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,2
* Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, S7N 5A8, Canada
Agriculture and Agri-Food Canada, Research Centre, Lethbridge, Alberta, T1J 4B1, Canada
2 Corresponding author: beauchemin{at}agr.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: ruminal pH • indwelling ruminal pH probe • acidosis • dairy cow
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Rumenocentesis and oro-ruminal probes have been used to collect ruminal fluid samples for measurement of ruminal pH under both experimental and field conditions (Oetzel and Nordlund, 1998; Garrett et al., 1999; Duffield et al., 2004). However, the usefulness of rumenocentesis in research studies is limited. Furthermore, the risk of peritonitis even with adequate surgical preparation may discourage the use of this technique (Keefe and Ogilvie, 1997; Duffield et al., 2004). Alternatively, various oro-ruminal probes are available, notably the one designed by Geishauser (1993); however, ruminal fluid samples collected through oro-ruminal probes are prone to salivary contamination leading to inaccurate ruminal pH values (Keefe and Ogilvie, 1997; Duffield et al., 2004). Direct ruminal fluid sampling via a ruminal cannula is still a common method for ruminal pH measurement in controlled research studies (Reis and Combs, 2000; Kononoff et al., 2003; Duffield et al., 2004). However, spot sampling techniques (rumenocentesis, oro-ruminal probes, direct sampling via a ruminal cannula) all have inherent limitations as they only indicate ruminal pH at one point in time. Additionally, characterizing ruminal pH variation over time using spot sampling techniques requires regular sampling with short intervals between samples, making it tedious and labor intensive.
Monitoring ruminal pH has been automated with the advent of continuous indwelling pH systems (Dado and Allen, 1993). Continuous indwelling systems have provided data allowing improved characterization of post-feeding ruminal pH variation, which has increased our understanding of the interactions between diet fermentability, meal size, eating behavior, and ruminal pH (Maekawa et al., 2002; Krause and Combs, 2003). Most continuous indwelling pH systems have inherent limitations because they restrict animal mobility and, thus, application is limited to tethered animals (Dado and Allen, 1993; Krause and Combs, 2003; Bevans et al., 2005). Recently, several stand-alone systems have been developed (Enemark et al., 2003; Graf et al., 2005). These stand-alone systems continuously measure reticular or ruminal pH without the use of external cables, thereby allowing the measurement of ruminal pH in grazing or loose-housed animals. Although stand-alone ruminal pH measurement systems have been developed, they are in limited use and a thorough validation of these systems is lacking. Therefore, the objectives of the present study were 1) to develop a stand-alone submersible ruminal pH measurement system for use in cattle and to evaluate its accuracy and precision by comparing its output to pH measurement of ruminal fluid samples collected via a ruminal cannula (Experiment 1); 2) to compare the stand-alone system to an older, well-documented indwelling ruminal pH measurement system (Experiment 2); and 3) to determine the required frequency of electrode standardization necessary to minimize changes in baseline millivolt readings between the start and end of the measurement period (Experiment 3).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Design of the LRCpH.
The LRCpH system was developed using a data logger (model M1b-pH-1KRTD, Dascor, Escondido, CA), a 9-V battery, and an electrode cable (model S653-ATC-20-BNC, Sensorex, Garden Grove, CA), which were housed in a watertight capsule constructed of polyvinyl chloride material (Figure 1
). The pH electrode (model S650-CDHF, Sensorex) was covered by a 38-mm diameter shroud with four 25-mm holes, which was designed to allow particle and liquid passage while preventing the electrode from contacting the ruminal epithelium. Two 900-g weights were fastened to the bottom of the electrode shroud to maintain the electrode in the ventral sac of the rumen. A 30-cm polyester cable was connected to the capsule and the ruminal cannula plug to aid in system location within the rumen and to help maintain the electrode in a vertical position.
