Biosorption and Bioprotective Potential of Levilactobacillus brevis in Mice Challenged by Lead-Induced Oxidative Stress.

Lead (Pb) poisoning is a widespread issue in both developed and developing countries that poses a significant public health challenge. Our study aimed to explore the impact of Levilactobacillus brevis strains on inflammatory and antioxidant gene expression in the liver and brain of mice exposed to oxidative stress caused by Pb. We began by evaluating Pb absorption by Levilactobacillus brevis strains (ARKA-CH-1 (A1) and ARKA-CH-6 (A6)) using the inductively coupled plasma mass spectrometry (ICP-MS) in vitro to identify the most effective strain. We then divided four groups of BALB/c mice into control and experimental groups and treated them for 30 days. The control group received a normal diet, while the experimental groups consumed lead-containing water (0.6 g/L) with or without Levilactobacillus brevis strains. Following the experiments, we collected blood samples to test liver markers, antioxidant enzymes, and immunoglobulins. We also used real-time PCR to examine the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) genes. The results showed that the A1 strain was the most effective in absorbing Pb. The Pb exposure led to an increase in liver enzyme values and a decrease in antioxidant enzyme activity and immunoglobulin factors. However, the combination of A1 and A6 strains had a greater effect in reducing inflammatory enzymes and increasing antioxidant enzymes. Furthermore, we observed a significant increase in iNOS gene expression and a notable decrease in SOD gene expression with Pb consumption. However, the combination of A1 and A6 strains had a synergistic effect in reducing iNOS and increasing SOD gene expression. In conclusion, Levilactobacillus brevis A1 strain alone or in combination with the A6 strain could be a promising strategy to mitigate the oxidative stress symptoms in mice challenged by lead-induced toxicity.

Microcapsules loaded with date seed extract and its inhibitory potential to modulate the toxic effects of mycotoxins in mice received mold-contaminated diet.

Mycotoxins are the secondary fungal metabolites generally produced by wide range of fungi including aflatoxins (AF), ochratoxin A (OTA), fumonisins (FB), zearalenone (ZEN), and deoxynivalenol (DON). Nowadays, they are main concern to food and agricultural commodities due to undesirable health and socio-economic effect. This investigation was designed to synthesized microcapsules loaded the bioactive compounds of date seed and evaluated its inhibitory activities in mice received mold-contaminated diet. The finding revealed that the developed microcapsule is homogenous and mostly spherical with size of 2.58 µm with acceptable PDI of 0.21. The main phytochemical has been confirmed by HPLC analysis were xylose, fructose, mannose, glucose, and galactose with the respective values of 41.95%, 2.24%, 5.27%, and 0.169%. The in vivo analyses manifested that the mice received date seed microcapsules significantly (p < 0.05) improved the average daily weight gain, feed intake, liver enzymes (ALT, ALP, and AST), and lipid peroxidation values compare to mice group received mycotoxin-contaminated diet. Furthermore, encapsulation date seed bioactive compounds notably up-regulated the expression of GPx, SOD, IFN-γ, and IL-2 genes while down-regulated the iNOS gene. Consequently, the novel microcapsules loaded date seed is suggested to be considered as a promising mycotoxin inhibitor.

Benefits and Challenges of Jatropha Meal as Novel Biofeed for Animal Production.

Jatropha curcas L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing various bioactive compounds, including saponins, phytic acids, trypsin inhibitors, lectins, phenolics, and flavonoids, which render it as a potential biofeed for animal production. The Jatropha meal demonstrated various biological activities, including antioxidant, antibacterial, and anti-inflammatory effects which enhance its property as a bio-feed. The levels of these bioactive compounds in the seeds are dependent on the genotypes. The J. curcas possessed different varieties which are either toxic or non-toxic according to the presence of phorbol esters. The presence of phorbol esters in the meal confirmed the toxic variety of Jatropha resulting in the limited application of meal as a biofeed. The Jatropha meal devoid of phorbol esters could be applied as a biofeed in the animal production industry, and for the toxic varieties, various techniques such as physicochemical and biological treatments have been introduced to the industry to remove the phorbol esters from Jatropha meal. Several studies employing various cells and animals confirmed the toxicity of the phorbol esters. The molecular mechanism of action of phorbol esters is through up-regulation of PKC-ß II gene, overexpression of down-stream proto-oncogenes resulted in inflammation and oxidative stress ending by apoptotic cell death. Despite the presence of valuable bioactive compounds in the Jatropha meal, its nutritional application is not recommended unless the phorbol esters are completely removed.

