Frequently, several hours can pass before piglets consume feed for the first
time after weaning. In addition to separation from the mother and confrontation
with piglets from another litter, the abrupt change in feed associated with
weaning places great demands on the ability of the animals to adapt (Mormede
and Hay, 2003). After weaning, piglet feed is abruptly changed from mainly
sow's milk to solid feed only which usually reduces the daily energy and nutrient
intake (Pluske et al., 1996). As a result of
the reduction in feed intake there is often a regression of the intestinal villi
with simultaneous deepening of the crypts which are thought to cause a decrease
in the digestive and absorptive capacity of the small intestine (Hampson,
1986; Li et al., 2001; Pluske,
2001). In addition, the change of diet may alter the gastrointestinal microbial
balance which increases the opportunities for pathogens to colonize the gut
(Fuller, 1989; Jensen, 1998).
In the 1st week after weaning there is frequently a depression in growth which
has wide-ranging consequences for the subsequent course of the nursery and finishing
period (King and Pluske, 2003).
To optimize performance of piglets at this stage, in-feed antibiotics have
been used as growth promotants and for therapeutic treatment of gastroin-testinal
diseases (Verstegen and Williams, 2002).
Because of concerns with residues in meat products and bacterial resistance
to antibiotics, investigating alternatives to in-feed antibiotics is required
(Cromwell, 2002). Thus there is an urgent need to remove
antibiotics from the list of feed additives allowed for food-producing animals.
In order to improve pig performance as well as to help pigs resist the common
diseases occurring in early-weaned period, the feed regulations in China allow
the use of Chinese herbal medicine as an alternative to the use of in-feed antibiotics.
The aim of this research was to study the impact of supplementing a piglet diet with different dose of one complex additive of Chinese herbal medicines, selected for their pharmacological properties on growth performance, gut environment and health status in post-weaning piglets. Researchers hypothesized that supplementation of a typical piglet diet with Chinese herbal medicines will improve growth performance and reduce the incidence of diarrhoea in weaned piglets.
MATERIALS AND METHODS
The study was carried out in accordance with the Chinese guidelines for animal
welfare and experimental protocol (Yin et al., 2004).
Composition of the Chinese herbal medicines additive: The Chinese Herbal Medicines additive (CHMD) used in this study is consisted of seven dried Chinese herbs including Astragalus Membranaceus, Scutellaria malt, Glycyrrhiza uralensis, Codonopsis pilosula, Poria cocos, Atractylodes macrocephala and the rate is 2:1:1:1:2:2:2. Before inclusion in the feed, they were mixed according the aforesaid ratio and crumbled to an ultra-fine powder with an average granule diameter of 30 μm, packed in hermetical plastic bags and stored at room temperature. All the medicinal herbs were purchased from WeiMing pharmacy in Hefei city of China.
Animals, diets and experimental design: At weaning, at 21 days of age, 144 crossbred (Duroc x Landrace x Yorkshire) piglets (72 females and 72 males) with a body weight of 5.86±0.24 kg were selected from 18 L (among the original 21 L) that were healthy and had not been treated by any antibiotics and were divided randomly into 4 groups balanced for sex, weight and litter origin. In each group, the piglets were divided randomly into 3 pens (12 animals per pen) and each group was fed one of 4 diets for 3 weeks.
The pens had concrete floors with no litter and each pen was equipped with a feeder and nipple drinker. The nursery had a temperature of 27.0°C in the 1st week after weaning. From week 2 until the end of the nursery, the temperature was decreased weekly by 0.5°C. The photoperiod was controlled to provide 12 h of light and 12 h of dark in the stall. The ventilation also was provided to ensure good air quality. All piglets were vaccinated against pasteurellosis, parathyphoid, asthma and hog cholera.
