Effect of sample volume and time on rumen juice analysis in cattle

Abstract

Background: Rumen juice analysis (RJA) involves analysis of a 10mL sample within minutes after sampling. However, it can be challenging to collect 10 mL of rumen juice (RJ) from some ruminants, and clinical circumstances can delay RJA.

Objectives: Quantify the effect of sample volume (2, 5, 10, 50, and 100 mL), and time-to-analysis (0, 30, and 60 minutes) on RJA.

Animals: Cannulated cow.

Methods: Observational experimental study. Two liters of RJ were collected at 26 separate times. The samples were subdivided into 2 duplicates of each sample volume at each sampling time; and analyzed at 0, 30, and 60 minutes after collection. Rumen juice analysis included pH measurement, methylene blue reduction time (MBRT), and protozoal motility.

Results: The pH of 2 and 5 mL samples was significantly (P = .01) higher than the pH of 50 and 100 mL samples at all time points. The MBRT was significantly lower (faster bacterial reduction) for 100 mL samples compared to all other samples at 0 minute and to 2, 5, and 50 mL samples at 30 min. The pH and MBRT at 60 minutes were significantly higher than at 0 minute for all volumes (P < .05 and P < .01, respectively). For large protozoa, small sample volumes (2 and 5 mL) had significantly lower protozoal motility (scores of 5 and 4.5, respectively) compared to 100 mL samples at 60 minutes (score of 4; P < .05).

Conclusions and clinical importance: Interpretation of RJA could be affected by small sample volumes and delays to analysis. Sample volumes of ≥10 mL analyzed within 30 minutes after collection are recommended.

Keywords: bovine; forestomach indigestion; protozoa motility; rumen fluid; rumen sampling.

FIGURE 1

Diagram summarizing the subsampling, storage, and timing of analysis of 26 RJ samples obtained from a rumen cannulated cow, and an overview of the RJA steps as performed. MBRT, methylene blue reduction time; RJ, rumen juice; RJA, rumen juice analysis; T, time; Temp, temperature.

FIGURE 2

Median (range) temperature of all RJ sample volumes obtained from a rumen cannulated cow, measured as part of a RJA after 0 minute (T ₀), 30 minutes (T ₃₀), and 60 minutes (T ₆₀). N = 26. All median values with the same superscript letter are not significantly different from one another. The median temperature of 100 mL (a) samples was significantly lower (P = .01) than the median temperatures of the 5, 10, and 50 mL samples (b) at each time point (T ₀, T ₃₀, and T ₆₀; 24.3°C vs 25.9°C, 26.8°C, and 26.7°C respectively at T ₀; 20.8°C vs 21.8°C, 21.9°C, and 21.7°C respectively at T ₃₀; 19.9°C vs 20.3°C, 20.5°C, and 20.3°C respectively at T ₆₀). The median temperatures of 2 mL samples (c) were significantly lower (P = .01) than 10 mL samples (d) at each time point (T ₀, T ₃₀, and T ₆₀; 24.6°C vs 26.8°C at T ₀; 21.6°C vs 21.9°C at T ₃₀; 20.4°C vs 20.5°C at T ₆₀). RJ, rumen juice; RJA, rumen juice analysis; T, time.

FIGURE 3

Median (range) pH of all RJ sample volumes obtained from a rumen cannulated cow, measured as part of a RJA after 0 minute (T ₀), 30 minutes (T ₃₀), and 60 minutes (T ₆₀). N = 26. Interrupted line: upper threshold of the normal pH range at 7.2 (lower threshold pH range is 6). All median values with the same superscript letter are not significantly different from one another. Median pH at T ₆₀ (b) was significantly higher than median pH at T ₃₀ (a) for 50 and 100 mL samples (P = .03 for 50 mL and P = .04 for 100 mL samples; 6.87 > 6.83 for 50 mL; and 6.78 > 6.76 for 100 mL). Median pH of the 2, 5, and 10 mL samples (d) were significantly higher than median pH of the 50 and 100 mL samples (c) at T ₀ (P = .01; 6.76, 6.79 and 6.67 > 6.60 and 6.61, respectively). Median pH of the 2 and 5 mL samples were significantly higher than pH of the 10, 50, and 100 mL samples at T ₃₀ and T ₆₀ (e > f [P = .01]; g > h [P = .01]; 7.03 and 7.0 > 6.81, 6.83, and 6.76 at T ₃₀; and 6.50 and 6.50 > 6.11, 6.44, and 6.37 at T ₆₀). RJ, rumen juice; RJA, rumen juice analysis; T, time.

FIGURE 4

Violin plot of the distribution of the pH of all RJ sample obtained from a rumen cannulated cow, measured as part of a RJA after 0 minute (T ₀). N = 26 for each sample volume. Within each violin, the dashed line represents the median, and the upper and lower dotted lines represent the 75th and 25th percentiles, respectively. The dashed lines outside of the violin plots represent the upper and lower threshold of the reference interval for pH range of 6 and 7.2. Violin plots with different letter superscripts are different at P < .05. RJ, rumen juice; RJA, rumen juice analysis.

FIGURE 5

Violin plot of the distribution of the pH of all RJ sample volumes obtained from a rumen cannulated cow, measured as part of a RJA after 30 minutes (T ₃₀). N = 26 for each sample volume. Within each violin, the dashed line represents the median, and the upper and lower dotted lines represent the 75th and 25th percentiles, respectively. The dashed lines outside of the violin plots represent the upper and lower threshold of the reference interval for pH range of 6 and 7.2. Violin plots with different letter superscripts are different at P < .05. RJ, rumen juice; RJA, rumen juice analysis; T, time.

FIGURE 6

Median (range) MBRT of all RJ sample volumes obtained from a rumen cannulated cow, measured as part of a RJA after 0 minute (T ₀), 30 minutes (T ₃₀), and 60 minutes (T ₆₀). N = 26. Dotted line: upper threshold of the normal MBRT range at 360 seconds (6 minutes). Gray box outlines sample volumes and time where MBRT remained normal. MBRT, methylene blue reduction time; RJ, rumen juice; RJA, rumen juice analysis; T, time.

FIGURE 7

Violin plot of the distribution of the MBRT of all RJ sample volumes obtained from a rumen cannulated cow, measured as part of a RJA after 0 minute (T ₀). N = 26 for each sample volume. Dotted line: upper threshold of the normal MBRT range at 360 seconds (6 minutes). The interrupted line within each violin represents the median, and the dotted line the 25th percentile. Violin plots with different letter superscripts are different at P < .05. MBRT, methylene blue reduction time; RJ, rumen juice; RJA, rumen juice analysis; T, time.