Introduction
The genus Aspergillus produces aflatoxins that are usually from the feed and food with moisture and warm atmospheres conditions. Extreme occurrence rates of contamination in cereal grains and animal feed have been reported in Asia and some diverse substitutes such as the potential use of fermented phytogenic products to decontaminates and influences in performances has been ahead drive in recently (Balasubramanian et al., 2017; Singh and Shrivastav, 2011a, 2011b; Singh, 2019). Therefore, the diet contaminations caused by mycotoxins and the compounds related to mycotoxins transmitted through the food have to be controlled properly. Production of buffalo is a crucial part of nation economical state and play an important role in affording of the increased quality and quantity food for the mankind. Few studies have suggested that the composition of milk, weight gain of the body, immune power and the reproduction capacity are affected in dairy ruminants by consuming aflatoxins contaminated feed (Battacone et al., 2003; Battacone et al., 2005; Xiong et al., 2015). The exposure of aflatoxins for a long period may lead to reduced body weight, reduced milk or production of egg, raised levels of susceptibility to diseases, lowered efficiency of feeding, teratogenicity and tumors (Streit et al., 2012). The aflatoxin B1 (AFB1) contamination feed has been given to the lactating animals that resulted in the production of contaminated milk contains with mono-hydroxylated derivative aflatoxins M1 (AFM1) compound, which classified as group 2B by International Agency for Research on Cancer (IARC), probable carcinogenic substance to the human beings (IARC, 2002). One of the most common forms of aflatoxicosis is chronic toxicity, which may be due to the consumption of toxic compounds in little amounts for a long period of time. Liver has been observed to be the major target organ for toxicity, in which the toxicity maybe metabolized into various metabolites (Di Gregorio et al., 2014). Probiotic yeasts are widely used in animal feeding because of their effects on activity of rumen microbes (Chaucheyras-Durand et al., 2008). Along with the exceptional dietary value of the yeast, the glucomannans from the cell walls of the yeast have been found to significantly reduce (approximately -60%) the concentration of AFM1 in the milk of cows consuming AFB1 (55 μg·kg-1) contaminated diets (Diaz et al., 2004). In our earlier report shown that the adverse effects of AFB1 through in vitro rumen fermentation (Singh et al., 2020). The adsorption of AFB1 by the products of Saccharomyces cerevisiae is not totally a linear fact and may vary according to several factors, especially the amount of β-d-glucans and the yeast cell wall conformation that varies among the strains of the yeast (Jouany and Diaz, 2005). Therefore, the aim of the present experiment was to examine the ameliorative effects of S. cerevisiae on adverse effects of AFB1 by in vitro rumen fermentation.
Materials and Methods
Production and determination of aflatoxin
The fungal strain of Aspergillus flavus, NRRL 6513 (U.S Department of Agriculture, Illinois, USA) produces aflatoxin, that can be used as inoculant and produced on liquid medium according to method described by Singh and Shamsudeen (2008). The culture was sub-cultured regularly on the slants of potato dextrose agar medium, in order to get the fresh spores and further stored at 5℃. Aflatoxin contents were finally quantified using UV-Spectrophotometry (Model no: 117, Systronics, Kolkatta, India). Plates were inspected for AFB1 in a chromate-viewer to find the components by fluorescence. Quantification of AFB1 was determined by visual comparison of fluorescence zone with the known quantity of zone formed by the standards of aflatoxin. AFB1 was separated by scratching the chromatogram.
Experimental design and substrate
Feed sample (wheat straw) was ground to go by a sieve of 1 mm and utilized for experimentation. The study categorized to one of the five groups as basal diet included with 0 (T1), 300 ppb of AFB1 (T2), and T2 mixed with 0.05% (T3), 0.1% (T4), 0.2% (T5) of S. cerevisiae for further analysis. The wheat straw was examined for dry matter (DM), ash, ether extract (EE), organic matter (OM) and crude protein (CP), while Van Soest et al. (1991) method has been used to determine the neutral detergent fiber (NDF) and acid detergent fiber (ADF) and mentioned in Table 1.
