Stability of the liquid markers chromium (III) and cobalt (III)-EDTA in autoclaved, clarified rumen fluid

ABSTRACT An ideal digesta marker for use in feeding studies is inert, unabsorbable, and moves with the portion of the digesta it is intended to mark. Both chromium (III) and cobalt (III) salts of EDTA (CrEDTA and CoEDTA, respectively) have been used as digesta liquid markers in studies with dairy cattle. Although a small portion of these markers is known to be absorbed and excreted in urine, the markers are assumed to remain ionically bound in the digesta. The degree to which these salts remain bound in solution can be determined through spectrophotometric measurement at the wavelength (λ) of peak absorbance of these colored compounds. The objective of this study was to evaluate whether CrEDTA and CoEDTA dissociate during incubation in autoclaved, clarified rumen fluid (ACRF), as indicated by changes in absorbance. In a complete block design with separate replicated analytical runs, approximately 40 mg/L of Cr from CrEDTA or Co from CoEDTA were incubated in 2 separate preparations of ACRF from 2 lactating Holstein cows, in water (CrEDTA), or in MES buffer (CoEDTA), and appropriate reagent blanks. Solution pH were approximately 6.0. Samples were incubated for 24 h at 39°C with absorbance measurements recorded at 0, 1, 2, 4, 6, 22, and 24. The CrEDTA was measured at λ = 541 nm, CoEDTA at λ = 535 nm, and both were measured with wavescans of λ = 330 to 700 nm. At their peak λ, both CrEDTA in water and CoEDTA in MES buffer maintained absorbance values throughout the incubation, whereas, in ACRF, CrEDTA absorbance decreased by 9% at 0 h, and by up to 14% by 24 h; CoEDTA showed a gradual decline over time, with approximately 4% loss in absorbance by 24 h. Responses differed by ACRF preparation. Both markers in ACRF showed increases and decreases over time in absorbance at λ = 330 and 380 nm, though the changes were more marked for CrEDTA; markers not in ACRF showed no change in absorbance at these λ. Changes in the absorbance values at λ = 330 and 380 nm suggest that soluble phenolic compounds may be involved in the exchange of metals with EDTA, but that does not preclude involvement of other components in rumen fluid. Both CrEDTA and CoEDTA incubated in ACRF showed declines over time in absorbance at their peak λ, suggesting that the metals dissociated from EDTA. The apparent dissociation indicates that these liquid markers are not inert as had been assumed.