Livestock plays a significant role in improving living conditions of the poor. The purpose of livestock keeping of small-scale farmers in the Southern region is for the draught power, food, cash income, and organic fertilizer from faeces (Samkol and Lukefahr, 2008). Recent outbreaks of Avian Influenza in birds have occurred in several countries in the world, such as in Italy, Spain, and Hong Kong. One study in Indonesia showed that 76% of human highly pathogenic avian influenza (HPAI) H5N1 cases were proven to be associated with poultry contact (Sedyaningsih et al., 2007). Other studies in Thailand, Vietnam, and China have suggested the association of human infection and poultry outbreaks with several risk factors, including demographic characteristics of humans and animals, i.e., human density and poultry density (Gilbert et al., 2008 and Tiensin et al., 2009).
So, Rabbit production is a new development in the region, which plays an important role in view of the economic risks by the spread of Asian bird flu (Otte et al., 2007). According to the FAO (2001), backyard rabbit keeping provides additional income and supplies additional protein for poor rural and urban households with low investment and labor inputs. Rabbits have small body size, short generation interval, high reproductive potential, rapid growth rate, genetic diversity, and the ability to utilize forages and by-products as major diet components that make the animal appropriate for small livestock keeping in developing countries (Cheeke, 1986). Nowadays, rabbit breeding has an increasing impact due to the nutritional value of meat and economic traits of breeding. Development in this animal industry also induces to search for new technologies to improve rabbit health and welfare.
The spread of enteric diseases and in particular epizootic rabbit enteropathy has negatively affected the health status and caused decrease in animal perfomance in rabbit farms and largely increased antibiotics use as a preventive method (Duperray et al., 2003). However, recent issues with bacterial resistance to these antibiotics urged scientists to find alternatives to the use of antibiotics (Smith et al., 2002). In addition, on the base of consumer demand, European legislation is banning the use of antibiotics as growth promoters and is working towards a reduction of therapeutic antibiotics for all livestock production, to avoid crossed resistance in humans and improve food safety.
Thus, Probiotics appears as possible alternative feed additives to modulate intestinal microflora and improve animal health (Medina et al., 2002). However, studies showing the effects of combination of different probiotics trains on gut health and growth of rabbits are also limited.
OBJECTIVES OF STUDY
The objectives of this study are investigation the effects of Lacto-bacillus in digestibility, ceacal fermentation, microbial protein synthesis and growth performance of weaning rabbits.
KEYWORDS (ENGLISH)
Weaning rabbits, digestibility, ceacal fermentation, growth performance
LITERATURE REVIEW
The digestive physiology of the rabbit
The digestive system of the rabbit
The digestive system of the rabbit is characterized by the relative importance of the caecum and colon when compared with other species (Portsmouth, 1977). As a consequence, the microbial activity of the caecum is of great importance for the processes of digestion and nutrient utilization, but also in the control of digestive pathologies. Furthermore, caecotrophy, the behaviour of ingestion of soft faeces of caecal origin, makes microbial digestion in the caecum more important for the overall utilization of nutrients by the rabbit (Carlos de Blas and Julian Wiseman, 2010).
Stomach: the first important compartment of the digestive system of the rabbit is the stomach; this has a very weak muscular layer and is always partially filled. The stomach pH ranges from 1 to 5, depending on site of determination, the presence or absence of soft faeces, the time from feed intake and the age of the rabbit (Carlos de Blas and Julian Wiseman, 2010).
Small intestine: the small intestine is the site where the greater part of digestion and absorption take place by passive or active transportation throughout the mucosa. Digestibility at the end of the ileum accounts for 0.8-1 of the total dietary amino acid and starch digestibility (Carabano et al., 2009). The pH of the small intestine is close to 7 (Nicodemus et al., 2002).