Manipulation of Rice Straw Silage Fermentation with Different Types of Lactic Acid Bacteria Inoculant Affects Rumen Microbial Fermentation Characteristics and Methane Production.

Bacterial inoculants are known to improve the quality of silage. The objectives of the present study were to evaluate the effects of different types of lactic acid bacteria (LAB; L. plantarum, L. salivarius, L. reuteri, L. brevi, and S. bovis) inoculation (106 cfu/ DM) on rice straw silage quality and to determine these effects on ruminal fermentation characteristics, digestibility and microbial populations in an in vitro condition. Inoculated rice straw was ensiled for 15 and 30 days. For the in vitro study, rumen fluid was obtained from three rumen-fistulated bulls fed on mixed forage and concentrate at 60:40 ratio twice daily. Inoculation with LAB improved (p < 0.05) the rice straw silage quality as indicated by higher dry matter and crude protein contents, decreased pH and butyric acid, and increased propionic acid and LAB numbers, especially after 30 days of ensiling. Results from the in vitro study revealed that starting with the addition of LAB to rice straw silage improved in vitro fermentation characteristics such as increased total volatile fatty acids and dry matter digestibility (p < 0.05). LAB treatments also decreased methane production and methane/total gas ratio after 15 and 30 days of ensiling. From the rumen microbial population perspective, cellulolytic, and fungal zoospores were enhanced, while protozoa and methanogens were decreased by the LAB treatments. Based on these results, it could be concluded that inoculating rice straw silage with LAB (especially for L. plantarum and S. bovis) improved silage quality, rumen fermentation parameters and microbial populations in vitro.

Oligosaccharides from Palm Kernel Cake Enhances Adherence Inhibition and Intracellular Clearance of Salmonella enterica Serovar Enteritidis In Vitro.

Salmonella enterica serovar (ser.) Enteritidis (S. Enteritidis) is a foodborne pathogen often associated with contaminated poultry products. This study evaluated the anti-adherence and intracellular clearance capability of oligosaccharides extracted from palm kernel cake (PKC), a by-product of the palm oil industry, and compared its efficacy with commercial prebiotics- fructooligosaccharide (FOS) and mannanoligosaccharide (MOS)-against S. Enteritidis in vitro. Based on the degree of polymerization (DP), PKC oligosaccharides were further divided into 'Small' (DP ≤ 6) and 'Big' (DP > 6) fractions. Results showed that the Small and Big PKC fractions were able to reduce (p < 0.05) S. Enteritidis adherence to Cancer coli-2 (Caco-2) cells at 0.1 mg/ mL while MOS and FOS showed significant reduction at 1.0 mg/mL and 10.0 mg/mL, respectively. In terms of S. Enteritidis clearance, oligosaccharide-treated macrophages showed better S. Enteritidis clearance over time at 50 µg/mL for Small, Big and MOS, while FOS required a concentration of 500 µg/mL for a similar effect. This data highlights that oligosaccharides from PKC, particularly those of lower DP, were more effective than MOS and FOS at reducing S. Enteritidis adherence and enhancing S. Enteritidis clearance in a cell culture model.

Effects of naturally-produced lovastatin on carcass characteristics, muscle physico-chemical properties and lipid oxidation and cholesterol content in goats.

This study investigated the carcass characteristics, physico-chemical properties, storage stability and cholesterol content of meat from goats fed with different levels of naturally-produced lovastatin used to mitigate enteric methane production. Twenty intact Saanen male goats of 5-6 months old with initial live weight of 25.8 ±â€¯4.0 kg were randomly allotted into four dietary treatments containing 0 (Control), 2 (Low), 4 (Medium) and 6 mg (High) per kg live weight (LW) of naturally-produced lovastatin for 12 consecutive weeks. No differences were found in all the parameters measured except for full LW, hot and cold carcass weight, shear force, color and cholesterol content among the treatment groups. Aging had significant effects on all the parameters measured in this study except a* (redness) of meat. Meat samples in the Medium and High treatments were of higher lightness and yellowness, more tender and lower cholesterol levels. We conclude that, in addition to mitigate enteric methane emissions, dietary supplementation of naturally-produced lovastatin at 4 mg/kg LW could be a feasible feeding strategy to produce tender meat containing lower cholesterol.

