Freshwater crayfish aquaculture is a rapidly growing sector, yet crayfish nutritional research lags behind other crustaceans. This necessitates exploring alternative and sustainable feedstuffs to replace expensive and dwindling fishmeal. Biofloc technology (BFT) offers a potential solution by producing microbial biomass (biofloc) that can be harvested and used as a feed ingredient. While BFT is increasingly used for various aquaculture species, its application in crayfish nutrition remains largely unexplored. The excess biofloc generated from BFT systems represents a significant waste stream. This study addresses the need for sustainable and cost-effective crayfish feed by investigating the potential of recycling this biofloc waste as a novel protein source. Specifically, the study aimed to (a) establish crayfish nutritional standards from existing literature given the limited available data; (b) understand growth trajectory and nutritional dependencies in crayfish; (c) evaluate the growth response of red swamp crayfish to graded levels of biofloc meal (BM) in their diet; (d) assess the risks of heavy metal bioaccumulation or mineral stress in crayfish fed BM; and (e) identify nutritional strengths and weaknesses of using BM in crayfish diets. The establishment of crayfish nutritional standards and the analysis of growth trajectory were crucial methodological steps to effectively interpret the results of the BM feeding trial and understand the implications of this novel feed source on crayfish growth and health.

The literature review synthesized information on the nutritional requirements of freshwater crayfish, a data-deficient group compared to other commercially important crustaceans like penaeids. Existing research on crayfish nutrition is limited, necessitating a meta-analysis of published studies to formulate optimal dietary requirements for macronutrients, essential amino acids, and essential minerals. The review also investigated the growth trajectory of crayfish, including the thermal growth coefficient (TGC) as a benchmark for evaluating growth performance under different dietary conditions. Furthermore, the review explored previous studies using biofloc as an unconventional protein source in aquaculture, noting its varying composition (protein, lipid, ash) depending on several factors. While several studies used biofloc in other crustacean diets, its use in crayfish feed has not been extensively researched.

The study involved a meta-analysis of published literature on crayfish nutrition to determine optimal dietary requirements. Data from 27 peer-reviewed articles were used to establish crayfish nutritional standards for macronutrients, essential amino acids (EAAs), and essential minerals, comparing these to existing standards for penaeid shrimp. Crayfish growth trajectory and nutritional dependencies were also modeled. For the growth trial, biofloc biomass was collected from an indoor BFT system using Nile tilapia. The biofloc was processed into biofloc meal (BM). A total of 120 juvenile red swamp crayfish were randomly assigned to four dietary treatments: control (0% BM), BM33 (33% BM), BM66 (66% BM), and BM100 (100% BM). Crayfish were reared for 9 weeks under controlled conditions. Body weight, survival rate, and feed utilization parameters (FCR, PER) were monitored every three weeks. At the end of the trial, muscle and hepatopancreas samples were analyzed for heavy metal and mineral content. Statistical analysis included normality tests, ANOVA (with post-hoc Tukey HSD), and Kruskal-Wallis tests (with Dunn’s post-hoc test and Bonferroni correction) to compare the different dietary groups. Generalized additive models were employed to analyze growth trajectories and identify nutritional dependencies on growth parameters. All statistical tests were performed using RStudio v1.2.5042.

Meta-analysis established crayfish nutritional standards that were then compared to penaeid shrimp standards. The 7-week growth trial showed that BM inclusion at 33% and 66% significantly improved crayfish growth compared to the control group. However, the BM100 group exhibited significantly reduced growth (lower TGC, LWG, and final body weight), size heterogeneity, and poorer feed utilization (higher FCR, lower PER). The BM100 group was characterized by a predominance of smaller individuals, suggesting a negative impact on growth uniformity. The study found no significant difference in survivability across dietary treatments. While heavy metal levels in crayfish fed BM were below critical limits, the hepatopancreas showed higher concentrations of mercury and other metals in the BM100 group, suggesting potential mineral stress. The high ash content in BM (>14%), particularly in the BM100 group, and arginine deficiency (14–20% lower than the optimum requirement) were identified as major factors hindering growth in groups with higher BM inclusions. The non-protein energy:protein ratio was also insufficient in the BM100 group, indicating inadequate energy for optimal protein utilization and growth. The FCR multiplied by the dietary arginine content seemed to correlate with the fulfillment of arginine requirement.

The results demonstrate that biofloc meal can be a valuable protein source for crayfish feed when used at moderate inclusion levels (33–66%). However, excessive inclusion leads to reduced growth due to nutritional imbalances and potential mineral stress. The crucial role of arginine, a limiting amino acid in BM, emphasizes the need for amino acid supplementation to improve the nutritional quality of BM. High ash content, beyond the physiological limit for crayfish, likely impacted growth by increasing metabolic energy expenditure in osmoregulation. The observed mineral and heavy metal stress, particularly in the hepatopancreas, underscore the need to carefully monitor and control the mineral composition of BM and to consider potential long-term effects on crayfish health. The study highlights the complexity of utilizing unconventional feedstuffs and the importance of considering synergistic effects of nutrient deficiencies and environmental stressors on growth performance.

This study provides valuable insights into the use of biofloc meal as a sustainable protein source for crayfish aquaculture. Moderate inclusion (33–66%) of BM enhanced growth, but high inclusion levels negatively impacted growth due to nutritional deficiencies and potential toxicity. Future research should focus on optimizing BM production to enhance its nutritional profile (specifically arginine content and reduction of ash), evaluating the long-term effects of BM on crayfish health, and exploring strategies for supplementing BM to overcome its limitations.

The study was conducted under controlled indoor conditions, which may not fully reflect the variability encountered in commercial crayfish farming. The use of a commercial fish feed as the basal diet, rather than a specifically formulated crayfish diet, might have influenced the results. The analysis of essential amino acids was incomplete as tryptophan data was excluded due to analytical error. Finally, the study focused primarily on growth parameters and didn't comprehensively assess other physiological aspects of crayfish health.