P.J. Van Soest’s research while affiliated with Cornell University and other places

Ideal digesta markers used for feeding studies are inert, unabsorbable, and move with the digesta they are intended to mark. Both chromium (III) and cobalt (III) salts of EDTA (CrEDTA and CoEDTA, respectively) are used as markers of liquid digesta in dairy cattle research. A small portion is absorbed and excreted in urine, but the markers are assumed to remain unreactive and as inert salts in the digesta and animal. The degree to which these colored salts remain bound in solution can be estimated through spectrophotometric measurement at their wavelength (λ) of peak absorbance. The objective of this in vitro study was to evaluate whether CrEDTA and CoEDTA dissociate under reducing conditions that could be experienced in the rumen. In a completely randomized design with 2 replicate analytical runs and samples in duplicate within run, approximately 26 mg/L Cr from CrEDTA or Co from CoEDTA was incubated in a 26-mL reaction volume containing 20 mL of Goering and Van Soest medium without tryptone, 3 mL of CoEDTA or CrEDTA solutions, or water (reagent blanks), and 3.0 mL of a combination of distilled water with 0, 0.25, 0.50, 0.75, or 1.00 mL of reducing solution (RedSol). After incubation for 0.5 h at 39°C, absorbance was read at λ = 535, 465, and 560 nm, the peak λ for EDTA salts of Co(III), Co(II), and Cr(III), respectively. Mean reagent blank values were subtracted from CoEDTA and CrEDTA data. The absorbance data at peak λ were analyzed by marker in models that included RedSol with analytical run as a random variable. Contrasts were used to detect linear through quartic effects of RedSol. Samples with RedSol had redox potentials of -250 to -328 mV, which are within the range of reported ruminal measures. As RedSol increased, CoEDTA showed a linear decline of 75% in ABS at 535 nm and a quadratic 4-fold increase followed by a 60% decline at 465 nm. These responses indicate a reduction of Co(III) to Co(II) and subsequent dissociation of Co(II)EDTA. The absorbance of CrEDTA at 560 nm showed a tendency for an 8% linear decrease as RedSol increased. Wavescans from λ = 330 to 700 nm showed CrEDTA retaining its characteristic 2-peak pattern as RedSol increased, whereas CoEDTA curves deformed entirely. We conclude that CoEDTA is not a stable, inert digesta marker under reducing conditions achievable in the rumen and is therefore unsuitable for use in studies with ruminants. Reexamination of the suitability of available liquid digesta markers is advised.

Although lignin has been negatively correlated with neutral detergent fibre (NDF) digestibility (NDFD) in ruminants and used to predict potential extent of NDF digestion of forages, selection of an analysis, Klason lignin (KL) or acid detergent lignin (ADL), to describe the nutritionally relevant lignin has not been resolved. Dismissed as an artifact is the difference between KL and ADL (∆L). A question is whether ∆L influences digestibility of NDF. We evaluated the relationships of ∆L, KL, and ADL with NDFD in order to determine the nutritionally homogeneous or heterogeneous nature of KL. Data sets from 2 laboratories (DS1, DS2) were used that included ADL, KL, and in vitro NDFD at 48 h (NDFD48). DS1 contained 7 C 3 grasses, 17 C 4 maize forages and 19 alfalfas, and DS2 had 15 C 3 grasses, 8 C 4 forages, and 6 alfalfas. Mean ∆L was greater than ADL in C 3 and C 4 samples, and less in alfalfas. Within forage type and laboratory, ∆L was not correlated with NDFD48 (r = -0.34 to 0.49; all P>0.17). ADL was more consistently correlated with NDFD48 (r = -0.47 to -0.95; P <0.01-0.21) than was KL (r = 0.03 to -0.91; P < 0.01-0.94). ∆L as a proportion of KL was correlated with NDFD48 in C 3 and C 4 samples (r = 0.44 to 0.76; P <0.01-0.08). The differing behaviors of ∆L and ADL relative to NDFD48 indicate that KL is a nutritionally heterogeneous fraction, the behavior of which may vary by forage type and ratios of ADL and ∆L present.

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.