Effects of corn supplementation on the antioxidant activity, selected minerals, and gene expression of selenoprotein and metallothionein in serum, liver, and kidney of sheep-fed palm kernel cake: urea-treated rice straw diets.

This study aimed to determine influence of corn inclusion on glutathion peroxidase (GPx) activity, selected minerals concentration, and gene expression in sheep-fed palm kernel cake (PKC) and urea-treated rice straw. Twenty-seven of Dorper sheep were divided into three groups and fed a basal diet of (20% rice straw and 80% concentrate) with addition of ground corn at either 0% (T1), 5% (T2), or 10% (T3), respectively. After 120 days feeding trial, all animals were slaughtered and tissue samples of kidney, liver, and muscles were taken for enzyme and mineral analyses. The results showed that Cu concentration in the liver was lower treatment T3 compared to the control and T2. The serum activity of GPx was higher in T2 than in T3 at day 120 of experiment. Serum malondialdehyde (MDA) concentrations decreased at day 80 in sheep on T3, whereas MDA of liver increased linearly with increasing corn supplementation. The qRT-PCR analyses revealed significant up-regulation of ATP7A and MIa genes in T3, while hepatic Cu/Zn SOD, GPx1, and GPx4 mRNA showed a higher expression in lamb hepatocytes in T3 compared to those on T1. Present study results suggest that feeding PKC as basal diet can increase antioxidant activity, but cause liver dysfunction in sheep. Inclusion corn was found to regulate transcriptional levels of the GPx family and metallothionein genes. These genes may play a role in the antioxidant protection response and reduce incidence of toxicity associated with Cu.

Oil supplementation improved growth and diet digestibility in goats and sheep fed fattening diet.

OBJECTIVE: This study evaluated the growth, digestibility and rumen fermentation between goats and sheep fed a fattening diet fortified with linseed oil. METHODS: Twelve 3 to 4 months old male goats and sheep were randomly allocated into two dietary treatment groups in a 2 (species)×2 (oil levels) factorial experiment. The treatments were: i) goats fed basal diet, ii) goats fed oil-supplemented diet, iii) sheep fed basal diet, and iv) sheep fed oil-supplemented diet. Each treatment group consisted of six animals. Animals in the basal diet group were fed with 30% alfalfa hay and 70% concentrates at a rate equivalent to 4% of their body weight. For the oil treatment group, linseed oil was added at 4% level (w:w) to the concentrate portion of the basal diet. Growth performance of the animals was determined fortnightly. Digestibility study was conducted during the final week of the feeding trial before the animals were slaughtered to obtain rumen fluid for rumen fermentation characteristics study. RESULTS: Sheep had higher (p<0.01) average daily weight gain (ADG) and better feed conversion ratio (FCR) than goats. Oil supplementation did not affect rumen fermentation in both species and improved ADG by about 29% and FCR by about 18% in both goats and sheep. The above enhancement is consistent with the higher dry matter and energy digestibility (p<0.05), as well as organic matter and neutral detergent fiber digestibility (p<0.01) in animals fed oil- supplemented diet. Sheep had higher total volatile fatty acid production and acetic acid proportion compared to goat. CONCLUSION: The findings of this study suggested that sheep performed better than goats when fed a fattening diet and oil supplementation at the inclusion rate of 4% provides a viable option to significantly enhance growth performance and FCR in fattening sheep and goats.

Naturally Produced Lovastatin Modifies the Histology and Proteome Profile of Goat Skeletal Muscle.

This study was conducted to examine the effects of different levels of lovastatin on the histological and sarcoplasmic proteome profile of goat skeletal muscle. A total of 20 intact male Saanen goats were randomly assigned in equal numbers to four groups and fed a total mixed ration containing 50% rice straw, 22.8% concentrates and 27.2% of various proportions of untreated or treated palm kernel cake (PKC) to achieve the target daily intake levels of 0 (Control), 2 (Low), 4 (Medium) or 6 (High) mg lovastatin/kg BW. A histological examination discovered that the longissimus thoracis et lumborum muscle of animals from the Medium and High treatment groups showed abnormalities in terms of necrosis, degeneration, interstitial space and vacuolization. Our preliminary label-free proteomics analysis demonstrates that lovastatin supplementation induced complex modifications to the protein expression patterns of the skeletal muscle of the goat which were associated with the metabolism of carbohydrate and creatine, cell growth and development processes and other metabolic processes. The changes in these biochemical processes indicate perturbations in energy metabolism, which could play a major role in the development of myopathy. In conclusion, the present study suggests that supplementation of naturally produced lovastatin above 4 mg/kg BW could adversely affecting the health and wellbeing of treated animals.