Large intestine: The caecum is characterized by a weak muscular layer and contents. The caecal contents are slightly acid (pH 5.4-6.8) (Garcia et al., 2002). Gut-associated lymphoid tissue (GALT) and specialized cells regulate the interaction of the gut mucosa with the microbiota and develop the mechanisms of tolerance and protection against pathogens.
Age-related changes in the morphology and function of the digestive system
The stomach glands are evident in late fetuses (26 days' gestation) and true villi and intestinal glands are observed at 29 days' gestation. During the suckling period, the mucosal glands are able to produce enzymes to digest the main components of the milk. Lactase activity is highest until 25 days of age and sucrase and maltase rise until reaching the adult level at around 28-32 days (Gallois et al., 2008b). At round 18 days of age the suckling rabbit begins to eat solid food and decrease its milk intake and the caecum and colon develop faster than the rest of the digestive tract (Gallois et al., 2005). From 3 to 7 weeks of age the caecum is filled by digesta and microbiota, and its contents reach a peak at 7-9 weeks of age. The pH of the caecum is also affected by age and decreases from 6.8 at 15 days of age to 5.6 at 50 days of age (Padilha et al., 1995).
The role of the intestinal flora in the digestion and absorption of nutrients
The presence of the microbial population in the caecum, together with caecotrophy, permits the rabbit to obtain additional energy, amino acids and vitamins. The main genus of the microbial population in the caecum of the adult rabbit is Bacteroides (Gouet and Fonty, 1973), which comprises 109-1010 bacteria g−1. During the first week of age, the digestive system of the rabbit is colonized by strict anaerobes, predominantly Bacteroides. At 15 days of age, the numbers of amylolytic bacteria seem to stabilize, whereas those of colibacilli decrease as the numbers of cellulolytic bacteria increase (Padilha et al., 1995). As a result of the fermentative activity of the microflora, VFAs are produced in the proportion of 60-80 mol of acetate, 8-20 mol of butyrate and 3-10 mol of propionate 100 mol−1 of VFAs (Garcia et al., 2002). However, these proportions change with increases in the acetate concentration from 15 to 25 days of age and a reversal of the propionate to butyrate ratio from 15 to 29 days of age (Padilha et al., 1995).
Fermentation patterns
The result obtained Gidenne and Bellier (1992) in adult animals showed that the VFA concentration in caecal contents depends on the time of feeding, rising to a maximum 5 h after feeding. Caecal VFA concentrations are greater during the hard faeces than during the soft faeces excretion period. According to Bellier et al. (1995), this increment could have two causes: (i) the greater flow of substrate to the caecum related to an increase in feed intake during this period; and (ii) enrichment of the microbial population as a consequence of antiperistaltic movements of the proximal colon. Caecal pH varies inversely to the increase in VFA concentration. Smaller values of caecal pH have been observed during hard faeces excretion.
Rate of passage
The passage of feed through the stomach of the rabbit and caecum is relatively slow and varies between 3-6 and 4-9 h, respectively (Gidenne and Poncet, 1985). However, transit is very fast in the small intestine. Estimated retention times in the jejunum and ileum are 10-20 and 30-60 minutes, respectively (Lebas, 1979). Taking into account the entire digestive tract, the mean retention time varies from 9 to 30 h (Laplace and Lebas, 1977).