A basal diet without antibiotics or probiotics was used as control and was
fed to one group (C). The other 3 groups were fed the control diet supplemented
with 0.5, 1 and 1.5% (wt/wt) CHMD (Treatment 1, T2 and T3). The basal diet mainly
contained maize, soyabean meal, expanded soybean, milk replacer, whey powder,
soybean oil and a premix of vitamins and minerals and the nutrient contents
met or exceeded nutrient requirements recommended by National
Research Council (1998). The piglets were fed ad libitum and had
free access to water. The diets were fed in meal form and the CHMD (Sealed,
placed in the cool dry place in separate bags) was mixed into the basal diet
Performance monitoring: The feed offered and refused was weighed daily to calculate daily feed intake. The piglets were weighed at the beginning of the research (at weaning) on day 21 and Body Weight (BW), Average Daily Feed Intake (ADFI), Average Daily Weight Gain (ADWG), Feed/Gain ratio (F/G, total feed consumed (kg) by per kg gain in weight) were measured in the whole period were calculated.
Assessment of severity of diarrhea: To ascertain the health status of
the pigs, faecal consistency was evaluated daily during 21 days after weaning
as follows: score 0: firm, dry faeces; score 1: pasty faeces; score 2: thick,
fluid faeces and score 3: watery faeces (De Cupere et
al., 1992; Kelly et al., 1990). The incidence
of diarrhoea (%) was calculated as the sum of the total number of diarrhoeal
piglets over the period divided by the number of piglet days in the period multiplied
by 100. The rate of diarrhoea (%) was calculated as the sum of the total number
of diarrhoeal piglets over the peirod by the sum of the total number of piglets
multiplied by 100. The index of diarrhoea was calculated as the sum of the diarrhoea
score over the period divided by the sum of the total number of piglets.
Digesta collection and histological measurements and bacterial enumerations: At the end of the 3 weeks study period, 1 pig selected at random from each pen was held under halothane general anesthesia and killed by an intra-cardiac injection of sodium pentobarbital. Stomach, small intestine and caecum, colon were removed and flushed with ice-cold physiological saline solution containing phenylmethyl sulfonyl fluoride (2 L of 0.9% saline, pH 7.4+2 mL of 100 mM phenylmethyl sulfonyl fluoride) to remove any excess blood and 20 mL each of digesta from the stomach and the small intestine were obtained for pH measurement. All pH measurements were made with an electronic pH meter (Accumet Basic, Fisher Scientific) which was standardized with certified pH 4 and 7 buffer solutions.
Approximately 1 g each of digesta sample from the caecum and the colon were obtained for microbial counts. The bacterial flora in digesta samples were estimated by culture methods using selective media. Digesta samples were dissolved in sterile normal saline (0.85%) in a 1:10 dilution. Secondary dilutions were from 10-4-10-5 for the digesta to estimate the E. coli population. For estimation of the lactobacilli population, the secondary dilutions were from 10-5-10-6 for samples. E. coli was cultured in MacConkey agar (MAC) and LAB was cultured in MRS agar (Mann, Rogosa and Sharpe). Each dilution was performed in duplicate and the result was the average of 2 dilutions. The digesta microbial enumerations were expressed as log 10 Colony Forming Units (CFU) in fresh matter.
After blotting the organs with an absorbent study, weight and length (small
intestine) were determined and 10 cm segments of the duodenum, jejunum and ileum
were taken and stored in 10% formalin to fix the villi and the crypts for subsequent
histological measurement according to the procedures described by Owusu-Asiedu
et al. (2003). Briefly, 6 cross-sections were obtained from each
formalin-fixed segment and processed for histological examination using the
standard hematoxylin and eosin method. Villous height was measured from the
tip to the crypt-villus junction and crypt depth was measured from the crypt-villus
junction to the base on 10 welloriented villi per specimen using a Zeiss photomicroscope
equipped with a Sony 3 chip CCD color camera (Carl Zeiss Canada Ltd., Toronto,
Ontario, Canada). Villus:crypt ratio was calculated by dividing villus height
by crypt depth. Duodenum sample was taken at 30 cm away from the stomach. Jejunum
sample was taken at 2 m before the ileal-cecal junction. Ileum was sampled at
30 cm before the ileal-cecal junction. The images were captured using Northern
Eclipse Image Processing Software (Empix Imaging, Inc., Mississauga, Ontario,
Statistical analyses: Data are presented as arithmetic means with standard deviation of the mean (Mean±SD). Differences among groups were compared by SPSS18.0 statistics software using one way ANOVA and LSD method test and p<0.05 was selected as significant standard, p<0.01 was selected as remarkably significant standard.
