Uv 1201

The 2 g/kg of chromium oxide (Cr₂O₃) as indigestible
marker were mixed with diets seven days before fecal sample collection in
order to determine the ATTD of nitrogen (N), dry matter (DM), and gross
energy (GE) of pigs. At the completion of the trial, we have selected one
gilt and one barrow per each pen for fresh fecal sample collection. The
samples were obtained by massaging a pig’s rectum. The samples were
directly put into a chilled box. Later on, we transported the samples to the
lab, and kept at a temperature of −20°C until they were
examined by trained personnel. Following 72 hours of drying at 70°C,
samples were finely powdered and sieved through a 1-mm screen. The AOAC
[25 ] methods were applied to
assess the digestibility of DM, N, and GE. The analysis of ATTD was
conducted using the method utilized in the previous research of Munezero and
Kim [26 (link)]. A UV absorption
spectrophotometric measurement was performed to measure chromium levels
(UV-1201, Shimadzu, Kyoto, Japan). A sample of 2 grams of faecal and feed
was analyzed using an oxygen bomb calorimeter (Parr, 6400 Instrument
Company, Moline, IL, USA). Moreover, in order to calculate the protein, the
N was assessed by using Kjeltec 8600 (Foss Tecator AB, Hoeganaes,
Sweden).

During days 35 to 41, a 0.20% chromium oxide (indigestible marker)-containing
experimental diet was used to feed animals for measuring the nutrient
utilization of dry matter (DM), nitrogen (N), and energy (E). After being
combined, feed specimens were taken from every treatment group. On day 42,
two randomly selected pigs from each pen were used to gather fecal samples
using the rectal massage technique. Then, feed and fecal specimens were
dried in an electric oven (70°C) for 72 h, and later they were
crushed to pass through a 1-mm sieve and collected. The DM, N, and E in feed
and feces were assessed using the AOAC [16 ] method. The concentration of chromium was determined using
ultraviolet spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan). The energy
was measured as the heat of combustion in the specimens, utilizing a bomb
calorimeter (Parr 6100; Parr Instrument, Moline, IL, USA). The indirect
ratio methods were used to calculate the apparent total tract digestibility
(ATTD) using Park et al. [17 (link)]’s procedure. ATTD (%) = [1 − (Nf × Cd)/ (Nd
× Cf)] × 100, where Nf denotes the nutrient concentration in
feces (% DM), Nd denotes the nutrient concentration in diet (% DM), Cd
denotes the chromium concentration in diet (% DM), and Cf denotes the
chromium concentration in feces (% DM).

Determination of lipid peroxidation was performed through measuring serum MDA level according to Nahar et al with some modification.¹¹ A mixture was prepared by mixing 100 μL of serum with 900 μL of 0.9% saline solution. A total of 2 mL of TBA reagent and 30 μL of 50 mM butylated hydroxyl toluene (BHT) had been added. After that, the prepared mixture was incubated for 15 minutes at 60°C and kept in ice water for another 5 min. Then, the samples were centrifuged for 10 minutes at 5000 rpm. Finally, the absorbance of the supernatant was determined spectrophotometrically at 535 nm using 1,1,3,3‐ tetraethoxy‐propane as standard.
In order to determine serum vitamin C (ascorbic acid), 5% TCA was mixed with isolated serum sample in a test tube and centrifuged for 10 min at 3000 rpm. The obtained supernatant was then preserved at ‐ 80°C for further study. UV spectrophotometer (UV‐1201, Shimadzu, Kyoto, Japan) was applied to determine the concentration of ascorbic acid.¹²