Collection of rumen liquor
Fistulated male buffalo, fitted with permanent rumen cannula, about 2 years-old having 250 kg body weight was used as donor animal for collection of rumen liquor. The animal was fed a basal diet of wheat straw offered ad lib and a standard concentrate mixture containing 20% CP and 70% TDN to compensate the nutrients required for the maintenance. The animal was given free access to clean drinking. The rumen liquor (about 300 mL), has been accumulated from the reticulo rumen of various depths and directions, followed by the transfer into thermos flask, which is pre heated and strained using a fourfold cloth of muslin and flushed with CO2. Rumen liquor was collected in the morning before feeding and watering of the animal as per standard procedure. The preparation of rumen fluid–medium mixture (inoculum) was carried out under constant flushing with CO2 to maintain anaerobic condition.
In vitro incubation of substrate and gas production (GP)
About 200 mg, in dry weight of every feed was taken into the calibrated syringes of 100 mL, followed by the incubation for 24 h at 39℃ with the mixed rumen inoculums (30 mL) and the simultaneous incubation of blanks has also been done (Menke et al., 1979). Each substrate was incubated in triplicate. The syringes were regularly shaken by hand during the incubation period for proper mixing of feeds with rumen inoculum. After the period of incubation for 24 h, the production of gas was recorded by the piston displacement for the duration of incubation period for test substrate and blank syringes. The net production of the gas, as the result of fermentation of substrate was evaluated by deducting the value of production of gas in the blank syringes from that of test substrates.
In vitro dry matter degradability (IVDMD)
The syringe content has been extracted by boiling in 100 mL of neutral detergent solution (NDS) for an hour, which is then filtered on the pre-weighed gooch crucibles followed by the washing with distilled water, which is hot along with the acetone for the proper recovery of undigested residue that is pure, by using the Van Soest et al. (1991) method. The above steps were carried out after the 24 h incubation period and the transfer of the obtained contents were done to the spotless beakers of 500 mL. The crucibles that contains the undigested residue has been subjected to overnight dry at 100℃ and then weighed for evaluating the true undigested residue. At 500℃, the residue was turned into ashes within 3 h in order to evaluate the true undigested organic matter (OM), that has been used for the blanks appropriately. After the 24 h of inoculation, a set of samples was evaluated in vitro truly degradable dry matter’s (TDDM) true digestibility and truly degradable organic matter’s (TDOM) true digestibility according to Van Soest and Robertson (1985) method, as the variation between incubated OM and the OM undigested that is recovered in the ND extraction residue. The microbial biomass production (MBP) and partitioning factor (PF) was evaluated as per the method of Blummel et al. (1997).
MBP = substrate truly degraded - (gas volume*stoichiometrical factor) (1)
The stoichiometrically factor was 2.20 for roughages.
Estimation of volatile fatty acid
After 24 h post incubation, 1 mL of the supernatant of each syringe content was taken in a micro centrifuge tube with 0.20 mL of metaphosphoric acid (25%, v/v) to analysis the total propionate, volatile fatty acids (TVFA), butyrate and acetate. The mixture was kept at the room temperature for 2 h followed by the centrifugation for 10 min at 5,000 g for 10 min to obtain the clear supernatant. The supernatant (1 μL) was injected into the flame ionization detector equipped gas chromatograph and chromosorb packed glass column according to the method described by Cottyn and Boucque (1968).
Statistical analysis
All data were evaluated in the statistical software SPSS, version 20.0 following one-way analysis. All the observations were observed at 95% (p < 0.05) level of significance.