Growth performance: The performance data measured during 3 weeks period in experiment are shown in Table 1. The initial BW was similar among diets at 5.86±0.24 kg (mean±SD). In the 3 weeks trial, piglets fed the diet containing 1% CHMD had greater (p<0.01) Final Body Weight (FBW), greater (p<0.01) Average Daily Weight Gain (ADWG), greater (p>0.05) Average Daily Feed Intake (ADFI) and lower (p<0.01) F/G compared to the control group. Piglets fed the diet containing 1.5% CHMD had greater (p>0.05) ADWG, greater (p>0.05) ADFI, compared to the control group but without affecting F/G (p>0.05). There were no differences in ADFI, ADWG and F/G between the 0.5% CHMD group and the control group (p>0.05).
Diarrhoea: During the 3 weeks post-weaning, some piglets in all groups
showed diarrhoea symptoms (Table 2). However, none of the
pigs had a diarrhoea score of 3 (watery faeces). In the 3 weeks trial, piglets
fed the diet containing 1% CHMD and 1.5% CHMD both had lower (p<0.01) rate
of diarrhoea, lower (p<0.01) incidence of diarrhoea and lower (p<0.01)
index of diarrhoea compared to the control group. Piglets fed the diet containing
0.5% CHMD had lower (p<0.01) incidence of diarrhoea and lower (p>0.05)
index of diarrhoea compared to the control group. Compared to the1.5% CHMD group,
the 1% CHMD had lower (p>0.05) rate of diarrhoea, lower (p>0.05) incidence
of diarrhoea and lower (p<0.01) index of diarrhoea. Compared to the 0.5%
CHMD group, the 1% CHMD group had lower (p<0.01) rate of diarrhoea, lower
(p<0.01) index of diarrhoea and lower (p>0.05) incidence of diarrhoea.
|| Effects of CHMD on the growth performance parameters of weaning
|In the same row values with different small letter superscripts
mean significant difference (p<0.05) and with different capital letter
superscripts mean extremely significant difference (p<0.01). The same
|| Effects of CHMD on diarrhea parameters of weaning piglets
|| Effects of CHMD on organ weights and digesta pH of weaning
|| Effects of CHMD on LAB and E. coli counts in different
intestinal segments of weaning piglets
Organ weights and digesta pH: Visceral organ weights (length for small intestine) and digesta pH results are shown in Table 3. In the 3 weeks trial, piglets fed the diet containing 1% CHMD had higher (p<0.01) stomach weight compared to the control group. Compared to the control group, the 0.5% CHMD group and the 1.5% CHMD group both had no effect (p>0.05) on stomach weight. Compared to the control group, the 1% CHMD group had longer (p>0.05) small intestine.
Compared to the control group, the 1% CHMD group and 1.5% both reduced the digesta pH of stomach (p>0.05), the 1% CHMD group reduced the digesta pH of duodenum (p>0.05), the 1% CHMD group and 1.5% both reduced the digesta pH of jejunum (p>0.05), the 1% CHMD group and 1.5% both reduced the digesta pH of ileum (p>0.05). The 1% CHMD group had lower (p>0.05) digesta pH of duodenum than the 0.5% CHMD group and 1.5% CHMD group had lower (p>0.05) digesta pH of jejunum than the 0.5% CHMD group had lower (p>0.05) digesta pH of ileum than the 0.5% CHMD group and 1.5% CHMD group.