Nutrient requirement of the growth rabbit
Table 1: Nutrient requirements of intensively rabbits, as concentration kg−1 corrected to a dry matter content of 900 g kg−1
Nutrients
Units
Growth rabbits
Sources
Digestive energy
Kcal
2400-2600
Lebas, 2004
Metabolizable energy
Kcal
2400
Lebas et al., 1986
Crude protein
g
150-170
Lebas, 2004
Ether extract
g
20-40
Lebas, 2004
NDF
g
≥320
Lebas, 2004
ADF
g
≥190
Lebas, 2004
Crude fibre
g
140
Lebas et al., 1986
Starch
g
≤140
Lebas, 2004
Lysine
g
75-80
Lebas, 2004
Methionine + Cysteine
g
55-60
Lebas, 2004
Threonine
g
56-58
Lebas, 2004
Calcium
g
7.0-8.0
Lebas, 2004
Phosphorus
g
4.0-4.5
Lebas, 2004
Sodium
g
2.2
Lebas, 2004
Chloride
g
2.8
Lebas, 2004
Introduction about probiotics
Definition of probiotics
A probiotic is generally deï¬ned as a live microbial food supplement which beneï¬cially affects the host by improving its intestinal microbial balance (Fuller, 1989). Microorganisms used in animal feed are mainly bacterial strains belonging to different genera, e.g. Bacillus (B. cereus, var. toyoi, B. licheniformis, B. subtilis), Lactobacillus (L. acidophilus, L. casei, L. farciminis, L. plantarum, L. rhamnosus), Enterococcus (E. faecium) and Pediococcus (P. acidilactici). Other probiotics are microscopic fungi, including Saccharomyces yeasts. Some probiotic microorganisms are normal residents in the digestive tract, while others are not (Guillot, 2009).
Some bacterial strains as probiotics
Lactic acid bacteria (add references)
Lactic acid bacteria (LAB) are non-sporeâ€forming rods and cocci that ferment carbohydrates forming lactic acid as the major endâ€product. Phylogenetically, the LAB belong to the clostridial branch of the gramâ€positive bacteria, includes genera such as Clostridium, Bacillus, Listeria, and Staphylococcus (Aguirre and Collins, 1993). However, the term lactic acid bacteria has become commonly associated with the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Enterococcus, and Streptococcus. Reduction of pH and removal of large amounts of carbohydrates by fermentation were considered the primary actions by which LAB inhibit foodâ€borne pathogens. LAB are capable of producing inhibitory substances other, even at refrigeration temperatures with no growth than organic acids with inhibitory activity to diferent microorganisms (Amezquita and Brashears, 2000).
To supply in animal as probiotics, LAB must be able to survive and metabolize in the intestine by resistibility to conditions encountered in the GI tract. Similarly, resistance of LAB to bile is an important characteristic that enables them to survive and grow in the intestinal tract. Bile entering the duodenal section of the small intestine has been found to reduce survival of bacteria, probably because all bacteria have cell membranes consisting of lipids and fatty acid, which are very susceptible to destruction by bile salts. Gilliland et al. (1984). In some studies, it has been suggested that the ability of L. acidophilus to cause a signiï¬cant increase in numbers of Lactobacillus in the intestinal tract may be critical for controlling growth of intestinal pathogens. Hence, the success of a probiotics also depend on the selected strain possessing bileâ€resistant qualities.
In the past few years, the LAB most commonly associated with antibiotic resistance have been strains of the genera Enterococcus, especially Enterococcus faecalis and E. faecium (Franz et al., 1999).
Bacillus
The species that have been most extensively examined these are Bacillus subtilis, Bacillus clausii, Bacillus cereus, Bacillus coagulans and Bacillus licheniformis. Bacillus are capable of producing spore coats that afford protection from heat, enzymatic degradation, and the acidic conditions of the stomach (Hong et al., 2005). Spores being heat-stable have a number of advantages over other non-spore formers (Simon M. Cutting, 2010). The spore is capable of surviving the low pH of the gastric barrier (Barbosa et al., 2005) so in principle a speciï¬ed dose of spores can be stored indeï¬nitely without refrigeration and the entire dose of ingested bacteria will reach the small intestine intact.
Bacillus probiotic for animal nutrition such as calves, poultry, rabbits and swine usually used Toyocerin® (B. cereus var. toyoi (NCIMB-40112/CNCM-1012) that is produced by Asahi Vet S.A., Tokyo (Head Off.), Japan (http://www.asahi-kasei.co.jp). In the rabbits, Hattori et al. (1984) observed increased body weight and a substantial reduction of diarrhoea when increasing the inclusion rate of B. cereus var. toyoi from 1 x 105 to 5 x 106 spores/g diet. The supplementation of B. cereus var. toyoi at 2x105 spores/g diet in rabbits does and suckling kits improved growth performance and reduced the morbidity, gave higher values for litter weight and litter size at weaning (25 d). (Trocino et al., 2005). Collado et al., 2007 indicated that combination of probiotics trains should consider to increase the beneficial health effects pompared with individual strains, because of their synergistic adhesion effecrs.