Effects of naturally-produced lovastatin on feed digestibility, rumen fermentation, microbiota and methane emissions in goats over a 12-week treatment period.

Twenty male Saanen goats were randomly assigned to four levels of lovastatin supplementation and used to determine the optimal dosage and sustainability of naturally produced lovastatin from fermentation of palm kernel cake (PKC) with Aspergillus terreus on enteric methane (CH4) mitigation. The effects on ruminal microbiota, rumen fermentation, feed digestibility and health of animal were determined over three measuring periods (4-, 8- and 12-weeks) and the accumulation of lovastatin in tissues was determined at the end of the experiment. The diets contained 50% rice straw, 22.8% concentrates and 27.2% of various proportions of untreated or treated PKC to achieve the target daily intake level of 0 (Control), 2, 4 or 6 mg lovastatin/kg body weight (BW). Enteric CH4 emissions per dry matter intake (DMI), decreased significantly (P<0.05) and equivalent to 11% and 20.4%, respectively, for the 2 and 4 mg/kg BW groups as compared to the Control. No further decrease in CH4 emission thereafter with higher lovastatin supplementation. Lovastatin had no effect on feed digestibility and minor effect on rumen microbiota, and specifically did not reduce the populations of total methanogens and Methanobacteriales (responsible for CH4 production). Similarly, lovastatin had little effect on rumen fermentation characteristics except that the proportion of propionate increased, which led to a decreasing trend (P<0.08) in acetic: propionate ratio with increasing dosage of lovastatin. This suggests a shift in rumen fermentation pathway to favor propionate production which serves as H+ sink, partly explaining the observed CH4 reduction. No adverse physiological effects were noted in the animals except that treated PKC (containing lovastatin) was less palatable at the highest inclusion level. Lovastatin residues were detected in tissues of goats fed 6 mg lovastatin/kg BW at between 0.01 to 0.03 µg/g, which are very low.

Chinese Herbal Medicines as Potential Agents for Alleviation of Heat Stress in Poultry.

Heat stress negatively affects the productivity of chickens in commercial poultry farms in humid tropics. In this study, the concentrations and types of the antioxidant compounds of eight Chinese herbal medicines, which have previously demonstrated promising effects on suppressing heat stress as a mixture, were investigated using reversed-phase High Performance Liquid Chromatography, spectrophotometry, Liquid Chromatography Mass Spectrometry, and Gas-Liquid Chromatography. Our results provided the levels of phenolic compounds, total amounts of sugars, and total unsaturated fatty acids in the herbal extracts. Apart from the detection and quantification of the active ingredients of herbs that have the potential to mitigate heat stress in poultry, results of this study also provide useful data for developing an efficient and accurate formulation of the herbs' mixtures in order to induce positive effects against heat stress in in vivo studies.

Lead biosorption of probiotic bacteria: effects of the intestinal content from laying hens.

This study investigated the effects and the possible mechanisms of intestinal content (IC) from laying hens on in vitro lead (Pb2+) biosorption of four probiotic bacterial strains (Bifidobacterium longum BB79, Lactobacillus paracasei Kgl6, Lactobacillus pentosus ITA23, and Lactobacillus acidipiscis ITA44). The total Pb2+ removal capacity of the four probiotic strains, with and without capsule polysaccharides (CPSs), increased in the presence of IC compared to the control (without IC). SEM imaging revealed certain unidentified particles from the IC adhered on the surface of bacterial cells sorted out using flow cytometry. Follow-up experiment showed an overall trend of increase in the Pb2+ removal capacity of the sorted bacteria, but statistically significant for L. pentosus ITA23 and B. longum BB79 after incubation with IC, particularly with the suspended solid portion of the IC. In addition, the Fourier transform infrared spectrophotometer data showed that functional groups such as C-H, O-H, C=O, and C-O-C which possibly associated with Pb2+ binding were mainly presented in the suspended solid portion of IC. Putting the above together, we postulated that the enhanced Pb2+ binding capacity the probiotic bacteria incubated in IC is due to the adherence of the yet to be identified particles which could much exist in suspended solid portion of IC containing negatively charged functional groups which bind with the positive Pb2+ ions.

