The protocols of the research were formally approved by the Animal Ethics Advisory Committee of Hue University, Vietnam (Approval No. HUVNO39.C; 20 March 2025).

Three hundred male yellow-feathered Rilai one-day-old chicks were purchased, individually weighed, and randomly assigned to six treatments with five replicate pens of ten birds each in a completely randomized design. The experiment was performed in the natural summer season in Central Vietnam in an open-sided house. For the first three days, all the chicks were fed on an anti-stress additive in drinking water. During the 90-day trial, the birds were freely exposed to commercial corn-soybean meal diets, which were formulated to meet the feeding requirements of the Vietnamese. The treatments included: (1) plain drinking water (negative control), (2) oxytetracycline at 100 mg/kg diet (positive control), or (3-6) LPFS supplemented in drinking water at 25, 50, 75, or 100 mg/L, respectively. The fermented extract was made fresh every morning and given ad libitum.

Fresh shallot (Allium cepa L.; GenBank ID: NC_057575.1) bulbs, which are harvested at 4-5 months of maturity and produced according to VietGAP biosecurity regulations (TCVN 11892-1:2017), were chosen as the starting material. The outer dry scales were removed, the bulbs were washed, and the edible part was homogenized to allow fermentation. The procedure described by (V. et al., 2024), with slight modifications, was followed for lactic acid fermentation. L. plantarum strain 1582 at the initial concentration of 1 x 108 CFU/mL was inoculated in shallot mash. The fermentation substrate contained 5% (w/v) NaCl and 3% (w/v) glucose, and it was incubated at 37 oC over 72 h under anaerobic conditions with mild agitation at 60 rpm. The fermented solution was then filtered to get the aqueous extract, which was then diluted in the drinking water and used. The final fermented extract had an estimated viable Lactiplantibacillus spp. of 2.7-3.0 x 108 CFU/mL. Major nutritional and bioactive components of the extract are shown in Table 1.

From the first day to the last day (day 35): The initial day and last day (day 35) were assigned to the birds as a control group. During the 36^th to 90^th day, they were fed on a finisher diet. In T1, the baseline diet was provided to the birds without antibiotics, and in T2, the birds were fed the baseline diet with the addition of 100 mg/kg diet of oxytetracycline. In T3 and T4, T5, and T6, the basal diet (no antibiotics) was administered to birds, and their drinking water was fed daily with fermented shallot extract at 25, 50, 75, and 100 mg/L. New dilutions were made daily in the morning and served ad libitum. The composition of the diet complies with the feeding standards of the Vietnam Ministry of Agriculture and Rural Development (TCVN 2265:2020) and is summarized in Table 2.

During the experiment, feed and water were given freely. Ambient dry-bulb temperature and relative humidity were measured at 30-minute intervals with a digital thermo-hygrometer set at bird height. In Figure 1, the temporal thermal pattern is depicted. Heat load was estimated using the temperature-humidity index according to Habeeb (2018): THI = Tdb - [(0.31 - 0.31 x RH) x (Tdb - 14.4)], where RH = RH%/100. Interpretation of THI values was made as follows: no heat stress, THI < 27.8; moderate heat stress, 27.8 <= THI < 28.9; severe heat stress, 28.9 <= THI < 30.0; and extremely severe heat stress, THI >= 30.0. The records of the environment revealed that there was extremely severe heat load in une and July and severe heat load in August, which confirmed the chronic natural summer heat challenge during the study period.

To measure the body weight gain and feed conversion ratio (FCR), the total feed intake and body weight for each replicate were recorded weekly on a replicate basis throughout the experiment by using standard procedures as described by (V. et al., 2025). One bird was randomly selected from each replicate pen at day 90, resulting in five birds per treatment (n = 5) and 30 birds in total. Blood samples were taken at the time of slaughter and examined for the lipid profile, liver function, acute phase proteins, corticosterone, and heat shock protein concentrations.