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For manual pH measurement (MANpH), the ruminal cannula plug was opened and a covered 20-mL container was submersed in the rumen contents. Upon submersion, the LRCpH electrode was located within the rumen and ruminal fluid was allowed to fill the container from the immediate location of the LRCpH electrode. The open end of the container was again covered, removed from the rumen, the ruminal cannula plug was replaced, and ruminal fluid pH was immediately measured using a portable pH meter (model IQ150, IQ Scientific Instruments Inc., San Diego, CA) with a glass electrode (model PHE-1411, Omega Engineering, Stamford, CT). The portable pH meter was calibrated with a 2-point calibration once daily using pH 4 and 7 buffer solutions. Recalibration occurred if readings obtained with pH buffer solutions were not within 0.02 pH units. The time of ruminal fluid collection was recorded to compare the MANpH method to results obtained from corresponding LRCpH measurement. During ruminal fluid collection, positioning of the LRCpH was noted. Ruminal fluid samples were collected on 4 d over a 2-wk duration. On d 1, ruminal fluid samples were collected from the 6 pregnant Holstein heifers every 30 min starting 1 h postfeeding until 3 h postfeeding, and sampling commenced again 8 h postfeeding and ending 10 h postfeeding. Thus, 66 ruminal fluid samples were collected in total on d 1. On d 2, the 3 lactating Holstein cows were sampled every 30 min starting 2.5 h postfeeding and ending 6.5 h postfeeding. In total, 21 ruminal fluid samples were collected. On d 3, ruminal fluid was again collected from the 3 lactating Holstein cows. However, 1 cow was omitted from this collection period because her ruminal contents were very dry, which prohibited adequate LRCpH placement and ruminal pH measurement. Ruminal fluid sampling began 6 h prefeeding and ended at 0.5 h prefeeding with samples collected every 30 min, resulting in a total of 24 samples. On d 4, ruminal fluid samples were obtained every 30 min from the 2 Black Angus heifers, starting at 2 h prefeeding with sampling ending at feeding. Sampling commenced again starting at 2.5 h postfeeding until 5 h postfeeding. In total, 20 ruminal fluid samples were collected. Across all experimental animals, a total of 131 ruminal fluid samples were obtained for determination of ruminal pH.
Experiment 2: Evaluation of a Continuous Indwelling Ruminal pH Measurement System
Animals and Management.
The 3 cannulated, lactating primiparous Holstein cows used in Experiment 1 were used in this experiment. These cows were selected for this experiment because a wide range of ruminal pH values was measured in these animals during Experiment 1. Diets and feeding management were the same as in Experiment 1.
Design of the Continuous Indwelling Ruminal pH Measurement System.
The design and use of the continuous indwelling ruminal pH measurement system (CIpH) system has been previously documented (see Maekawa et al., 2002; Beauchemin and Yang, 2005; Bevans et al., 2005). Briefly, the CIpH system comprised an industrial microprocessor-based pH controller (model PHCN-37, Omega Engineering). The pH electrodes (S650-CDHF, Sensorex) were connected to the pH controller with a 9-m cable (PHEH-65-30-ATC, Omega Engineering) suspended above the cows. The cable passed through a ruminal cannula plug and extended approximately 50 cm into the rumen. The cable was protected from the ruminal environment with a plastic hose. A shroud was constructed around the pH electrode with four 25-mm holes, which allowed material to percolate through but prevented the electrode from contacting the ruminal epithelium. Two 900-g weights were attached to the electrode shroud to maintain positioning within the ventral sac.
Measurement of Ruminal pH.
Continuous measurements of ruminal pH were collected over 2 consecutive 24-h collection periods using the CIpH. Ruminal pH was measured every 5 s. For the CIpH system, the 5-s ruminal pH readings were averaged over 5-min intervals and recorded by a data logger (model CR10, Campbell Scientific, Logan, UT). Averaging the 5-s pH readings over 5-min intervals has previously been reported in our laboratory (Beauchemin and Yang, 2005). The 5-min averages corresponded to actual ruminal fluid sampling times used for MANpH (i.e., when the rumen cannula plug was open) and were used to determine the relationship between CIpH and MANpH. In this experiment, pH electrodes were standardized as described for the LRCpH system in Experiment 1.
For manual pH measurement, ruminal fluid samples were collected using procedures already described in Experiment 1. During ruminal fluid collection, positioning of the CIpH was noted. Ruminal fluid samples were collected at 30-min intervals over 48 h. In the first 24 h, ruminal fluid samples were collected starting at 5.5 h prefeeding, ending at 3.5 h postfeeding, and recommencing at 7 h postfeeding, and ending at 10 h postfeeding. During the second 24-h interval, ruminal fluid samples were collected starting at 4.5 h prefeeding and ending at 1 h prefeeding. Thus, 32 ruminal fluid samples were collected from each of the 3 cows resulting in a total of 96 data pairs.
Experiment 3: Frequency of Electrode Standardization
Animals and Management.
The 6 cannulated, pregnant Holstein heifers used in Experiment 1 were selected for use in this experiment. Animals were housed individually in tie-stalls. Diets and feeding management were the same as in Experiment 1.
