| Abstract | Rotifer, Brachionus sp. has the potential to serve as food, and to deliver nutrients for early¬stage fish larvae. Size reduction, nutritional enrichment, followed by consistent mass production could make the rotifer more suitable food for first feeding of grouper larvae. This study was designed to understand the potential for rotifer size reduction, the effect of enrichment and cryopreservation on rotifers and embryos. In total, 4 studies were carried out. The first study examined the life history characteristics of small egg-bearing females (SEF) and their offspring. The life span of their first offspring was only 5.11 ± 0.35 days (d) (mean ± SEM), the reference population's (REF) longevity was higher, 9.65±0.4 d. The length of reproductive periods of offspring from the SEF was 2.58±0.38 d, shorter than that of the REF population (6.83±0.58 d). Although the rate of egg production was not significantly different between SEF and REF, the total number of eggs produced by the SEF's first offspring was lower (4.78±0.60 individuals) than that of the REF (11.83 ± 1.26 individuals). The SEFs' first offspring had a higher egg to lorica length ratio (76%) than the REF (57%) population, indicating a relatively higher investment in reproduction. The mean lorica length of the SEF at the time of collection was 120±6 um (SD), and this mean-length did not increase over their life time. The SEFs' first offspring reached only 125±6 um in 36 hours, and further culture of their offspring over 35 days resulted in a mean offspring size of 163±11 um, similar to the REF population (159±16 um). This suggests that size is not a heritable trait in rotifers, and thus, the stable production of small-size rotifers through intensive selection is unlikely to be successful. The second study assessed the efficacy in the loading of three dietary components, PUFA-rich marine-oil (PUFA), L-ascorbic acid phosphate (AA) and synthetic astaxanthin into rotifers. Experimental emulsions containing combinations of PUFA, AA and/or astaxanthin were used to enrich rotifers. Following enrichment, the PUF A, AA and astaxanthin levels were measured in the rotifers. Treatment with 10 ppm astaxanthin (195.8 ug g⁻¹ DW) of astaxanthin was compared to 1 ppm (73.9 ug g⁻¹DW), 24 h after enrichment. Astaxanthin was not detected in the the baker's yeast Sacharomyces cerevisae and detected only in minute quantities in the microalgae, Nannochloropsis sp. Treatment with 100 ppm of AA resulted in 1.52 mg g-l DW AA in the rotifers and only 0.50 mg g⁻¹DW of AA in the control (fed with baker's yeast only). Similarly, PUFA treatment (l00 ppm) resulted in 13.9 mg g⁻¹DW of DHA and 7.6 mg g⁻¹DW of EPA in the rotifers, compared to 0.39 and 0.67 mg g⁻¹DW of DHA and EPA measured in the control. The study showed not only that PUF A, AA and astaxanthin are incorporated into the rotifer, but also the level of uptake depends on the enrichment medium. The third study was concerned with the cryopreservation of rotifers. If the rotifers could be successfully cryopreserved, this would provide a ready supply of start-up populations for the mass production of improved rotifer stock. To improve the stable mass production of rotifers for early larval feeding, this third study aimed to understand the possibility of ultrasound enhanced cryoprotectants prior to rotifers or embryo cryopreservation. The hatch and survival of rotifer embryo were examined following exposure to ultrasound with or without dymethylsulfoxide (DMSO). The rotifers and embryo were first placed in an ultrasonic bath and subjected to cavitation level ultrasound. After exposure the rotifers and embryo were placed in an incubator to observe hatch and survival. Hatch decreased by 12% when the exposure duration was increased to 60 minutes (min). Increased DMSO, from 5 to 15% (V N), and increased treatment duration, from 5 to 25 min, reduced embryo hatch from 69.3±3.8 to 49.3±4.9%. The highest survival (89.8±1.25 and 86.0±2.1 %) of rotifers was recorded after 5 min exposure to ultrasound at 420 V and 440 V, and this decreased to 59.7± 1.9% when the voltage was increased to 520 V and exposure time to 25 min. Similarly, the highest hatch percentage of embryo (88.2 ± 1.5%) was obtained with 480 V for 15 min duration. This study showed that the rotifer and its embryo can survive to a threshold level of ultrasound and DMSO exposure. Further study on aplication of ultrasound to enhance DMSO transport is needed before cryopreservation trials can be carried out. In the fourth study the benefits of feeding enriched rotifers to early-stage (1 to 14 d) grouper larvae was tested. The humpback grouper, Cromileptis altivelis larvae were reared from hatch to 14 day post hatch (14dph) with rotifer enriched with the three dietary components described in the second study. The mean larvae survival ranged from 48.9±5.7% to 54.9±5.4% for all treatments, and there was no difference between the treated grouper and the control. The total mean length ranged from 5.15±0.55 to 5.55±0.63 mm. However, larvae reared with enriched with rotifers more resilient to stress. Only 10.0±5.8 to 13.3±6.1 % of the larvae did not withstand the salinity stress test compared to 53.3±6.7% in the control (fed Nannochloropsis sp. only). Although, attempts to reduce size was not successful, the studies on the application of ultrasound and DMSO prior to cryopreservation shows potential. Moreover, nutritional enrichment has shown to improve larvae survival during stressful conditions. |