## 4 Discussion
Assessing enzyme activity is important for understanding how the organism adapts to dietary composition. Under cold stress, shrimp tend to rely more on proteins and lipids as energy sources [55]. A previous study reported enhanced protease activity in the hepatopancreas with FPE supplementation at 0.1% or 0.15% [29]. However, in the present study, no effect of FPE supplementation was observed on the activity of digestive enzymes. This absence of response may be associated with the chronic exposure to a suboptimal temperature (22 \({}^{\circ}\)C), as cold stress is known to reduce metabolic activity [47] and suppress the expression and secretion of digestive enzymes, thereby limiting the organism's capacity to respond to functional dietary stimuli. An effect was also observed regarding the total bacterial count in the gastrointestinal tract of the shrimp. Higher numbers of heterotrophic bacteria and lower _Vibrio_ spp. counts were found in the STD and STD+1 treatments, whereas the opposite trend was clearly observed in the STD+2 group. _Vibrio_ bacteria occur naturally in marine and estuarine waters and are considered opportunistic pathogens that can become harmful when shrimp are under stress. They are mesophilic organisms, reproducing optimally between 20 and 35 \({}^{\circ}\)C, with reduced activity below 20 \({}^{\circ}\)C [56; 57]. Nevertheless, no signs of vibriois were observed, and survival rates remained above 95% in all treatments. The observed alteration in bacterial composition in the STD+2 group may be related to the higher inclusion level of FPE, which contains free amino acids, soluble peptides, and nucleotides. One hypothesis is that this nutritional profile may have selectively favored the proliferation of _Vibrio_ spp. over other heterotrophic bacteria. Alternatively, a possible overstimulation of palatability mechanisms at high FPE doses may have caused digestive imbalance, which, in turn, could have altered the gut microbial environment. Although these explanations remain speculative, they highlight the importance of evaluating microbiota shifts when formulating functional feeds at higher inclusion levels. A study conducted on shrimp farms in northeastern Brazil identified 145 _Vibrio_ spp. strains, with 62 strains isolated from water and 56 strains isolated from shrimp. Colony counts ranged from 2.00 \(\times\) 10\({}^{2}\) to 1.42 \(\times\) 10\({}^{8}\), but no positive correlation was found between the presence of _Vibrio_ and shrimp mortality [58]. Differences exist between the abundance of _Vibrio_ in the gastrointestinal tract and their virulence, as shown in studies by [PERSON] et al. (2015) [59] and [PERSON] et al. (2014) [60], who demonstrated that AHPND-causing strains vary in their pathogenic potential. Monitoring _Vibrio_ spp. abundance remains essential for maintaining the health of _P. vannamei_, particularly when quantification is associated with lesions, clinical signs, or increased mortality. Shrimp survival after the low-salinity shock challenge was similar across all treatments. In a previous study conducted at 29 \({}^{\circ}\)C, the same additive (FPE at 1 and 2 g kg\({}^{-1}\)) clearly improved shrimp survival under low-salinity shock [30]. However, in that study, the salinity stress was less intense, as shrimp were exposed to freshwater for only one minute. In the present study, the physiological stress was more prolonged: animals were abruptly transferred from a salinity of -32.1 g L\({}^{-1}\) to 2.8 g L\({}^{-1}\) and maintained under these conditions for 24 hours before being returned to full-strength seawater. Hemolymphs were collected from shrimp before and after the low-salinity shock to compare parameters at 0 h and 24 h after the stress. The effect of the challenge was evident in all measured hemato-immunological parameters, except for the agglutination titer. Hemocytes are well-known immune defense cells in shrimp, acting as the first barrier through phagocytosis to prevent the invasion of foreign particles.