Continuous Measurement of Ruminal pH and Electrode Standardization.
Continuous ruminal pH measurement was conducted using the LRCpH system as already described. At the beginning of the experiment, 6 new pH electrodes (model S650CD-HF, Sensorex) were installed. In this experiment, pH electrodes were assigned to a random sequence of 3 treatments: baseline millivolt readings after 24, 48, or 72 h in the rumen. Millivolt readings in pH 4 and 7 buffer solutions for each pH electrode were recorded immediately before pH electrodes were placed into the rumens of experimental animals. Corresponding pH electrodes were removed from the rumen at 24, 48, or 72 h, and millivolt readings in pH 4 and 7 buffer solutions were again recorded for each pH electrode. Baseline reading difference was defined as the difference in millivolt readings between the start and end of each treatment duration.
Statistical Analyses
Experiments 1 and 2.
Paired data for ruminal pH from the LRCpH (1- and 5-min averages) and MANpH and from the CIpH (5-min averages) and MANpH were analyzed using the MIXED procedure of SAS (version 9.13, SAS Institute, Inc., Cary, NC) with repeated measures to calculate the correlation coefficient (Hamlett et al., 2004). The correlation coefficient was then used to calculate the concordance correlation (Lin, 1989, 1992). Also used in this calculation were the overall mean and variance for each ruminal pH measurement method. The correlation coefficient calculated to account for repeated measures was used to determine precision by determining the deviation of the data from the best-fit linear line. The concordance correlation coefficient was used to determine accuracy by determining how much the best-fit line deviated from the line y = x. The data from the experimental method (LRCpH, 1- and 5-min averages and CIpH, 5-min averages) were plotted on the y-axis and the data obtained from MANpH were plotted on the x-axis. Data were also analyzed separately for 2 cows (1 with very dry and 1 with very liquid ruminal contents) using the same procedures as above. This was conducted to determine if consistency of ruminal contents (due to DM content) had an effect on accuracy and precision.
Experiment 3.
To evaluate the frequency required for electrode standardization, the change in baseline millivolt readings between the start and end of each treatment duration was calculated for each electrode. These data were analyzed as a double Latin square design using the MIXED procedure of SAS. The model included the fixed effect of treatment with period and electrode considered random effects. Differences among treatments were compared using Fisher’s protected LSD test adjusted with the Tukey-Kramer option. Differences were considered significant at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Relationships between the LRCpH and CIpH continuous ruminal pH measurement systems and manual sampling via the ruminal cannula are shown in Table 1. Because the CIpH averaged ruminal pH measurements over 5-min intervals, the LRCpH ruminal pH measurements were also averaged over 5-min intervals. This allowed direct comparison of the 2 systems. In addition, ruminal pH measurements from the LRCpH were averaged over 1-min intervals to determine if averaging readings over a shortened interval affects accuracy and precision. Overall, mean ruminal pH values obtained using the LRCpH (1- and 5-min averages) and the CIpH systems were numerically lower (0.03 and 0.05 pH units, respectively) when compared with mean ruminal pH values obtained using the MANpH method (Table 1). In other studies, mean ruminal pH values were 0.11 pH units (Dado and Allen, 1993) and 0.06 to 0.18 pH units (Graf et al., 2005) lower when measured using an indwelling pH electrode system that continuously monitored ruminal pH compared with direct measurement in ruminal fluid samples obtained via ruminal cannula. With manual sampling of ruminal fluid through the ruminal cannula, there is usually a delay until ruminal fluid pH is actually measured. Smith (1941) postulated that this delay might allow the escape of CO2 from ruminal fluid samples, thus elevating ruminal fluid pH. In the current study, continuous ruminal pH measurement occurred while the ruminal cannula plug was open. Thus, the release of carbon dioxide and disruption of the ruminal mat during manual sampling may have increased the values of the continuous measurements. In the current study, the effects of opening the ruminal cannula plug on ruminal fluid pH were not of concern, because we did not intend to characterize ruminal pH as a function of diet. Rather, the objective was to evaluate the accuracy and precision of 2 continuous ruminal pH measurement systems by comparing the output to MANpH. This study confirms results from other laboratories (Dado and Allen, 1993; Graf et al., 2005) indicating that mean pH from manual sampling methods are higher than mean pH values obtained from continuous pH systems although our mean differences were lower than previously reported values. However, caution should be used when evaluating mean ruminal pH differences between 2 systems because means indicate very little about the relationship between the 2 methods.
