Sampling procedure
The present cross-sectional study was carried out across all the 22 districts of Punjab State of India during the period of July 2019 to February 2020. A total of 120 bovines [cattle (n = 82) and buffalo (n = 38)] were selected in the study (Table 1). The consent of the dairy farmers for enrollment in the study was obtained and the samples were taken in presence of regional veterinary doctors. The ethical permission for the collection of rumen liquor samples was taken from the Institutional Animal ethics Committee (IAEC) (IAEC/2019/188-221).
From each animal, 50 ml of milk and 15 ml of rumen liquor samples were collected aseptically under the supervision of field veterinarians. The milk collection was done in sterile collection tubes which were transported to the laboratory in ice-box and stored at -200C until further analysis. The rumen liquor was collected in sterile collection tubes by needle puncture of rumen at the left paralumbar fossa using 20 ml syringe with 16-gauge needle under aseptic conditions. The collected samples were strained through double layer of muslin cloth as described by Lengemann and Allen (1955) and labeled properly as ‘strained rumen liquor’ (SLR) with unique identification number. The preservation of rumen liquor was done in saturated solution of mercuric chloride and in 8% formaldehyde solution separately. For analysis of rumen metabolites, 2-3 drops of mercuric chloride were added per 4-5 ml of rumen liquor sample and for rumen microflora count, 1 ml of formaldehyde was added per 1 ml of rumen liquor sample (Singh et al. 2018). The samples were immediately stored in ice box after collection and in -20oC upon reaching laboratory for further analysis. The district wise details of the collected samples are presented in Table 1.
AFM1 detection in milk samples by ELISA
All the collected milk samples (n=120) were subjected to commercial competitive Aflatoxin M1 ELISA (EuroProxima B.V., The Netherlands) having the limit of detection (LOD) of 5 pg/ml for milk samples. All the procedures were performed as per the instructions provided by the manufacturer. The milk samples were prepared for ELISA testing by carrying out centrifugation at 2000×g for 10 min at 4⁰C after being thawed to room temperature and the separated fat layer was removed by using spatula. The optical density (O.D.) values for each sample were recorded immediately at 450 nm by using microplate spectrophotometer. The concentration of AFM1 in milk samples was calculated by using regression equation obtained from calibration curve built by plotting values of % maximal absorbance of standards versus the analyte equivalent concentration (pg/ml).
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The O.D. of the standards and samples were averaged and corrected by subtracting the mean O.D. of blank wells before computation. The O.D. is inversely proportional to the AFM1 concentration in the sample.
Analysis of rumen liquor
To study the correlation of aflatoxins on rumen fermentation, the rumen liquor parameters selected as indicators of rumen fermentation were: (1) Physico-chemical parameters [colour, odour and consistency, pH, Methylene Blue Reduction Time (MBRT), Sedimentation Activity Time (SAT)]; (2) Rumen metabolites [Total Volatile Fatty Acids (TVFA’s) and ammonia nitrogen]; and (3) Rumen microflora count [Total Bacterial Count (TBC) and Total Protozoal Count (TPC)].
Physico-chemical parameters:
1. Colour, odour and consistency
After the collection of rumen liquor, the colour, odour and consistency of rumen liquor samples were immediately recorded as described by Garry (2002). The observations were grouped into 2 categories: normal and abnormal (Supplementary Table 1). The normal group included, greenish-yellow to yellowish-brown colour, aromatic odour and slightly viscous consistency. The observations recorded other than normal were grouped under abnormal category.
2. pH of rumen liquor
The pH of rumen liquor was determined immediately after collection by digital pH meter.
3. Methylene blue reduction test (MBRT)
Methylene blue reduction test was performed as per the method described by Dirksen (1979). A total of 0.25 ml of 0.03% methylene blue solution was added in 5 ml of freshly collected rumen liquor in a test tube and incubated in water bath at room temperature. The time taken for the reduction of methylene blue by the microbial constituents of rumen liquor, i.e., for discoloration of sample, was recorded by using a plain rumen fluid as a basis for comparison.