Bacterial counts: Table 4 shows data relative to microbiota
in the digestive tract characterized as the lactobacilli and E. coli
counts in the middle caecum and in the middle colon. Pigs fed the diets with
1% CHMD had a higher cecal and colonic lactobacilli count (p<0.01), a lower
cecal and colonic Escherichia coli count (p>0.05) and had a higher
LAB: E. coli ratio in the middle caecum (p<0.01) in the middle colon
(p>0.05) than pigs fed the diets without CHMD. Pigs fed the diets with 1.5%
CHMD had a higher cecal and colonic lactobacilli count (p>0.05) and had a
higher LAB: E. coli ratio in the middle caecum (p>0.05) than pigs
fed the diets without CHMD.
|| Effect of CHMD supplemented diets on intestinal morphology
in early-weaned piglets
Pigs fed the diets with 1% CHMD had a higher cecal lactobacilli count
(p>0.05), a higher LAB: E. coli ratio in the middle caecum (p>0.05)
than pigs fed the diets with 0.5% CHMD.
Morphology of the small intestine: Table 5 showed the structural characteristics of the mucous membrane in the small intestine segments. Among dietary treatments various interactions were observed. Diets with 1% CHMD resulted in a greater VH in the duodenum (p<0.01) in the jejunum (p<0.01) and in the ileum (p>0.05), a lower CD in the duodenum (p<0.01) in the jejunum (p<0.01) and in the ileum (p>0.05), a greater (p<0.01) calculated VH:CD ratio in the duodenum, in the jejunum and in the ileum, compared to the control group. Diets with 1.5% CHMD resulted in a greater (p>0.05) VH , a lower (p>0.05) CD and a greater VH:CD ratio (p>0.05) in the duodenum, compared to the control group. Compared to the 0.5% CHMD group, the 1% CHMD group had a greater VH in the duodenum (p<0.01), in the jejunum (p<0.01) and in the ileum (p>0.05), a lower CD in the duodenum (p<0.01) in the jejunum (p>0.05) and in the ileum (p>0.05), a greater VH:CD ratio in the duodenum (p<0.01) in the jejunum (p<0.01) and in the ileum (p>0.05). Compared to the 1.5% CHMD group, the 1% CHMD group had a greater VH in the duodenum (p>0.05), in the jejunum (p>0.05), a lower CD in the duodenum (p>0.05), a greater VH:CD ratio in the duodenum (p>0.05) and in the jejunum (p>0.05).
The inclusion of the Chinese herbal medicines additive in the current experiment
increased FBW, ADWG, ADFI and decreased F/G of the pigs throughout the duration
of the experiment (Table 1). This is in general agreement
with previous studies in weaned piglets given a herbal plant extract or a mixture
of Chinese herbal ultra-fine powder (Kong et al.,
2007), a single Chinese herbal medicine BaZhen (Lien
et al., 2007) or a single Chinese herbal medicine Acanthopanax
senticosus extract (Kong et al., 2009). This
study showed that 1% CHMD supplement in the diet was enough to achieve the most
beneficial effects. This positive response to CHMD maybe due to a number of
Firstly, the improved performance may be attributable to an increase in the
lactobacilli population and a reduction in E. coli populations in the
gut of the CHMD fed pigs (Table 4). Lactobacillus sp.
are known to produce lactic acid, proteolytic enzymes and cellassociated polysaccharide
depolymerases which can enhance nutrient digestion in the gastrointestinal tract
(Veizaj-Delia et al., 2010; Dillon
et al., 2010). Lactobacilli can colonize and adhere to the gastrointestinal
tract epithelium forming a protective membrane against pathogenic microorganisms
while at the same time modulate immunity with stimulating epithelial lymphocytes
(Yu et al., 2008). Lactobacilli sp. are
in abundance post weaning rapidly converting lactose to lactic acid (Pierce
et al., 2006) through fermentation in the hindgut. This further causes
a reduced intestinal pH and thereby unfavorable conditions for coliform bacteria.
Lactobacilli represent the largest group of microorganisms in the small intestine
and are considered important to maintain good intestinal health because of their
ability to control potentially pathogenic groups such as E. coli (Blomberg
et al., 1993; Canibe and Jensen, 2003) and
to optimize immune response (Perdigon et al., 2001).