Lactobacillus (Lactic acid bacteria)
Lactobacilli are found in the normal intestinal microbiota of animals and humans (Schneider et al., 2004), and have been identiï¬ed as responsible for controlling some effects of pathogens. Some lactic acid bacteria (LAB) are capable of colonizing the intestinal tract of mice, remaining in it without affecting feed intake, and protecting animals against Salmonella dublin DSPV 595T (Frizzo et al., 2006). In addition, LAB can colonize the gastrointestinal tract of calves without translocating to the internal organs (Frizzo et al., 2009). LAB could play a role in specific age groups, specifically neonates or the elderly (L.M.T. Dicks and M. Botes, 2010).
Lactobacillus casei and Lactobacillus rhamnosus have been associated with bacteremia and endocarditis (Cannon et al., 2005). A few strains of LAB have been associated with urinary tract infections, wound, tissue and other infections (Vankerckhoven et al., 2008). In immunocom promised patients some lactobacilli have been associated with arthritis and meningitis (Vankerckhoven et al., 2008).
The effects of probiotics in livestock
Mode of action of probiotics
The beneï¬cial modes of action include: (i) reduction of toxin production; (ii) stimulation of enzyme production by the host; (iii) production of some vitamins or antimicrobial substances; (iv) competition for adhesion to epithelial cells and increased resistance to colonization; and (v) stimulation of the immune system of the host (Falçao-e-Cunha et al., 2007).
A number of studies mention the production of a range of antimicrobial substances by probiotic bacteria. Among such substances are organic acids, hydrogen peroxide and bacteriocins, which can kill other microbes, alter their metabolism, and/or reduce their production of toxins (Rolfe, 2000).
Improving growth rate
In animal nutrition microorganisms used as probiotics were linked with a proven efficacy on the gut microflora. Administration of probiotic strains separately and in combination was significantly improved feed intake, feed conversion rate, daily gain and total body weight in animals (Casey et al., 2007). Probiotics have a positive effect on various digestive processes, especially cellulolysis and synthesis of microbial protein (Yoon and Stern, 1995). Probiotics were stabilizers of ruminal pH and lactase, increase the absorption of some nutrients and displayed a growth-promoting effect that was comparable to avilamycin treatment (Mountzouris et al., 2007).
Nutrient digestibility
Saccharomyces cerevisiae supplementation was associated with an increased flow of microbial protein leaving the rumen and enhanced supply of amino acids entering the small intestine (Erasmus et al., 1992). Putnam et al. (1997), however observed no effect with yeast on the passage of nitrogen fraction and amino-acids to the small intestine. Amber et al. (2004), working with Lact-A-Bac (Lactobacillus acidophilus), got improvements in the digestibilities of energy and of most analytical fractions (DM, CP, EE), including crude fibre.
Caecal fermentation
The literature reveals that Saccharomyces cerevisiae does not have an obvious effect on total VFA concentrations, methane production and ammonia content (Chaucheyras et al., 1997). The effect of yeast supplementation on pH stabilisation seems to be strongly dependent on the type of diet tested. The research in stear of Ghorbani et al., 2002 showed that probiotics do not affect to VFA concentration, but have effect in ruminal NH3-N value. Proboitics inproved rumen acetate concentration in the horse and swine (Swyers et al., 2008; Shen et al., 2009).