Results and Discussion
Mycotoxins are secondary toxic metabolites formed by various hurtful fungi species, which are caused potential health effects on livestock animals and humans (Jeong et al. 2018a, 2018b). The average figures pertaining to TDDM, TDOM, GP, MBP, and PF as influenced by various treatments are presented in Table 2. The data pertaining total TVFA production are presented in Table 3. The TDDM and TDOM values in AFB1 contaminated group (T2) was reduced (p < 0.05) and higher in control (T1) when compared to that of the other treatment groups. Further, the TDDM and TDOM values in T3 group was lesser (p < 0.05) when compared to that of T4 and T5. The TDDM and TDOM values in T4 group was lesser (p < 0.05) when compared to that of T5. The results have indicated that inclusion of AFB1 300 ppb in feed has decreased DM and OM degradability, in a significant (p < 0.05) manner when compared to that of the control. This finding was in concurrence with that of Westlake et al. (1989) who also suggested that the IVDMD of alfalfa hay was decreased by 50% with the addition of 1 μg·mL-1 AFB1. Also, Mojtahedi et al. (2013) reported that the significantly reduced IVDMD (p < 0.05), in case of AFB1 addition to the culture medium, and hence both the lowest IVDMD values as well as the highest IVDMD values were detected in AFB1 treatments (900 and 0 ng·mL-1) as 0.54 vs. 0.68 respectively. Decreased IVDMD values with AFB1 inclusion can be endorsed to the conciliated functions of rumen function by fibre digestion reduction and production of TVFA (Fehr and Delage, 1970; Helferich et al., 1986a; Helferich et al., 1986b).
However, some studies have suggested that the AFB1 has no effect on in vitro hay DM disappearance (Pettersson and Kiessling, 1976; Jiang et al., 2012). Yeanpet et al. (2018) reported that the IVDMD and IVOMD were not significantly affected by AFB1. In the present study, inclusion of S. cerevisiae in feed at any level significantly (p < 0.05) improved the TDDM and TDOM in a dose dependent manner as the S. cerevisiae has the binding ability towards aflatoxin. However, inclusion of S. cerevisiae in feed even at highest level (0.2%) could not reverse the TDDM and TDOM equivalent to that of control. This result was in agreement with earlier reports wherein Wu et al. (2009) suggested that S. cerevisiae has the capacity to bind to AFB1 due to the oligomannanes presence in the cell wall of yeast. The chemical structure of oligomannanes has been found to have the 95% capability of binding to AFB1. Bueno et al. (2007) has also stated that the enhanced capacity of S. cerevisiae binding to the AFB1 because of the high availability of sites for binding. It has been understood that, the removal of toxins happens through the cell wall component adhesion, rather than by the covalent binding or the degradation of metabolites, even though the non-viable cells are binding to the toxins still (Shetty and Jespersen, 2006). Also, Devegowda et al. (1998) detected that the cell wall, which has been extracted from the yeast, S. cerevisiae was capable of binding to the wide range of mycotoxins, in vitro.
The GP value in the control group (T1) was elevated (p < 0.05) and T2 group was lesser (p < 0.05) when compared to that of other treatment groups. The GP value in T3 was lesser (p < 0.05) than those of T4 and T5. The GP value in T4 group (p < 0.05) was lower (p < 0.05) than that of T5. The results indicated that the AFB1 contamination of wheat straw at 300 ppb level reduced the GP significantly (p < 0.05) when compared to that of control (T1). The present result was in agreement with other reports wherein Mojtahedi et al. (2013) reported that, the raise of AFB1 levels from 0 to 900 ng·mL-1 resulted in the reduction of production of gas from 0.071 to 0.051 as well as the cumulative production of gas has also reduced from 196.4 to 166.0 mL·g-1 DM. Similarly, Jiang et al. (2012) and Helferich et al. (1986a; 1986b) also stated that, the parameters of production of gas were decreased on the addition of AFB1. These reductions in the production of gas production show that populations of microbes have been influenced by the contaminated feed that contains AFB1. In the present study, inclusion of S. cerevisiae to the contaminated feed by aflatoxin improve the undesirable effects of aflatoxin on GP in a dose dependent manner. However, even highest level (0.2%) of S. cerevisiae could not reverse the gas production value equivalent to that of control. With respect to MBP, in control group (T1) was elevated (p < 0.05) when compared to T2, T3, and T4, statistically similar to that of group T5. The MBP value in T2 group was reduced (p < 0.05) when compared to the other treatment groups.
Similarly, the PF value in control group (T1) was elevated (p < 0.05) and lesser (p < 0.05) in AFB1 contaminated group (T2) when compared to that of T3 and T4. The PF value in AFB1 contaminated group (T2) was lesser (p < 0.05) when compared to that of treatment groups. The PF value in group T3 was lower than those of T4 and T5. The PF value between T4 and T5 groups did not vary significantly. The PF value of group T5 was statistically similar to that of control (T1). The findings of the current examination exposed that inclusion of AFB1 to the feed at 300 ppb level resulted in significant decrease in the MBP and PF compared to that of control. Inclusion of S. cerevisiae at the highest level (0.2%) reversed the MBP and PF values equivalent to that of control.
