Population growth and cyst production of the rotifer Brachionus plicatilis (Monogonta: Brachionidae) fed with three different diets

This study compares the effect of different diets on population growth, fecundity rate, production, and cyst hatching efficiency of the rotifer Brachionus plicatilis . Microalgae were cultured using filtered and sterilized seawater at 25 and 26°C. The volume of the containers for algae, bread yeast, and rotifer was duplicated daily from 31mL to 16L in nine days. Rotifers were cultured at 25°C and 35‰ salinity. The mean values of daily population growth rate for the rotifers fed on C. muelleri , I. galbana and bread yeast were 0,91, 0,89 and 0,87 daily counts, respectively. After 10 cultured days, fecundity was 72%, 63% and 36% using C. muelleri , I. galbana and bread yeast, respectively. The highest mean value for cysts production was 4 064 000 cysts found from 63 243 (1,55%) rotifers fed on C. muelleri . Hatching efficiency after 36 hours of cysts from rotifers fed C. muelleri was 51 500 (94,3%) new borns, followed by 45 000 (93,3%) with I. galbana , and 31 000 (92,6%) with bread yeast. C. muelleri is a good food source for B. plicatilis .

In order to expand the practice of marine n sh larvae rearing, it is necessary to increase and stabilize the production of live feed. The technology is based on the use of highly concentrated algal biomass as food for the rotifers. Villegas, Millamena and Escritor (2008) studied the e ects of three selected algal species, Tetraselmis tetrahele, Isochrysis galbana, and marine Chlorella sp., on the population growth of Brachionus plicatilis after three, ve and seven days of culture. The rotifers fed on T. tetrahele showed growth with a mean peak density of 92,5 individuals per mL. This result was superior to those fed on I. galbana (48,2 individuals per mL) and Chlorella sp. (47,2 individuals per mL) in ve days. Rotifers are mass-cultured at 10 000-30 000 individuals mL-1 by feeding them exclusively on condensed freshwater Chlorella. The cultures are generally supplied with O 2 at a constant rate; and the pH is adjusted to 7 by addition of HCI to avoid an increase of free ammonia in the culture system, substituted rotifers for Artemia in larval sh culture. He is using a new, continuous culture system that appears to be reliable and stable. It is still in the development phase, but he is averaging about 8Kg of wet weight rotifers per day. Rotifer production has very signi cant economies of scale. Labor cost is almost independent of production levels. He is using yeast and other nutrients in a continuous production system with L-type rotifers. Apparently large rotifer production systems will solve the cost of producing live Artemia nauplii; hence, it is important to know the biology of rotifers and their nutritional requirements to optimize their culture. Since the supply of rotifers does not always meet the demand, it is necessary to apply techniques to produce high quality cysts that can be used to initiate rotifer culture systems on a large scale.
The aim of this study was to compare the population growth rate of B. plicatilis, production of cysts, fecundity rate (Release eggs/Eggs in each female in the sample), and e ciency of hatching when the rotifers where fed on the microalgae Chaetoceros muelleri Lemmermann, or Isochrysis galbana Bruce, Knight and Parke, or baker´s yeast Saccharomyses cerevisiae strain Lalvin EC1118 (Prise de mousse) as food.