Diarrhoea
Probiotics enhanced animals diarrhea by protection against pathogens: The indigenous intestinal bacterial inhibit pathogens by competition to colonization sites and nutritional source and production of toxic or stimulation of the immune system (Parvez et al., 2006). Probiotics can significantly protect pigs against diarrhea (Corr et al., 2007). Probiotics have been shown to be involved in protection against a variety of pathogens in chicken including Escherichia coli (Chateau et al., 1993), Salmonella and Campylobacter (Stern et al., 2001), Clostridium and Eimeria (Dalloul and Lillehoj, 2005). Probiotic activity was largely inhibitory since the probiotics bacteria can reduce the level of Escherichia coli O157 carriage and faecal shedding in cattle and calves (Brachears et al., 2003) and decreased the severity and duration of diarrhea in Escherichia coli O157:H7-infected infant rabbits (Casey et al., 2007). Probiotic was reduced gastric inflammation and bacterial colonization in Helicobacter pylogy-infected animals (Johnson-Henry et al., 2005). Probiotics tended to improve the health status and fertility of sows (Alexopoulos et al., 2004). Probiotic can alter the balance of gastrointestinalmicroflora in healthy cats (Marshall-Jones et al., 2006) and shown to be effective in preventing antibiotic associated diarrhea (Hawrelak et al., 2005).
Balance of the gut microbiota
The rabbit is both a monogastric and an herbivore. Its digestive system is adapted to fermentation of vegetable in colon and cæcum. In young rabbits, the gut microflora is not completely stabilised until 30-40 days of age (Fortun-Lamothe and Boullier, 2007).
The rabbits facultative anaerobic bacteria, mainly Streptoccus sp. and Escherichia coli, reached a maximum level at the 2nd or 3rd week of life and then decreased to be residual or absent after weaning. The epithelial cells, gut associated lym-phoid tissue (GALT) and commensal flora interact with each other forming a dynamic but delicate equilibrium. A balanced system is necessary to ensure both nutrient absorption and protection against pathogens. .
Application of probiotics in livestock
Pigs
All stress factors of the weaning and post-weaning periods can lead to increased susceptibility to gut disorders, infections and diarrhea (Modesto et al., 2009). An interdisciplinary research study on the modes of action of probiotics in swine, showed that E. faecium NCIMB 10415 reduced the pathogenic bacterial load of healthy piglets and sows (Lodemann, et al., 2006). Enterococcus faecium was also found to reduce in the colon of weaned pig population of Enterococcus faecalis which is responsible for the onset of some cases of post-weaning diarrhoea (Vahjen et al., 2007). Supplementation of the diet of neonatal pigs with a strain of Lactobacillus plantarum resulted in an increase in total gut populations of lactobacilli in weaned pigs (Takahashi et al., 2007). An improved daily weight gain was also observed in the animals that received probiotic Lactobacillus sobrius compared to the control fed group (Konstantinov et al., 2008). B. animalis subsp. lactis affected positively the growth performance in weaning piglets and the ratio of biï¬dobacteria to E. coli in the gut (Modesto et al., 2009).
Poultry
Kalavathy et al., 2003 found that a supplementation of twelve Lactobacillus strains in broiler diets improved the bodyweight gain, feed conversion rate and was effective in reducing abdominal fat deposition. Lactobacilli were also successful in decreasing mortality due to necrotic enteritis from 60% to 30% in a challenge trial, when they were given orally at day 1 of life (Hofacre et al., 2003). Mountzouris et al., 2007 indicated that probiotic cultures modulated the composition and the enzymatic activities of the chickens cecal microflora, resulting in a signiï¬cant probiotic effect. Probiotics increase egg production and quality (Panda et al., 2008). Vila et al. (2009) showed that Lactobacillus-based probiotic cultures signiï¬cantly reduced Salmonella enteritidis recovery in challenged neonatal broiler chicks.
