The Animal Conservation and Utilization Committee of the Jinzhou Medical University approved the animal use agreement (Grant No. JZMULL2022011).

The test waterline had a length of 60 meters, with two test cages at the front, end, and 30 meters in the middle, resulting in a total of six test cages per waterline. Four waterlines comprising a total of 24 test cages, were selected for the experiment. A total of 480 healthy 1-day-old AA+ broilers weighting 46.77 ± 0.50 g were randomly divided into four groups with six replicates of 20 birds per group. The broilers were housed in test cages along each waterline. The experiment lasted for 42 days. In the control group, tap water was provided through the waterline, while in the test groups, the water lines were supplemented with NaDCC at concentrations of 10, 30, and 50 mg/L. The test group consumed tap water containing NaDCC on specific days during the experiment. For the rest of the time, the treatment was the same as the control group. It should be noted that long-term use of chlorinated water can damage the waterline and may have some impact on the broiler organism.

The NaDCC used in this experiment was provided free of charge by Jinzhou Zhongke Genetic Testing Service Co. It was in powder form and had an effective chlorine content of 60.35%.

All birds were housed in experimental cages measuring 1.25 × 0.80 × 0.50 m³ per cage, located in a dedicated test broilers room. The test broilers room was fully enclosed and equipped with an automatic environmental control system to maintain optimal temperature and humidity conditions. The temperature was initially set at 33°C and gradually reduced by 3°C every week until reaching 22°C, while the relative humidity was maintained at 65%. The birds had unrestricted access to feed and water. The immunization program for the test broilers include vaccination against Newcastle disease and avian influenza at 7 days of age, followed by a Newcastle disease vaccination at 21 days of age administered through the water. The diets provided to the broiler were formulated to meet the nutritional requirements recommended by the NRC (1994) and were modified to suit the specific nutritional needs of Chinese broilers. The diets were provided in mash form and their composition is detailed in Table 1.

On days 21 and 42 of the experiment, 10 mL of NaDCC-free drinking water was collected from the nipple drinker above each test cage. Additionally, 1 g broiler fecal samples was collected from each replicate and immediately transported to the laboratory on ice, following the method described by Dang et al. (28). The number of E. coli, Salmonella, S. aureus and Moulds in the water samples was determined using the plate count method. The number of microorganisms in the waterline was expressed as log₁₀ colony forming units per mL of water. Similarly, the number of microorganisms in the fecal samples was expressed as log₁₀ colony forming units per gram of feces.

The data was analyzed using a completely randomized grouping design. Multiple comparisons of significant differences in means were conducted using the one-way ANOVA LSD method in SPSS 25.0, and the results were visualized using Graphpad Prism 8. The results were presented as the mean and standard deviation, with a significance level of p < 0.05 indicating a significant difference. Correlation analysis was performed using R (V4.0.2), and the results were displayed using the R (V4.0.2) corrplot package. Pearson correlation analysis was carried out for the 21 and 42-day indicators, and correlations with a significance level of p < 0.05 and an absolute correlation coefficient greater than 0.6 or less than-0.6 were considered significant.

As shown in Table 2, the addition of 50 mg/L NaDCC to the waterline significantly increased ADG in broilers from 1 to 42 days (p < 0.05). The addition of NaDCC at 10 mg/L and 30 mg/L also increased ADG in broilers compared to the control, but not at a significant level (p > 0.05). The addition of NaDCC to the waterline had no significant effect on ADFI, F/G, and AWC in broilers (p > 0.05). NaDCC supplementation in drinking water had a negative linear effect on F/G in 22–42 day old broilers (p = 0.041) and a positive linear effect on ADG in 1–42 day old broilers (p = 0.012), with a quadratic curve effect (p = 0.027).

As shown in Figures 1, 2, the addition of NaDCC to the waterline did not have a significant effect on antibody potency and various biochemical parameters in broiler serum (p > 0.05).

As shown in Figure 3, NH₃ emissions from broiler feces continue to rise over time, while H₂S emissions increase initially and then decline. Additionally, both NH₃ and H₂S emissions were consistently lower in the treatment group compared to the control group.

According to Figure 4, the addition of NaDCC to the waterline significantly reduced the levels of E. coli, Salmonella, S. aureus, and Moulds in the waterline (p < 0.05).

As shown in Figure 5, the addition of NaDCC to the waterline resulted in significantly lower levels of E. coli, S. aureus and Moulds in broiler feces at 21 and 42 days (p < 0.05). The addition of NaDCC to the waterline reduced Salmonella levels in broiler feces at 21 days, but not to a significant level (p > 0.05), while it significantly reduced Salmonella levels in broiler feces at 42 days (p < 0.05).