Total serum cholesterol, triglycerides (TG), and high-density lipoprotein cholesterol (HDL) were measured using an automatic analyzer (Automatic analyzer 902, Hitachi, Germany), and very low-density lipoprotein cholesterol (VLDL) and low-density lipoprotein cholesterol (LDL) were estimated using the Friedewald equations: LDL = Total cholesterol - HDL - VLDL, where VLDL = Triglycerides/5 (Friedewald et al., 1972). The following parameters for liver function were determined using a BA400 biochemical and turbidimetry analyzer from Spain (Manual code TEUS00048-07-EN): alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and aspartate aminotransferase (AST).

Using commercial enzyme-linked immunosorbent assay (ELISA) kits (QAYEE-BIO, China), the concentrations of serum amyloid A (SAA), alpha-1-acid glycoprotein (AGP), ceruloplasmin (CP), and heat shock protein 70 (HSP 70) in each blood serum sample were measured. Although the working phases of all the ELISA kits are similar, each kit uses a unique set of standards with known concentrations. A microplate reader (Bio-Rad Microplate Reader, USA) was used to determine optical density (OD) at 450 nm. The standard curve linear regression equation was established using the concentration of the standards and the corresponding OD values, and the sample concentration was computed using this same equation.

Data were analyzed by one-way analysis of variance (ANOVA) using SPSS version 21. For growth performance traits, the replicate pen was considered the experimental unit (five replicates per treatment). Weekly measurements were collected throughout the trial for management and monitoring purposes; however, the inferential analysis reported in Table 3 focused on biologically meaningful phase endpoints, namely day 35 (starter phase) and day 90 (overall final performance). For serum biochemical and stress-related measurements, one bird sampled from each replicate pen was used as the experimental observation (n = 5 per treatment). Because the primary objective was to compare treatment effects at these prespecified phase endpoints rather than model repeated trajectories over time, Time/Week was not included as a fixed factor in the primary model. When treatment effects were significant, means were separated using Tukey’s post hoc test. Results are presented as mean ± SE, and differences were considered significant at p<0.05.

The effects of LPFS supplementation on the growth performance of Rilai chickens reared under chronic natural summer heat exposure are presented in Table 3. The sustained thermal load documented by the environmental profile (Figure 1) adversely affected the control group (T1), which exhibited the lowest body weight gain and the poorest overall feed efficiency. LPFS supplementation significantly mitigated these negative effects (p<0.05). During the starter phase, treatment differences were already evident for final body weight and body weight gain, whereas the clearest treatment-related improvements in cumulative feed conversion became apparent by the final 90-day evaluation. Overall, birds receiving the fermented extract used nutrients more efficiently and grew better than untreated birds despite a sustained summer heat challenge.

As shown in Figure 2, heat load was associated with unfavorable changes in serum lipid profile and liver-related enzymes. The control group exhibited the highest levels of total cholesterol, triglycerides, and LDL-cholesterol. In contrast, LPFS supplementation dose-dependently ameliorated these parameters. Groups T5 and T6 showed a significant reduction in serum cholesterol and triglycerides compared to the control (p<0.05), while HDL-cholesterol levels tended to increase. Additionally, serum activities of AST and ALT, which are markers of hepatocellular damage, were significantly elevated in the control group but were effectively maintained at lower levels in the T5 and T6 groups (p<0.05), suggesting a hepatoprotective effect of LPFS against heat-induced oxidative damage.

The impact of LPFS on physiological stress and oxidative status is summarized in Figure 3. Chickens in the control group exhibited significantly elevated serum Corticosterone and Heat Shock Protein 70 (HSP70) levels (p<0.05), confirming a state of acute heat stress. However, supplementation with LPFS significantly downregulated these stress biomarkers, with the lowest levels observed in the T6 group. Regarding oxidative stability, the malondialdehyde (MDA) content (or TBARS value), a marker of lipid peroxidation, was significantly lower in the LPFS- treated groups compared to the control (p<0.05). At the same time, antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) were enhanced, especially in T5 and T6. Together, these responses indicate that the fermented extract strengthened endogenous antioxidant defense in chickens exposed to chronic summer heat.