Stevioside

The animal experiments were performed in accordance with the Animal Care and Use Committee of Nanjing Agricultural University, Nanjing, China (PZ2019064). A total of 192 one-day-old male Ross 308 broiler chicks with similar hatching weights were purchased from a local commercial hatchery. Broilers were randomly allocated to four treatments. Each treatment contained six replicates (cages) of eight broilers per replicate in each group. The present experiment lasted for 21 d (from 1 to 21 d of age). The basal diet used in this study was according to National Research Council (1994) (Table 1). The four experimental treatments were as follows: (1) non-challenged broilers fed a basal diet (CON); (2) non-challenged broilers fed a basal diet supplemented with 250 mg/kg stevioside (STE); (3) LPS-challenged broilers fed a basal diet (LPS); (4) LPS-challenged broilers fed a basal diet supplemented with 250 mg/kg stevioside (LPS + STE). Stevioside used in this study were purchased from Macklin Inc (Shanghai, China) with a purity of more than 98%. The supplemental stevioside level was optimized according to previous studies [11 (link),14 (link),19 (link)]. All broilers were housed in four-level cages in a temperature- and light-controlled room with continuous light in Nanjing Agricultural University. All broilers had ad libitum access to mash feed and water. The temperature of the room was maintained at 32 to 34 °C for a week, and it was then gradually decreased by 1 °C every 2 d until a final temperature of 26 °C was achieved. Furthermore, all broilers were inoculated with a Newcastle disease vaccine on 7 d and with an inactivated infectious bursal disease vaccine on 14 d. The experiment consisted of a 2 × 2 factorial design. The main factors were (1) Lipopolysaccharide (LPS)-challenge, injection with LPS or saline, and (2) diet, basal diet with 0 or 250 mg/kg stevioside. LPS from Escherichia coli (L2880, Sigma Aldrich Inc., St. Louis, MO, USA) was dissolved in 0.9% sterile saline solution. At 7:00 am of 17, 19, and 21 d, LPS-challenged groups received an intraperitoneal injection of LPS solution at a dose of 1 mg/kg, whereas the CON and STE groups received a sterile saline injection. The dosage and injection of LPS were referred to as available findings [2 (link),22 (link)]. At 17 d and 21 d, all broilers were weighed to calculate average daily gain (ADG). The feed consumption by the broilers in a replicate (cage) was recorded to calculate average daily feed intake (ADFI). The spilled feed was carefully collected and weighed in order to correct the final data of ADFI. Feed conversion rate (FCR) was defined as ADFI: ADG.
Provided the following % per kilogram in completed diet: vitamin A, 12,500 IU; vitamin D3, 2500 IU; vitamin E, 80 mg; vitamin K, 2.65 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; nicotinic acid, 50 mg; pantothenic acid, 20 mg; vitamin B6, 4 mg; folic acid, 1.25 mg; vitamin B12, 0.025 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; niacin, 50 mg; Fe, 80 mg; Zn, 75 mg; Mn, 100 mg; Cu, 8 mg; I, 0.35 mg; Co, 0.2 mg; and Se, 0.15 mg.

SGs (steviol glycosides stevioside and rebaudioside A) were separated and quantified using high-performance liquid chromatography (HPLC) by the modified method [53 (link)]. An Agilent 1200 series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) with a diode array detector was used. Samples were filtered through a syringe filter with a PVDF membrane (pore diameter, 0.22 µm) and separated on a reversed phase column (Purospher STAR RP-18e 5 µm Hibar 2 × 250 mm, Merck, Germany) with a precolumn. Injection volume was 10 µL at 70 °C column temperature. Isocratic elution with a mobile phase consisting of 70% deionized water acidified with HCl to pH 2.75 and 30% acetonitrile was used for separation, with an additional washing step with 50% acetonitrile at a flow rate of 0.25 mL min⁻¹. Stevioside and rebaudioside A were detected at the wavelength of 210 nm. Identification of stevioside and rebaudioside A in the samples was done by means of retention time and UV spectra. Calibration was done by plotting the peak area responses against the concentration values in the concentration range from 1 to 1000 µg mL with linear dependence for both analytes. Each analysis was repeated three times, and the mean value was used. Working under an isocratic mode, less than 10 min was required to separate the components of interest without affecting resolution.

Trophozoites were incubated for 24 h at 37 °C with stevioside at a concentration of 9.53 mM leaving trophozoites under normal culture conditions. After the incubation, trophozoites were harvested by cooling at 4 °C for 15 min. Trophozoites (2.5 × 10⁵) were seeded in 24-well plates treated with 100 μg fibronectin. Adhesion assays were carried out at 15, 30, and 60 min. Cell viability was evaluated by trypan blue, and the percentage of adhered cells was determined by a light microscope.

The measurement of the cytopathic effect was performed according to [11 (link)]. Briefly, monolayers of the CaCo2 cell line were cultured, and after the end of the incubation time these cells were interacted with trophozoites previously treated with 9.53 mM stevioside for 24 h and untreated trophozoites for 15, 30, and 60 min. After the interaction times, as many trophozoites were removed as possible, and the cells were washed with cold 1× PBS. Subsequently, the cells were fixed with 2.5% paraformaldehyde. The remains of the monolayers were stained with methylene blue to later be subjected to chemical lysis of the cells via dye extraction and quantified by spectrophotometry at a 660 nm wavelength. Also, CaCo2 cells were attached in silanized coverslips, interacted with trophozoites as mentioned above, fixed with 2.5% paraformaldehyde, and washed with 1× PBS, then stained with Hematoxylin & Eosin (H&E) to be analyzed with a light microscope.