Dietary n-6:n-3 Fatty Acid Ratios Alter Rumen Fermentation Parameters and Microbial Populations in Goats.

Revealing the ruminal fermentation patterns and microbial populations as affected by dietary n-6:n-3 PUFA ratio would be useful for further clarifying the role of the rumen in the lipid metabolism of ruminants. The objective of the present study was to investigate the effects of dietary n-6:n-3 PUFA ratios on fermentation characteristics, fatty acid (FA) profiles, and microbial populations in the rumen of goats. A total of twenty-one goats were randomly assigned to three dietary treatments with different n-6:n-3 PUFA ratios of 2.27:1 (low ratio, LR), 5.01:1 (medium ratio, MR), and 10.38:1 (high ratio, HR). After 100 days of feeding, all goats were slaughtered. Dietary n-6:n-3 PUFA ratios had no effect (P > 0.05) on rumen pH and NH3N concentration. Goats fed HR diet had lower (P < 0.05) propionate and total volatile fatty acids and higher (P < 0.05) butyrate compared with those fed the MR and LR diets. The proportion of C18:0 decreased (P < 0.05) as dietary n-6:n-3 PUFA ratios increased. The proportions of C18:1 trans-11, C18:2n-6, cis-9 trans-11 CLA, and C20:4n-6 were greater in the HR goats compared with the MR and LR goats. Lowering dietary n-6:n-3 PUFA ratios enhanced (P < 0.05) the proportion of C18:3n-3 and total n-3 PUFA in the rumen fluid of goats. The populations of R. albus and R. flavefaciens decreased (P < 0.05) as the n-6:n-3 PUFA ratios increased in diet. Diet had no effect (P > 0.05) on the ruminal populations of F. succinogenes, total bacteria, methanogens, total protozoa, Entiodinium, and Holotrich. The population of B. fibrisolvens was lower (P < 0.05) in the LR goats compared with the MR and HR goats. It was concluded that HR would increase the concentration of cis-9 trans-11 CLA and C18:1 trans-11 in the rumen. However, LR whould decrease the B. fibrisolvens population, which is involved in the BH process in the rumen. Further research is needed to evaluate the potential role and contribution of rumen microbiome in the metabolism of FA in the rumen.

Conjugated linoleic acid: A potent fatty acid linked to animal and human health.

Conjugated linoleic acid (CLA) is a mixture of isomers of linoleic acid (C18:2 n-6), which is mostly found in the ruminant meat and dairy products. The CLA is known to have many potential health benefits, and considered a potent powerful fatty acid, which is linked to animal and human health. The present work aims to discuss the source and production, mechanism of action, and effects of CLA on humans, poultry, and ruminants by reviewing the recent studies carried out on CLA. Despite most of the recent studies indicating beneficial effects of CLA on improving body weight control parameters, its effects on reducing risk factors of cardiovascular diseases (CVD), inflammation, blood glucose, and insulin are still controversial, and need to be further studied in different hosts.

Manipulation of Rumen Microbial Fermentation by Polyphenol Rich Solvent Fractions from Papaya Leaf to Reduce Green-House Gas Methane and Biohydrogenation of C18 PUFA.

Different solvents (hexane, chloroform, ethyl acetate, butanol, and water) were used to identify the effect of papaya leaf (PL) fractions (PLFs) on ruminal biohydrogenation (BH) and ruminal methanogenesis in an in vitro study. PLFs at a concentration of 0 (control, CON) and 15 mg/250 mg dry matter (DM) were mixed with 30 mL of buffered rumen fluid and were incubated for 24 h. Methane (CH4) production (mL/250 mg DM) was the highest (P < 0.05) for CON (7.65) and lowest for the chloroform fraction (5.41) compared to those of other PLFs at 24 h of incubation. Acetate to propionate ratio was the lowest for PLFs compared to that of CON. Supplementation of the diet with PLFs significantly (P < 0.05) decreased the rate of BH of C18:1n-9 (oleic acid; OA), C18:2n-6 (linoleic acid; LA), and C18:3n-3 (α-linolenic acid; LNA) compared to that of CON after 24 h of incubation. Real time PCR indicated that total protozoa and total methanogen population in PLFs decreased (P < 0.05) compared to those of CON.

Diploid and triploid African catfish (Clarias gariepinus) differ in biomarker responses to the pesticide chlorpyrifos.

