Acid insoluble ash (AIA) has been used as a marker for nutrient digestibility determination in various animals with conflicting results. Lack of reliability for AIA analysis has been hypothesized as a cause. For validation, the present study compared AIA with yttrium oxide for determining apparent digestibility coefficients (ADCs) of nutrients in rainbow trout and hybrid striped bass, using a newly developed method for AIA measurement. Twelve diets were made by spiking a reference mixture with Y 2 O 3 at 0.1% and Celite at 0, 0.3 and 0.6%, and mixing each with soybean meal, distillers' dried grains with solubles, or fishmeal at a 70:30 ratio, respectively. Trout and bass were grown in 36 tanks, three tanks per diet. Feces were collected by stripping. With both fish species, ADCs of dry matter, protein, fat, energy and phosphorus, based on yttrium retention were consistent and realistic. However, ADCs by AIA were significantly lower and variable, with many being negative, even though the issue with AIA analysis had been addressed. Further investigation revealed that yttrium content as % total ash in feces was higher than that in diets, but AIA as % total ash in feces was lower than that in diets. The observed low or negative ADCs by AIA likely resulted from slower AIA movement than nutrient digesta through fish gastrointestinal tracts, which was compounded by using stripping for fecal collection. Therefore, further studies incorporating methods of collecting naturally defecated feces are needed before definitive conclusions on AIA reliability as a marker can be made. • AIA was compared with yttrium oxide for determining ADCs of nutrients in rainbow trout and hybrid striped bass. • The combination of using yttrium oxide as a marker and stripping for fecal collection was effective for ADC determination. • The combination of AIA as a marker and stripping for fecal collection was ineffective for ADC determination. • In determining nutrient ADCs in fish, effects of inner markers and fecal collection methods and their interactions need to be taken into consideration. • Further studies incorporating methods of collecting naturally defecated feces are needed before a definitive conclusion on AIA reliability as a marker can be made.

Intensive farming in crowded cages increases antibiotic treatments, promoting antimicrobial resistance (AMR) in fish-associated bacteria. Combined with environmental pollution, this accelerates AMR emergence, impacting food safety and ecosystems. To better understand AMR dynamics in aquaculture, this study examined antibiotic resistance genes (ARGs) in farmed gilthead sea bream (Sparus aurata) and in environmental samples, including seawater, sediments, and feed. Sampling was conducted across the full production cycle in three Mediterranean countries (Italy, Greece, and Spain) with wild S. aurata specimens also collected in Italy and Greece as a comparative baseline. Shotgun metagenomics revealed distinct ARG profiles between farmed fish and their surrounding environment, with notable shifts from land-based phases (eggs to juveniles) to open sea farms (juveniles to adults). Beta-lactam resistance dominated in adult farmed specimens across all sites, underscoring its prevalence in Mediterranean aquaculture. Interestingly, Greek wild fish samples lacked beta-lactamases. This study emphasizes the effectiveness of metagenomics-based approaches for tracking ARGs in fish farming, and underlines its potential to mitigate risks to human health, preserve aquatic ecosystems and make aquaculture more sustainable.