As shown in Figures 6, 7, a significant positive correlation (p < 0.05, R > 0.6) was observed between the microbial content (E. coli, Salmonella, S. aureus and Moulds) in the waterline and feces. However, the microbial content in the waterline and manure showed a negative correlation with production performance indicators (ADG, ADFI, F/G and AWC), although these correlations did not reach significant levels (p > 0.05).

Drinking water and drinking water systems play an important role in the overall health and performance of broiler chickens (29). This pilot study found that the addition of NaDCC to broiler drinking water systems significantly reduced the levels of E. coli, Salmonella, S. aureus, and Moulds in both the waterline and broiler feces. Correlation analysis revealed a significant positive correlation between microbial levels in the waterline and feces. Generally, bacterial infections in broilers are caused by microorganisms commonly found in the poultry environment. In this context, colibacillosis caused by E. coli (30), paratyphoid infection caused by Salmonella (31), necrotizing enteritis caused by Clostridium perfringens (32), as well as S. aureus infection (33) and Pseudomonas aeruginosa infection (34) could be important contaminants leading to issues in 3–6 week old chickens.

NaDCC is a highly effective disinfectant containing two active chlorine atoms that disrupt cell membranes, nucleic acids, and proteins, thereby causing oxidative degradation of microorganisms (35). It has been reported to immediately kill E. coli, S. aureus, and fungi (36). Jang et al. (37) demonstrated that a 0.3% NaDCC solution effectively kills Salmonella even at −10°C after only 1 min of treatment, while a 0.1% glutaraldehyde solution requires 10 min of treatment at 25°C. Ferreira et al. (38) found that using a disinfectant containing 0.67 g/L sodium dichloroisocyanurate for 15 min effectively kills Pseudomonas aeruginosa, E. coli, Salmonella, and Clostridium perfringens in disinfection tests. Increasing the concentration to 0.93 g/L and treating for 20 min eliminated all Staphylococcus aureus. In this experiment, the addition of NaDCC to the drinking water of broilers demonstrated a reduction in the number of pathogenic bacteria entering the broiler by reducing the amount of these bacteria in the waterline, consequently leading to a decrease in pathogenic bacteria in the feces.

Drinking clean and hygienic water is essential for the welfare of broilers. This pilot study found that drinking water containing NaDCC did not affect the normal water intake of broilers. However, it significantly increased the ADG when consumed by broilers at a concentration of 50 mg/L. Nonetheless, at this stage, there is insufficient information to confirm that NaDCC can directly increase the body weight of broilers. Based on the results of this experiment, it is hypothesized that the potential cause of weight gain in broilers is the addition of NaDCC to their drinking water, which eliminates pathogenic microorganisms in the drinking water and waterline biofilm. When chickens consume healthy and clean water, it reduces their intake of pathogenic bacteria, leading to an increase in beneficial bacteria in their gut. By improving the micro-ecological environment of the gut, broilers tend to have increased feed intake and subsequently gain more body weight. However, the exact mechanism underlying this hypothesis requires further in-depth study.

It has been demonstrated that birds can tolerate free chlorine residues of 50 ppm in drinking water without any adverse effects (39, 40). Similar results were obtained in this experiment, as the broiler serum antibodies and biochemical parameters were not affected by the drinking water containing NaDCC. More-Bayona et al. (41) reported an increase in macrophages and a decrease in CD8+ lymphocytes in the peritoneal cavity of broilers that consumed well water containing a mixture of biological preparations for an extended period. They also observed varying degrees of inflammatory responses in the organism caused by fungi and bacteria present in the water. Therefore, occasional consumption of chlorinated drinking water is necessary for the health of broilers.

Under anaerobic conditions, microbial decomposition of feces produces certain gaseous compounds, mainly from carbohydrates and proteins (42). In this experiment, drinking water containing NaDCC significantly reduced NH₃ and H₂S emissions from feces. Previous reports have shown that when the number of E. coli and Salmonella in feces is reduced, fecal NH₃ and H₂S emissions are also reduced (43). Prolonged exposure of broilers to NH₃ has been shown to affect the structure of their cecal flora and, consequently, their growth performance (44). Additionally, sudden releases of H₂S from manure stored in chicken houses can increase the concentration of toxic substances in the room, leading to the death of many animals and humans (45). While birds are not as sensitive to the toxic effects of hydrogen sulfide as mammals, high concentrations of this gas in the air can still negatively impact production parameters in laying hens and broilers (46). Therefore, improving the farm environment is equally important for the healthy growth of broilers. Based on the results of this experiment, NaDCC is highly effective in waterline treatment. Further research can focus on optimizing the dosage to reduce resource consumption while maintaining efficacy.