Dm4000b

The alkaline comet assay performed according to the modified OECD TG 489 protocol. Triplicate slides were prepared per treatment. Sperm sample (20 μL) containing 5 × 10⁵ sperm per mL were suspended in 180 μL of 0.5% (w/v) low melting agarose and 55 μL was immediately pipetted onto a comet slide. These slides were incubated for 30 min. The slides were immersed in 250 mL chilled lysis solution with 10 mM DTT for 1 h in a refrigerator. After lysis, the slides were washed twice with DW for 10 min. The slides were then incubated in alkaline buffer (0.3 M NaCl, 1 mM EDTA) for 20 min in the dark at 4°C. Electrophoresis as performed at 21 V and 300 mA for 20 min. After that, the slides were washed twice with DW for 5 min. The slides were then immersed for 5 min in ethanol. Slides were airdried at room temperature in the dark. Immediately before scoring, slides were stained with 55 μL SYBR green (1 : 10,000 dilution of liquid concentrate). Slides were analyzed using a fluorescence microscope (DM-4000B, Leica Microsystems) at 40× magnification. For each sample, 50 comets per slide were analyzed, with three slides scored per sample. The percentage of tail DNA and OTM were measured according to the DNA damage degree using computer software (Komet 5.5, Kinetic Imaging Liverpool) with a fluorescence microscope (DM-4000B, Leica Microsystems).

To investigate the intestinal morphology of the selected chickens, 3 cm segments from the duodenum, jejunum, and ileum were removed carefully without chyme and were fixed in a 4% paraformaldehyde solution. Then, the segments were successively placed into an ascending gradient of ethanol for dehydratation. After xylene clearing, these samples were embedded in paraffin wax and processed into slices followed by hematoxylin-eosin staining. The villus height and crypt depth were observed and measured under a positive fluorescence microscope (DM4000B, Leica Microsystems, Wetzlar, Germany), and the ratio of the villus height to crypt depth (VCR) was calculated.