Ruminants
In young calves, incorporating live yeasts into the grain, reduced the number of days with diarrhoea (Galvao et al., 2005). With regard to animal performance, improved weight gain and rumen development have been reported in young calves with several bacterial and yeast strains supplementation (Adams et al., 2008). In dairy ruminants, live yeasts have been shown to improve performance, the most consistent effects being an increase in dry matter intake and milk production (Stella et al., 2007). A recent meta-analysis study conducted by Desnoyers et al. (2009) showed that yeast supplementation, in ruminants, increased dry milk intake, milk yield, rumen pH, rumen volatile fatty acids concentration, and organic matter digestibility. Other beneï¬ts have been related to greater total culturable ruminal bacterial population densities and cellulolytic microorganisms (Chaucheyras-Durand et al., 2008) and increase ï¬ber digestibility (Marden et al., 2008).
Rabbits
The husbandry of rabbits in intensive system can cause physiological and environmental stress, contributing to the development of digestive problems and consequent reduction of performance. There are naturally fewer studies with probiotics in rabbits than in other monogastric farm species. Several studies exist nevertheless, which are limited to the assessment of the effect on growth, feed conversion, reproduction and mortality; sometimes caecal activity and digestibility are studied too (Falçao-e-Cunha et al., 2007).
Bacillus cereus var. toyoi (Toyocerin®), has been found to improve the performance and/or health of growing rabbits (Trocino et al., 2005). However, rarely study has been performed to evaluate the contribution of the dietary inclusion of this product either on caecal fermentation of growing rabbits in the last decade, or on the nutrient digestibility and faecal N excretion (Pascual et al., 2008). In the trial of Yamani et al. (1992), Lacto-Sacc (a complex product containing microorganisms % Lactobacillus acidophilus, Streptococcus faecium and yeasts % but also enzyme activities % protease, cellulases, amylase) improved crude fibre digestibility at 8 and 12 weeks.
Since probiotics can influence gut microbiology, several authors looked at their effects upon the caecum microbiota, either by counting bacteria (Amber et al., 2004) or their products, VFA in particular (Maertens et al., 1994). In the study of Amber et al. (2004), the probiotic significantly increased cellulolytic bacteria counts (cfu/ml). In this study, caecal pH was unaffected by the probiotic.
MATERIALS AND METHODS
Experimental protocols will be approved by Chulalongkorn University, Thailand.
Framework is followed by the figure 1:
6. Statitical analysis and write the thesis
Prepare equipments
2. Collect animals
3. Conduct experiment
4. Collect the samples
5. Analyse the samples
7. Register for conference or journal
8. Defend the thesis
Figure 1: The framework of the experiment
Animals and experimental design
A total of 72 rabbits (Newzealand white) weaned at 28 d of age are randomly assigned to 4 treatments: 1) basal diet (BD) (without antibiotic or probiotic), 2) low level of probiotic (LP), 3) middle probiotic (MP) and 4) high level of probiotic (HP). Each treatment has 3 replications with 6 rabbits per experimental unit (balance sex). At weaning, rabbits ase chosen from among those born the same day in the farm from multiparous does, move from the maternal sector to the fattening sector and put in the cages. The six rabbits in each experimental unit come from different litters and had similar live weight.
Table 2: Ingredient and chemical composition of the basal diets
Ingredient composition, g/kgDM
%
Corn/maize
Ground paddy rice
Rice brain
Soybean meal
Fish meal
Grass meal
L-lysine HCL
DL-methionine
Bone meal
Sodium chloride (NaCL)
Calcium oxide (CaO)
Vitamin/mineral premix
Chemical composition
Content (g/kg raw basis)
DE, MJ/kg DM
ME, MJ/kg DM
CP
EE
NDF
ADF
CF
Lysine
Methionine + Cysteine
Threonine
Calcium
Phosphorus
Sodium
Chloride
DE: digestive energy, ME: metabolizable energy, NDF: neutral detergent fibre, ADF: acid detergent fibre, CF: crude fibre, EE: ether extract, CP: crude protein.