4. Sedimentation activity test (SAT)
Sedimentation activity test was performed according to the method described by Dirksen (1979). The freshly collected rumen liquor was observed in glass test tubes kept in water bath at 390C. The time required for sedimentation and floatation was referred to as sedimentation activity time.
Rumen metabolites:
1. Total volatile fatty acids (TVFA)
Total volatile fatty acids concentration in rumen fluid was estimated by the method of Barnett and Reid (1957). A total of 1 ml of SRL was transferred into Markham’s micro-Kjeldahl distillation apparatus and 1 ml of scaribrick buffer (10% potassium oxalate and 5% oxalic acid in equal volumes) was added to it. The cup was made air tight with stop cork and by adding some water in it. The steam distillation was carried and approximately 75 ml distillate was collected. To the distillate, few drops of phenolphthalein indicator were added and titrated against standard 0.01N NaOH solution.
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2. Ammonia nitrogen (NH3-N)
The Conway micro diffusion technique (Conway 1957) was used to estimate NH3-N in SRL. In the inner chamber of Conway cell, 1 ml of 2% boric acid solution containing mixed indicator was taken and 1 ml of clear SRL was pipetted into the outer compartment. Further, 1 ml of 50% potassium carbonate solution was added slowly into the outer compartment opposite to SRL. After covering the micro-diffusion cell, it was gently rotated clockwise and anticlockwise at a horizontal plain to mix contents of outer chamber followed by incubation for 1 hour. Thereafter, contents of the inner chamber were titrated against standard 0.01N H2SO4 solution. Simultaneously, a blank of 1 ml distilled water was also titrated against standard acid.
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Rumen microflora count:
1. Total bacterial count
Total bacterial count was determined by using Nigrosin slide technique as per the method described by Gall et al. (1949). In brief, the preserved sample of SRL was thawed and shaken vigorously in order to separate microbes from feed particles and to break microbial clumps. The thawed rumen liquor sample was centrifuged at 3000 rpm for 5 min and the supernatant was serially diluted in 1:10000 ratio with distilled water. The diluted bacterial suspension was mixed well and 0.01 ml of diluted suspension was taken onto a clean grease free glass slide. A loopful of saturated Nigrosin stain was added to 0.01 ml of diluted suspension. The sample was mixed and uniformly spread over 2×2 cm area of glass slide with the platinum loop. The smear was dried immediately over a preheated (about 60°C) hot plate. The bacterial counting was done in total 30 microscopic fields from 2x2 cm area of stained smear under 100X objective of microscope. The total bacterial count per ml of rumen liquor was calculated by the formula given below:
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2. Total Protozoal count (TPC)
The rumen protozoal count was done as per the method described earlier by Naga and El-Shazly (1969). A total of 5 ml of sample was taken through wide bore (3.5 mm) pipette into a test tube. Then 15 ml of normal saline solution (0.85%) was transferred and thereafter 5 ml Lugol’s iodine was added. The solution was mixed gently and 0.1 ml of sample was transferred swiftly to a dry clean slide and spread under a glass cover of known area (24×60 mm). A total of 30 fields were counted per slide both for ease and accuracy and total protozoal count per ml of rumen liquor was calculated by the formula given below:
![](https://myfiles.space/user_files/87796_83ef146087a3bf32/87796_custom_files/img1626185831.PNG)
Statistical analysis
All the data entry and the computation of mean, standard deviation, maximum and minimum concentrations were carried out using Microsoft® Excel 2010. The correlation analysis was taken as the measure for the degree of association between AFM1 excretion in milk and indicators of rumen fermentation computed as Pearson Correlation Coefficient (r) by using Microsoft® Excel 2010. The correlation coefficient was categorized into weak (0-0.25), fair (0.25-0.5), good (0.5-0.75) and excellent (0.75-1) correlation based on r value as described by Cohen (2013).