Accordingly, researchers use the LAB: E. coli ratio as an index of intestinal
equilibrium (Hillman et al., 1995). Xu
et al. (2003) reported that Chinese herbs could reduce the density
of enterotoxigenic E. coli and increase the density of Bacillus acidi
lactici or Bacillus bifidus. The results from the present study also
suggest the potential of CHMD in suppressing pathogenic bacteria and enriching
Secondly, CHMD supplementation exerted a positive influence on intestinal morphology
in the weaned piglets of the experiment (Table 5). The gut
plays an important role in the digestion, absorption and metabolism of nutrients.
Findings from the previous (McCracken et al., 1999;
Fang et al., 2009) study demonstrated that piglets showed a decrease
in villus height and an increase in crypt depth during the weaning process,
villus height and crypt depth are indirect indications of the maturity and functional
capacity of enterocytes and larger villi and crypts are associated with a greater
number of enterocytes (Hampson et al., 1985).
Both VH and CD are related to the absorptive capacity of the mucous membrane
(Buddle and Bolton, 1992). The results indicated that
dietary supplementation with CHMD effectively increased villus height in the
duodenum, jejunum and ileum and decreased crypt depth in the gut, compared with
the control group. The above findings suggest that CHMD could effectively improve
the recovery of stress-induced damage to the gut morphology which might be a
potential mechanism by which CHMD increases the nutrient uptake from intestine
and result in improved growth performance in weaned piglets.
In addition, gastrointestinal pH was measured to provide an indication of the
effect of feeding CHMD-supplemented diets on indicators of gastrointestinal
health. The pH values in the current study are in close agreement with those
reported previously (Owusu-Asiedu et al., 2003).
Decreased pH values in the stomach reduce the gastric emptying rate (Francois,
1962). Higher intestinal pH is thought to provide an optimal environment
for enterotoxigenic E. coli to colonize the villi, leading to diarrhea
(Smith and Jones, 1963). A lower pH, on the other hand
may favor development of beneficial bacte bacteria and/or inhibit development
of harmful bacteria (Fuller, 1977) and has been shown
to have a beneficial effect on nutrient digestibility (Canibe
and Jensen, 2003; Lyberg et al., 2006).
A novel and important finding from the present study is that dietary supplementation
with the CHMD reduced the incidence of diarrhea, the rate of diarrhoea and the
index of diarrhoea in piglets throughout the duration of the research (Table
2). The lower diarrhoea parameters in piglets fed diets supplemented with
CHMD suggests a more healthy gut. Obviously, the anti-diarrhea effect of the
CHMD was great which noted that the antibiotic effect of the CHMD was good.
Diarrhea results from an increase in water secretion from the intestinal epithelial
cells and/or a decrease in the absorption of water and nutrients from the intestinal
lumen. Thus, it is likely that the CHMD regulates these two physiological processes
by improving the metabolism of amino acids and glucose (the major fuels) and
enhancing the anti-oxidant activity in the small-intestinal mucosa (Wu
et al., 2004). Alternatively, the dietary supplementation with the
Chinese herbs enhances the intestinal immune function thereby reducing inflammation
in the small-intestinal mucosa that often occurs in weanling piglets (Nabuurs,
1995). Although, the precise mechanisms are not clear, the results demonstrate
the feasibility of using the Chinese herbs as natural green dietary additives
for early-weaned piglets to replace feed antibiotics.
In summary, these findings suggest that CHMD as a dietary additive could enhance gastrointestinal health by regulating the microbiota composition and maintaining a normal morphology in weaned piglets, thereby decreasing the incidence of diarrhea resulting from weaning stress and results in improved growth performance.
This research was jointly supported by grants from the key scientific and technological project of AnHui province (Grand No. 08010301079), the school young scientists fund of AnHui Agriclutral University (Grand No.2009zr01), the school initial fund for the newly-enrolled scholars of AnHui Agriclutral University (Grand No. yj2008-31).