During the 8-wk feeding period, all rabbits are housed in a temperature-controlled nursery room (25 to 27°C). Feed and water are provided ad libitum. The diets are similar to common commercial diets for growing rabbits and formulate using commercial raw materials currently adopt by Thai feed producers. They do not contain antibiotics, additives, growth promoters or coccidiostatics.
Prior to the eperimental period (before weaning), both suckling rabbits and their mothers will be given ad libitum access to a commercial feed. From 28 (weaning) to 84 days of age rabbits are offered the experimental diets.
At the end of experiment, two rabbits per each experimental unit will be killed by a lethal injection of sodium pentobarbital to obtain digesta samples in the caecum. The representative digesta from fresh caecum are determine pH immediately, gently squeezed into a preweighed tube and then store in a freezer at −80°C until analysis for VFA and N-NH3 concentrations.
Records
At weaning, the rabbits are given identification marks on the ear, put into bicellular cages and fed the experimental diets. Individual live weight and cage feed intake are recorded weekly. Mortality is controlled daily throughout the experimental period. Daily feed intake are calculated taking into account the effective number of animals per cage per day, thus excluding the intake of dead animals.
Measurements
Morbidity and mortality
In the calculation of morbidity, the ill rabbits are counted only once, independently of the duration of illness. The dead animals are not considered in the morbidity calculation. The health risk is calculated as the sum of morbidity and mortality (Bennegadi et al., 2000).
Feed intakes and growth performance
Feed intakes and BW will be measured at the beginning and end of the experiment to determine daily feed intake (DFI), average daily gain (ADG), and feed conversion ratio (FCR):
Dairy feed intake = feed offer - feed refuse;
Average daily gain = (weight in initial - weight in final)/number of days;
FCR = DFI / ADG.
Chemical analysis of feed and feces
Feed samples are collected at the start of experiment. Feed and fecal samples are dried in an oven (65°C) and ground to pass through a 1-mm sieve. Each sample is analyzed for DM, OM, CP (N - 6.25) according to the standard methods of AOAC, 2000; NDF and ADF (Van soest et al., 1991), Gross energy (GE) is determined by an automatic adiabatic oxygen bomb calorimeter (Parr 1281 Automatic Energy Analyzer, Moline, IL).
Apparent digestibility
The experimental design of digestibility is similar to that of the feeding period, however, the 10-week old rabbits are used to determine the faecal apparent digestibility of ME, DM, OM, CP, NDF and ADF; retention energy (Toschi et al., 2004). Following a 7-day adaptation according to the method of Gómez-Conde et al. (2006). Dry matter intake and total faecal output are recorded daily for each experimental unit over a 5-day collection period. On each day, approximately 50 g of fecal sample is collected from each cage into sterile plastic bottles and immediately store at −20°C until further analysis for nutrient content. Feces produce daily are collected in labeled polyethylene bags and store at −20°C. Urine is also collected for nitrogen analysis to calculate the nitrogen retention and determine purine derivatives (PD).
Apparent digestibility (%)
=
Nutrient in feed - Nutrient in feces
x
100
Nutrient in feed
Retention nitrogen = Nitrogen in feed - (Nitrogen in feces + Nitrogen in urine)
Retention energy (RE) = (0.58 MEI - 252.4), where MEI: Metabolizable energy intake (Toschi et al., 2004).
Determine urinary excretion of purine derivatives (PD)
Only allantoin and uric acid, not xanthine and hypoxanthine, are present in the rabbits urine. PD = Allantoin + acid uric (Balcells et al., 1998).
Allantoin is determined by method of Young and Conway (1942),
Acid ucid is analysed according to the method of Fujihara (1987).
Ceacal fermentation
Volatile fatty acid concentration in digesta from the caecum is determined by a gas chromatographic method following the procedures of Franklin et al. (2002). About 3 g of caecal chyme are homogenized with 4.5 ml metaphosphoric acid (4.16%), then centrifuged at 10,000 x g for 10 min and filtered. 2-ethyl-butyrate (FLUKA Chemie GmbH, Buchs, Switzerland) is used as the internal standard. Parameters: Nukol 30 m x 0.25 mm x 0.25 µm capillar column (Supelco, Bellefonte, PA, USA), FID detector, 1:50 Split ratio, 1 µl injected volume, helium 0.84 ml/min. Parameters of the detector: air 400 ml/min, hydrogen 47 ml/min, temperature: injector 250°C, detector 250°C, column 150°C.
