2.4. Chemical and microbial analyses
Analysis of DM and CP concentration in the experimental
diets, excreta and probiotic products was done according to
AOAC (1990) methods (930.05 and 976.05, respectively). The
GE was measured by using the bomb calorimeter (model
1261, Parr Instrument Co., Moline, IL), and chromium
concentration was determined with an automated spectrophotometer
(Jasco V-650, Jasco Corp., Tokyo, Japan) according
to the procedure of Fenton and Fenton (1979).
The microbiological assay of faecal samples (d 14 and 28)
and intestinal digesta (d 28) was conducted by culturing in
different media for the determination of total anaerobic
bacteria (Tryptic soy agar), Bifidobacterium spp. (MRS agar),
Lactobacillus spp. (MRS agar+0.02% NaN3+0.05% L-cystine
hydrochloride monohydrate), Clostridium spp. (TSC agar) and
coliforms (violet red bile agar). The microbiological assay of
probiotic products was also carried out by culturing technique.
The L. acidophilus was enumerated using MRS agar+0.02%
NaN3+0.05% L-cystine hydrochloride monohydrate, B. subtilis
by using plate count agar, S. cerevisiae and A. oryzae by potato
dextrose agar. The anaerobic conditions during the assay of
anaerobic were created by using gas pack anaerobic system
(BBL, No. 260678; Difco, Detroit, MI). The tryptic soy agar (No.
236950), MRS agar (No. 288130), violet red bile agar (No.
216695), plate count agar (No. 247940), and potato dextrose
agar (No. 213400) used were purchased from Difco Laboratories
(Detroit,MI), and TSC agar (CM0589) was purchased from
Oxoid (Hampshire, UK). The pH of probiotic products was
determined by pH meter (Basic pH Meter PB-11, Sartorius,
Germany).
2.5. Small intestine morphology
Three cross-sections for each intestinal sample were
prepared after staining with azure A and eosin using standard
paraffin embedding procedures. A total of 10 intact, welloriented
crypt-villus units were selected in triplicate for each
intestinal cross-section as described previously (Jin et al.,
2008). Villus height was measured from the tip of the villi to
the villus crypt junction, and crypt depth was defined as the
depth of the invagination between adjacent villi. All morphological
measurements (villus height and crypt depth)
were made in 10-μm increments by using an image processing
and analysis system (Optimus software version 6.5,
Media Cybergenetics, North Reading, MA).
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