MATERIAL AND METHODS
This study was performed at the Aquaculture Laboratory, Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa, using two rooms: one, to culture microalgae, which was provided with 13 white-light lamps from 40 to 75W, and the other, for the culture of rotifers. Each room has a water aeration system for culturing C. muelleri (CHGRA) (Lora-Vilchis, Cordero & Voltolina, 2004), I. galbana microalgae and rotifers.
The seawater for culturing microalgae was ltered successively through lters of 5,0, 1,0, and 0,45µm (Reyes, 2003), ultraviolet radiation was used to sterilize the water; controlled temperature between 25° and 26°C was maintained to permit the rotifer population grow adequately. The rotifers were initially maintained in 500mL asks at a temperature of 25°C and salinity of 35‰ and fed on microalgae or bread yeast. C. muelleri measures between 4 and 7µm in diameter by 6 to 8µm in length, whereas I. galbana measures 8µm in diameter by 11 to 13µm in length (Ortega, 1984;Trujillo & Voltolina 1994). The production of microalgae according to Matthiessen and Toner (1966) method, doubling the volume of the containers daily from 31mL to 16L in nine days.
The rotifer culture started 10 to 20organisms/mL. The growth of the rotifer population was assessed by daily counts according to Yúfera and Pascual (1980) with the formula Ke = (ln Nt -ln N0)/ T, where N0 is the number of organisms at the start of a period of T days, and Nt the number after T days. The population growth was assessed in triplicate each sampling. The rotifers were fed each one of the two selected microalgae at a density of 1x10 6 cells/ mL, and bread yeast dissolving 1g in 20L seawater each day. The volume of the rotifers culture was doubled daily from 31mL tubes to 16L containers in nine days. Each day the rotifers were concentrated in a sieve and transfer to the next volume containing the algae's and the bread yeast.
The fecundity rate A f = H/n was estimated from the number of eggs released per eggs in each female, where H is the total number of eggs released and n is the number of females which have eggs in the sample ( Table 1).
Production of cysts from three essays: rotifers were ltered into 18L ask 2x10 6 cels/ml density of the designed microalgae for rotifers to feed, daily, also rotifers were ltered in 1g of bread yeast in 18L ask, until density reached more than 100rot/mL, then feeding stopped; sexual reproduction started (mictic), 5 days later rotifers were ltered using a 60µm mesh, one 20 th part of the volume was evaporated by light sun rays then cysts were cleaning using brine water and mixed with the  To evaluate the percentage of hatching, groups of 100 well preserved cysts were settled in a Petri dish with sterilized water, a 60W lamp was placed at 60cm for 24h, and the neonates were counted and separated until no more were born. The cysts were also decapsulated to obtain more neonates; they were settled with a mixture of 12mL of sodium hypochlorite with 0,6mL of sodium hydroxide at 40% for 15min, then watered, and 3mL of sodium thiosul te was added to neutralize them, and then they were watered again (US Patent 7258890 -Process for decapsulating crustacean or rotifer eggs) ( Table 2). E ciency and rate of hatching: 0,1g of cysts was settled in a 1L ask with light and aeration for 24h at 25°C; using a 1mL micropipette, neonates were count and extrapolate to 1g cysts. The rate of hatching after 12h, the rst count was made each hour in three samples until 90% of cyst hatched. T0 (10%) = time when the rst neonate hatched, until T 90 (90 %) T s = T 90 -T 0 ( Table 2) where Ts is the synchronized time ( Table 2).
The ANOVA and Kruskal Wallis statistical methods were used.
The highest mean production of cysts was 63 243(1,55%); C. muelleri was used as food. The percentage of hatching was higher at 24h time intervals. E ciency of hatch extrapolate to 1g of cysts from rotifers fed with C. muelleri was 51 500(94,3%) neonates at 36h (Table 2). ANOVA showed no signi cant di erences between C. muelleri and bread yeast, or between C. muelleri and I. galbana (F2,27, P<0,05   from 92,6% fed rotifers bread yeast to 94,3% fed C. muelleri. This percentage decreases from about 90 in 15 days to less than 60 in 60 days.
Fecundity and cyst production showed that C. muelleri to be better food.

DISCUSSION
The size of the buccal cavity of a rotifer depends on the overall body size. The relationship between maximum size of the food ingested (Tm) and lorica length (L) is as follows: Tm= 0,090L-0,033 (Hino & Hirano, 1980). For rotifers between 250 and 300 µm, the maximum food ingested measured between 22 and 27µm. In the present study, B. plicatilis had good results fed on C. muelleri, which in this case measured less than 10 µm (Trujillo & Voltolina, 1994).
The rate of ingestion increases with the concentration of the food up to a maximum level, then the ingestion rate remains constant (Hirata & Mocawa, 1983). In the present study the hatching rate was 94,3% when food was C. muelleri. Minko , Lubzens and Kahan (1983) mentioned that at 9‰ salinity, hatching of B. plicatilis was optimal (40-70%) when the temperature was 10-30° C and decreased linearly with rise in incubation temperature. Light was obligatory for termination of dormancy. At 15°C resting eggs incubated over a salinity range of 9-40‰ showed optimal hatching at 16o/oo. In the present study at 25ºC and salinity of 35‰ cysts were given light and aeration for 24h.
Frequent densities of rotifers in commercial cultures are 2 500rotifers/mL in recirculation system (Suantika, Dhert & Sorgeloos, 2003), although ultra densities of more than 1,6x105/mL have been reported (Yoshimura, Tanaka & Yoshimatsu, 2003). In the present study, the highest densities reached were of 254rotifers+ eggs/mL fed on C. muelleri, and the lowest was 172rotifers+eggs/mL fed with bread yeast after 9 days of culture in 16L. C. muelleri showed to be better food.