The high PF in the feed, indicates that increased amount of degraded matter has been incorporated in the mass of microbes, which shows that efficiency of protein synthesis capacity of the microbe’s id higher. More the PF in roughages, more the intake of DM has been observed (Harikrishna et al., 2012). The TVFA, acetate (A), propionate (P) and butyrate (B) values in control group (T1) was elevated (p < 0.05) when compared to that of the other treatment groups i.e. T2 to T5. The TVFA, A, P and B value in AFB1 contaminated T2 group was lesser (p < 0.05) when compared to that of the treatment groups. The TVFA value in T3 was reduced (p < 0.05) when compared to that of T4. The TVFA value in T4 was reduced (p < 0.05) when compared to that of T5. The A and P value of T3 was reduced (p < 0.05) when compared to that of T4 and T5. The A and P value between groups T4 and T5 did not vary significantly. The B value in group T3 was lesser (p < 0.05) when compared to that of T5. The B value between groups T3 and T4; and between T4 and T5 did not vary significantly. The A : P ratio in control group (T1) was reduced (p < 0.05) when compared to that of other treatment groups i.e. T2 to T5. The A : P ratio in T2 was higher (p < 0.05 when compared to that of other treatment groups i.e. T1, T3, T4 and T5. The A : P value of group T3 was higher (p < 0.05) than those of T4 and T5. The A : P value between T4 and T5 groups did not vary significantly. The findings showed that the inclusion of AFB1 at 300 ppb in feed significantly decreased the TVFA, A, P, and B production compared to that of control. This finding of reduced VFA due to aflatoxin concentration was in agreement with Jiang et al. (2012) who also stated that the concentration of VFA reduced with the raise of dosage levels of AFB1. Degradation of cellulose, synthesis of VFA and ammonia and proteolysis were lowered by 0.2 - 0.8 mg·kg-1 body weight of AFB1 in acute bovine aflatoxicosis (Cook et al., 1986). Along with this, the VFA production has also been found to be inhibited irrespective to the nature of the substrate on raising the dosages of AFB1 that has been consistent through the asymptotic gas volume reduction. The VFA level suppression along with the production of gas and ammonia has been concerned with the activity of microbes irrespective of the substrate. Contrastingly, a study by Edrington et al. (1994) has reported that, there have been no variations in the VFA concentrations of the rumen in the lambs growing with the given feed of AFB1 (2.5 mg·kg-1 of diet). Another study by Helferich et al. (1986a) also stated that, the AFB1 at the levels 60 - 600 ppb did not cause variations in the VFA production in steers. In the follow-up study, of 0.714 µmol ingestion of AFB1 per animal has not altered the VFA production in the rumen, in the goats that are lactating (Helferich et al., 1986b). In the present study, inclusion of S. cerevisiae to the aflatoxin contaminated feed partially ameliorated the adverse effects of AFB1 on VFA production. However, inclusion of S. cerevisiae to the AFB1 (300 ppb) contaminated feed caused reduction in A : P ratio. Increased A : P ratio due to aflatoxin contamination indicated that the fermentation has diverted towards more methanogenesis.
It was concluded that aflatoxin contamination of wheat straw based feed at 300 ppb level significantly affected the in vitro rumen fermentation in terms of reduced TDDM and TDOM, GP, MBP, PF, TVFA concentration and increased A : P ratio. Inclusion of S. cerevisiae to the contaminated feed partially to completely ameliorate the adverse effects of AFB1 on in vitro rumen fermentation parameters.
Acknowledgements
The authors are thankful to Buffalo Nutrition Division, ICAR- Central Institute for Research on Buffaloes, Hisar, India for providing the necessary facility and The authors BB, JK and SP would like to extend their sincere appreciation to the National Research Foundation of South Korea for support through the Basic Research Project (Grant number: 2018R1C1B5086232) by Ministry of Education, Science, and Technology.
