conical tanks of 100 l with dark smooth walls (polyethylene) are set up in shaded conditions. The culture medium consists of diluted seawater of 25 ppt kept at 25°C. No water renewal takes place during the 4-day culture period. Air stones are installed a few cm above the cone bottom of the tank to allow sedimentation and possible flushing of waste particles. Food flocculates are trapped in pieces of cloth which are suspended in the water column (Fig. 3.6a.), or in an air-water-lift trap filled with sponges (Fig. 3.6b.).
Figure 3.6.a. Piece of cloth to trap the floccules in the rotifer tank.
Figure 3.6.b. Air-water-lift filled with sponges to trap the floccules in the rotifer tank.
Table 3.2. Feeding regime for optimal rotifer culture in function of the rotifer density using the formulated diet Culture Selco®.
|
Rotifer density.ml-1 |
Culture Selco® per 106
rotifers.day-1 |
Culture Selco® per
m3.day-1 |
|
(L-strain) |
(in g) |
(in g) |
|
100 - 150 |
0.53 |
53 - 80 |
|
150 - 200 |
0.47 |
70 - 93 |
|
200 - 250 |
0.40 |
80 - 100 |
|
250 - 300 |
0.37 |
92 - 110 |
|
300 - 350 |
0.33 |
100 - 117 |
|
350 - 400 |
0.30 |
105 - 120 |
|
400 - 450 |
0.27 |
107 - 120 |
|
450 - 500 |
0.23 |
105 - 117 |
|
> 500 |
0.25 |
125 |
|
> 1200 |
0.20 |
240 |
Applying this standard culture strategy a doubling of the population is achieved every two days, reaching a harvest density of 600 rotifers.ml-1 after four days only (Table 3.3.), which is better than for the traditional technique using live algae (and baker’s yeast). There is no high variation in production characteristics among the various culture tests and crashes are rarely observed, which most probably is due to the non-introduction of microbial contaminants and the overall good water quality over the culture period. In this respect, it should be emphasized that hygienic precautions should be taken to avoid contacts among different rearing units. All material used during the production (i.e. glass ware) can be disinfected in water baths with NaOCl, HCl or other disinfectants. After each production cycle (4 days) the tanks, airstones and tubing need to be disinfected thoroughly. In order to avoid crashes it is recommended that after approximately one month of culture that the complete system be disinfected and the cultures started again using rotifers from starter cultures.
In commercial hatcheries, peristaltic pumps are not always available. In this case the artificial diet can be fed on a daily basis at a concentration of 400-600 mg/10-6 rotifers, and administered in 4 to 6 rations with a minimum quantity of 50 - 100 mg.l-1 culture medium. Analogous production outputs are achieved under upscaling conditions in commercial hatcheries (Table 3.3.).
Table 3.3. Growth and reproduction characteristics of rotifers reared on CS under experimental and upscaled conditions.
|
Experimental |
Batch 1 |
Batch 2 |
Batch 3 |
|
Age of the population |
|
Number of rotifers per ml |
|
|
Day 1 |
200 |
200 |
200 |
|
Day 2 |
261 ± 13 |
327 ± 17 |
280 ± 12 |
|
Day 3 |
444 ± 65 |
473 ± 42 |
497 ± 25 |
|
Day 4 |
581 ± 59 |
687 ± 44 |
681 ± 37 |
|
Growth rate.day-1 |
0.267 |
0.308 |
0.306 |
|
Doubling time |
2.60 |
2.25 |
2.27 |
|
Commercial |
Batch 1 |
|
Age of the population |
Number of rotifers per ml |
|
Day 1 |
200 |
|
Day 2 |
285 |
|
Day 3 |
505 |
|
Day 4 |
571 |
|
Day 5 |
620 |
Figure 3.8. Illustration of the drip-feeding technique which can be applied when no sophisticated pumping devices are available.
Although high density rearing of rotifers increases the risk for more stressful rearing conditions, and an increased risk of reduced growth rates due to the start of sexual reproduction, promising results have been obtained in controlled cultures. The technique is the same as the one used for the mass culture on Culture Selco® but after each cycle of 4 days the rotifer density is not readjusted. The feeding scheme is adjusted to 0.25-0.3 g/10-6 of rotifers for densities between 500 and 1500 rotifers.ml-1 and to 0.2 g for densities above 1500 rotifers.ml-1. Rearing rotifers at high stocking densities has a direct repercussion on the egg ratio (Fig. 3.9.). This latter is dropping from an average of 30% at a density of 150 rotifers.ml-1 to 10% at a density of 2000 rotifers.ml-1 and less than 5% at densities of 5000 rotifers.ml-1. Maintaining cultures with this low egg ratio is more risky and thus the system should only be used under well controlled conditions.
Figure 3.9. Effect of high density rotifer culture on the egg ratio.
High density cultivation of Brachionus is also being performed in Japan. In this technique Nannochloropsis is being supplemented with concentrated fresh water Chlorella, baker’s yeast and yeast containing fish oil. Freshwater Chlorella is being used for vitamin B12 supplementation (± 12 mg.l-1 at a cell concentration of 1.5.1010 cells.ml-1). In continuous cultures the rotifer population doubles every day. Half the culture is removed daily and replaced by new water. Using this system average densities of 1000 rotifers.ml-1 are achieved with peaks of more than 3000 animals.ml-1.
Small-scale harvesting of rotifers is usually performed by siphoning the content of the culture tank into filter bags with a mesh size of 50-70 µm. If this is not performed in submerged filters the rotifers may be damaged and result in mortality. It is therefore recommended to harvest the rotifers under water; concentrator rinsers are very convenient for this purpose (Fig. 3.10.). Aeration during the concentration of rotifers will not harm the animals, but should not be too strong so as to avoid clogging of the rotifers, this can be very critical, specially after enrichment (see Fig. 3.6.4.).