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) and, consequently, the correlation coefficient that accounted for repeated measures and concordance correlation coefficients were similar between the LRCpH and MANpH when LRCpH output was averaged over 1 or 5 min. The LRCpH (1-and 5-min averages) correlation coefficients that accounted for repeated measures (0.98 and 0.97, respectively) were numerically higher than that calculated for the CIpH (0.88; Table 1). The concordance correlation coefficient was numerically higher for the LRCpH when compared with the CIpH. Dado and Allen (1993) previously reported a Pearson correlation coefficient of 0.85 for the relationship between a continuous indwelling ruminal pH measurement system and manual ruminal pH measurement. The Pearson correlation coefficient indicates how closely the results are related between 2 methods but does not indicate how the plotted data deviates from the line y = x (Lin, 1992). Thus, in our study, a correlation coefficient that accounted for repeated measures was used to replace the Pearson correlation coefficient and was also used to calculate the concordance correlation. The repeated measures correlation is used to evaluate precision because it determines the deviation of the data set from the best-fit linear line whereas the concordance correlation is used to evaluate accuracy by determining the deviation of the best-fit linear line to the line y = x.
To our knowledge, no other studies have reported the correlation coefficient, accounting for repeated measures, or the concordance correlation coefficient when evaluating the accuracy and precision of a continuous ruminal pH measurement system. The correlation coefficient results from this study suggest that the LRCpH more closely reflected the MANpH results than did the CIpH, thus indicating higher accuracy and precision. The improvement in accuracy and precision can be attributed to the design of the system and not the pH recording interval because the LRCpH had similar correlation coefficients and concordance correlation coefficients regardless of whether the data were averaged over 1- or 5-min intervals corresponding to ruminal fluid collection time for MANpH (Table 1).
The location shift for the relationship between LRCpH and MANpH (0.07 and 0.09 for the 1- and 5-min averages, respectively) was lower than that of CIpH and MANpH (0.16). The line y = x would have a location shift of zero and the location shift indicates how the y-intercept of the plotted data differs from the y-intercept of the line y = x. Thus, for both systems the y-intercept was less than zero indicating that MANpH results are slightly higher than results from the LRCpH or CIpH. The scale shift was similar for the relationship between the LRCpH and MANpH and between the CIpH and MANpH; however, the shift occurred in opposite directions. The scale shift indicates a discrepancy in slope between the plotted data and the line y = x. The plotted data for the LRCpH (1- and 5-min averages) vs. MANpH, and CIpH vs. MANpH appear in Figures 2, 3, and 4, respectively.
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Experiment 3: Frequency of Electrode Standardization
No electrode failure occurred in the current study. Mean baseline millivolt readings in pH 4 (P = 0.94) and 7 (P = 0.23) buffer solutions were not different for pH electrodes after 24, 48, or 72 h of ruminal incubation (Table 2). Furthermore, the changes in baseline millivolt readings for all treatments did not differ (P > 0.05) from zero (Table 2). However, electrode drift for individual probes did not always occur in the same direction for any of the treatment durations (data not shown). Previously, Nocek et al. (2002) noted a requirement for electrode standardization; however, that study only examined the effect of probe cleansing and standardization on pH readings after 4, 6, 8, 12, and 24 h in the rumen. Enemark et al. (2003) maintained electrodes in the reticulum for 8 d with minimal electrode drift; however, only 2 electrodes were used. Results from the current study suggest that new electrodes can be maintained in the rumen for at least 72 h without having a significant impact on millivolt readings. Using millivolt readings to calculate pH values indicated that the mean error that occurred by not recalibrating electrodes and not correcting data for changes in baseline millivolt readings within a 72-h period was 0.03 pH units. The maximum possible error found in this study between consecutive standardizations was 0.13, 0.18, and 0.10 pH units after 24, 48, and 72 h in the rumen, respectively, within a pH range of 4.5 to 7.0. Based on the results of this study, there is no requirement for daily removal and standardization of new electrodes; however, there may be a requirement for regular inspection of electrode function, because malfunction would result in a loss of data. Unlike the CIpH, the LRCpH does not have a visual display indicating millivolt or pH readings and electrode failure would not be diagnosed until the time of electrode standardization. More research is required to determine how electrode usage over time influences baseline millivolt reading stability between standardizations. It can be concluded that daily standardization of new electrodes is not required and the duration between consecutive standardizations could be extended to 72 h.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Received for publication August 12, 2005. Accepted for publication January 26, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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