Markers of liquid digesta are an essential tool for determination of in vivo nutrient digestibility and passage as they move with and mark passage of the liquid fraction through the gut. Such markers are ideally biologically and chemically unreactive so they do not affect the digesta or animal. Two issues encountered with the liquid marker chromium (III)(Cr)ethylenediaminetetraacetic acid (EDTA; Cr-EDTA): 1)the Cr acetate·monohydrate required for preparation of the crystallized lithium salt of Cr-EDTA (Li·Cr-EDTA; Udén method; J. Sci. Food Agric. 31, 1980, 625–632)has become largely unavailable, and 2)as an ionic salt, the degree of complexation of Cr and EDTA in solution of an uncrystallized preparation of Cr-EDTA has not been evaluated. Accordingly, the objectives of this study were to develop a method for preparing Li·Cr-EDTA using Cr (III)acetate hydroxide, and to compare the degree to which Cr is complexed with EDTA in solutions made with crystallized Li·Cr-EDTA and in preparations in which Cr-EDTA is not crystallized for purification (Binnerts method; Vet. Rec. 82, 1968. 470). The new Li·Cr-EDTA preparation method which uses Cr acetate hydroxide gave product yields of 901 to 870 g actual/kg theoretical which was similar to the 900 g actual/kg theoretical yield reported for the original Udén method. The Cr concentrations were 928–961 g actual/kg theoretical for the salt. Solutions of both the crystallized preparation of Li·Cr-EDTA and the liquid preparation of the Binnerts method gave visibly similar absorbance patterns at wavelengths from 300 to 750 nm and showed maximum light absorption at 541 nm, the reported wavelength of maximum absorption for Cr-EDTA. Comparison of absorbances at 541 nm to a Li·Cr-EDTA generated standard curve showed that uncrystallized Cr-EDTA produced with the Binnerts method gave a recovery of Cr in Cr-EDTA of approximately 993 g/kg, which did not differ from the 992 g/kg value for preparations of Li·Cr-EDTA; this indicates that Cr and EDTA for both preparation methods were almost completely complexed in solution. In conclusion, the new method for preparing Li·Cr-EDTA gave results comparable to the Udén method, and Cr and EDTA were ionically bound to a high degree and comparably in both the uncrystallized solution of the Binnerts method and solutions of crystallized Li·Cr-EDTA. Verification of the degree of Cr and EDTA complexation in Binnerts preparations can be accomplished with spectrophotometric comparison against a purified standard such as Li·Cr-EDTA.

you can find the full SMPR on the AOAC web site:- http://www.aoac.org/aoac_prod_imis/AOAC_Docs/SMPRs/SMPR_2018_001.pdf

The influence of stage of lactation and corresponding diets on rates of protein degradation (kd) is largely unstudied. Study objectives were to measure and compare in situ ruminal kd of crude protein (CP) and estimate rumen CP escape (rumen-undegradable protein; RUP) of selected feeds by cows at 3 stages of lactation fed corresponding diets, and to determine the incubation times needed in an enzymatic in vitro procedure, using 0.2 units of Streptomyces griseus protease per percent of true CP, that predicted in situ RUP. Residue CP was measured after in situ fermentation for 4, 8, 12, 24, 36, 48, and 72 h of 5 protein sources and 3 total mixed rations, which were fed to the in situ cows. Two nonlactating (dry) cows and 2 cows each at 190 (mid) and 90 (peak) days of lactation were used. Each pair of cows was offered free-choice diets that differed in composition to meet their corresponding nutrient requirements. Diets had decreasing proportions of forages and contained (dry matter basis) 11.9, 15.1 and 16.4% CP and 54.3, 40.3 and 35.3% neutral detergent fiber, for dry, mid, and peak TMR (TMR1, TMR2, and TMR3), respectively. Intakes were 10.3, 21.4, and 23.8 kg of dry matter/d, respectively. Kinetic CP fractions (extractable, potentially degradable, undegradable, or slowly degradable) were unaffected by treatment. Lag time and kd varied among feeds. The kd was faster for all feeds (0.136/h) when incubated in dry-TMR1 cows compared with mid-TMR2 (0.097/h) or peak-TMR3 (0.098/h) cows, and no differences in lag time were detected. Calculated RUP, using estimated passage rates for each cow based on intake, differed between dry-TMR1 (0.382) and mid-TMR2 (0.559) or peak-TMR3 (0.626) cows, with a tendency for mid-TMR2 to be different from peak-TMR3. Using the average kd and lag time obtained from dry-TMR1 to calculate RUP for mid-TMR2 and peak-TMR3 cows using their passage rates reduced RUP values by 6.3 and 9.5 percentage units, respectively. Except for that of herring meal, in vitro residue CP at 6, 12, and 48 h of enzymatic hydrolysis was correlated (r = 0.90) with in situ RUP of peak-TMR3, mid-TMR2, and dry-TMR1, respectively. Although confounded within treatments, stage of lactation, diet, and intake appeared to affect CP degradation parameters and RUP. Using kd from nonlactating cows, or the RUP calculated from them, may bias diet evaluation or ration formulation for lactating cows. In addition, enzymatic in vitro predictions of RUP should be measured using incubation times that are appropriate for lactating cows.