The impacts of environmental stressors on polyploid organisms are largely unknown. This study investigated changes in morphometric, molecular, and biochemical parameters in full-sibling diploid and triploid African catfish (Clarias gariepinus) in response to chlorpyrifos (CPF) exposures. Juvenile fish were exposed to three concentrations of CPF (mean measured µg/L (SD): 9.71 (2.27), 15.7 (3.69), 31.21 (5.04)) under a static-renewal condition for 21days. Diploid control groups had higher hepatosomatic index (HSI), plasma testosterone (T), and brain GnRH and cyp19a2 expression levels than triploids. In CPF-exposed groups, changes in HSI, total weight and length were different between the diploid and triploid fish. In contrast, condition factor did not alter in any of the treatments, while visceral-somatic index (VSI) changed only in diploids. In diploid fish, exposure to CPF did not change brain 11ß-hsd2, ftz-f1, foxl2, GnRH or cyp19a2 mRNA levels, while reduced tph2 transcript levels compared to the control group. In contrast, 11ß-hsd2 and foxl2 expression levels were changed in triploids following CPF exposures. In diploids, plasma T levels showed a linear dose-response reduction across CPF treatments correlating with liver weight and plasma total cholesterol concentrations. In contrast, no changes in plasma cholesterol and T concentrations were observed in triploids. Plasma cortisol and 17-ß estradiol (E2) showed no response to CPF exposure in either ploidy. Results of this first comparison of biomarker responses to pesticide exposure in diploid and polyploid animals showed substantial differences between diploid and triploid C. gariepinus.

Lovastatin production by Aspergillus terreus using agro-biomass as substrate in solid state fermentation.

Ability of two strains of Aspergillus terreus (ATCC 74135 and ATCC 20542) for production of lovastatin in solid state fermentation (SSF) using rice straw (RS) and oil palm frond (OPF) was investigated. Results showed that RS is a better substrate for production of lovastatin in SSF. Maximum production of lovastatin has been obtained using A. terreus ATCC 74135 and RS as substrate without additional nitrogen source (157.07 mg/kg dry matter (DM)). Although additional nitrogen source has no benefit effect on enhancing the lovastatin production using RS substrate, it improved the lovastatin production using OPF with maximum production of 70.17 and 63.76 mg/kg DM for A. terreus ATCC 20542 and A. terreus ATCC 74135, respectively (soybean meal as nitrogen source). Incubation temperature, moisture content, and particle size had shown significant effect on lovastatin production (P < 0.01) and inoculums size and pH had no significant effect on lovastatin production (P > 0.05). Results also have shown that pH 6, 25°C incubation temperature, 1.4 to 2 mm particle size, 50% initial moisture content, and 8 days fermentation time are the best conditions for lovastatin production in SSF. Maximum production of lovastatin using optimized condition was 175.85 and 260.85 mg/kg DM for A. terreus ATCC 20542 and ATCC 74135, respectively, using RS as substrate.

Correlation coefficients between different methods of expressing bacterial quantification using real time PCR.

The applications of conventional culture-dependent assays to quantify bacteria populations are limited by their dependence on the inconsistent success of the different culture-steps involved. In addition, some bacteria can be pathogenic or a source of endotoxins and pose a health risk to the researchers. Bacterial quantification based on the real-time PCR method can overcome the above-mentioned problems. However, the quantification of bacteria using this approach is commonly expressed as absolute quantities even though the composition of samples (like those of digesta) can vary widely; thus, the final results may be affected if the samples are not properly homogenized, especially when multiple samples are to be pooled together before DNA extraction. The objective of this study was to determine the correlation coefficients between four different methods of expressing the output data of real-time PCR-based bacterial quantification. The four methods were: (i) the common absolute method expressed as the cell number of specific bacteria per gram of digesta; (ii) the Livak and Schmittgen, ΔΔCt method; (iii) the Pfaffl equation; and (iv) a simple relative method based on the ratio of cell number of specific bacteria to the total bacterial cells. Because of the effect on total bacteria population in the results obtained using ΔCt-based methods (ΔΔCt and Pfaffl), these methods lack the acceptable consistency to be used as valid and reliable methods in real-time PCR-based bacterial quantification studies. On the other hand, because of the variable compositions of digesta samples, a simple ratio of cell number of specific bacteria to the corresponding total bacterial cells of the same sample can be a more accurate method to quantify the population.