Amonia (N-NH3) concentration in the caecum will be determined by using Kjeldahn method (AOAC, 2000).
The pH value of the fresh caecal content was determined by a manual automatic pH meter (OP-110, Radelkis, Hungary).
Determine coliforms
According to the literature (Gouet and Fonty, 1973) the caecal microflora in rabbits is found to consist of simple, non sporulated, strictly anaerobic, Gram-negative Bacteroides. For the microbiological examinations of the caecal contents, strictly anaerobic bacteria are cultured on Schaedler's agar (Sharlan Chemie, Barcelona, Spain), the selectivity of which was increased by the addition of esculin (Merck, Darmstadt, Germany), neomycin (Merck, Darmstadt, Germany) and Fe-ammonium citrate (Sharlan Chemie, Barcelona, Spain). Subsequently the samples are incubated in anaerobic conditions at 37°C for 96 hours. Coliforms are cultured on a Chromocult differentiation medium (Merck, Darmstadt, Germany). The samples are incubated at 37°C, under aerobic conditions, for 24 hours. Total aerobe germ count is determined on blood agar after incubation at 37°C, under aerobic conditions, for 48 hours. After the incubation time had elapsed, the colonies are counted with a Titriplaque colony counter (LMIM, Esztergom, Hungary). The colony counts are expressed in log10 colony forming units (CFU) related to 1 g of sample.
Statitiscal analysis
Statistical analysis of the data obtained are carried out by the SAS (SAS Inst. Inc., Cary, NC) using the version 9.0 (2002). Effect of treatments are analyzed by the analysis of variance (ANOVA). The significance of differences between treatments will be tested by the Tukey mothod at α = 0.05.
The following model will be used:
yij = µ + τi + εij i = 1, 2,...; j = 1, 2, ...,ni
where:
yij = an observation j in treatment i
µ = the overall mean
τi = the effect of treatment i
εij = random error with mean 0 and variance σ2
The Chi-Square Test (SAS 9.0) will be used to compare the mortality and morbidity between the treatments
DURATION OF STUDY
Activities
Months
3
4
5
6
7
8
9
Start experiment
********************
Sample analysis
***************
Report and paper preparation
************
PLACES OF STUDY
The experiment will be conducted in experimental farm of Chulalongkorn university.
EQUIPMENTS
The equipments of the experiment include digital balances, balance meters, pH meter, nylon bags, plastic cans, plastic nets and chemical includes acid sulfuric solution.
BUDGETS
Granted fund is supported by Chulalongkorn University with the detail is showed in table 3
Table 3: The detail in the budgets
No.
Names
Units
Quantity
Price/unit, Baths
Total price,
Baths
1
Animals
heat
2
Cages
/heat
3
Feeds
Corn/maize
kg
Ground paddy rice
kg
Rice brain
kg
Soybean meal
kg
Fish meal
kg
Grass meal
kg
L-lysine HCL
kg
DL-methionine
kg
L-Threonine
kg
Sodium chloride
kg
Calcium oxide
kg
Vitamin/mineral premix
kg
Probiotics
kg
4
Mixing and pellet the feed
kg
5
Vaccine and medicine
/heat
6
Equipments
/heat
7
Chemicals for analysis
Exp.
8
Electricity
Exp.
9
Car fuel for transfortation
Exp.
10
Farm depreciation
Exp.
11
Potential cost
Exp.
ADVANTAGES OF STUDY
After this experiment, we will know the effects of probiotics in experimental parameters and conclude which level of probiotics is promising to use for the weaning rabbits.
