Estimating the undegradable intake protein content of two forages by different commercial proteases

We evaluated the potential of several commercially available proteases for use in predicting the undegradable intake protein (UIP) concentrations o f alfalfa and prairie hay. Protease s differed in their estimates o f the rate of forage protein breakdown an d the amounts of different forage protein fractions . At least one protease appeared to yield acceptable predictions of UIP via a short-term, single time-point assay. Assays of this type deserve further consideration for commercial application.


Introduction
Current feeding systems for ruminants require knowledge of the proportion of forage protein degraded in the rumen (degradable intake protein = DIP) versus that escaping the rumen (undegradable intake protein = UIP).Measurin g the DIP or UIP content using animals (i.e., vi a in vivo or in situ techniques) requires maintenance of int estinally or ruminally fistulated an imals, which are expensive, require special care, and are frequently unavailable in commercial laboratory settings.
In vitro procedures using semipurified proteolytic enzymes have show n promise as routine laboratory tech niques for estimating UIP, but in most cases, only concentrates and protein supplement s have been tested extensively.Informatio n about how these proteases work with forages is needed.
Therefore, our objective s were to evaluate the potential of several commercial proteases for determining protein degradability, size of protein fractions, and the UIP content of forages.Values obtained using the proteases were compared with those obtained b y in situ and in vivo methods in a previous experiment.

Experimental Procedures
Experiment 1. Four co mmercially available proteases were used to measure protein degradabilit y in alfalfa and prairie hay.The protease s were fro m Streptomyces griseus (SGP) , Aspergillus oryzae (AOP) , Ficus glabrata (ficin), or bromelain from pineapple stem (BR).
For the SGP procedure, hay samples containing 14 mg N (.52 g of alfalfa or 1.64 g of prairie hay, air-dry basis) wer e incubated for 1 hour at 39EC in 40 ml of borate-phosphate buffer (pH 8.0).For the AOP, BR, and ficin procedures, .5 ml of triton X-100 and 20 ml of 1:1 mixture o f in vitro rumen buffer (pH 6.8) and macromineral solution were added to hay samples .One ml of sodium azide (1% w/v) was added to all flasks as an antimicrobial agent.Following the 1-hour buffer incubation, 10 ml of SGP at .33 units/ml, AOP at 3.5 units/ml , BR at 5.0 units/ml, or ficin at 2.15 units/m l were added, and samples were incubated for .25,.5 , 1, 2, 4, 8, 12, 24, and 48 hours.The 0-hour incubations were those subjecte d only to the 1-hour buffer incubation.
Following exposure to the protea s e, samples were filtered, residues were washed with 400 ml of deionized water, and nitrogen (N) content s of the residues were measured.Fraction s and rates obtained were used to calculat e the UIP contents of th e forages using passag e rates measured in a previou s in vivo trial (average for both forages was approximately 2.9%/hour).
Experiment 2. Alfalfa and prairie hay samples were incubated at 3 9EC for 1 hour in an appropriate buffer solution, followed by addition of 10 ml of SGP solution containing .33,3.3 , or 33 units/ml; BR solution containing .5, 5.0, or 50 units/ml; or ficin solution containin g .215,2.15, or 215 units/ml.Based on results observed in Exp. 1, the AOP enzyme was not used in Exp. 2. Samples were incubated for 2, 4, or 48 hours.Residual N was considered to represent the U I P content and was expressed as a percentage of total protein.

Results and Discussion
Experiment 1.The size of forage protein fractions and degradation rates (Table 1) estimated with different proteases were similar in some instances to those obtained by a standard in situ procedure.However, none replicate d in situ methods consistently.These results agree d with other reports indicating lack of consistency betwee n in situ methods and those based on protease enzymes.In contrast, combining degradation rates and fractions to estimate the UIP content yielded UIP estimates that, for the SGP, BR, and ficin proteases, were similar to those det ermined in animals (in vivo).
The UIP estimates from the AOP enzyme were significantly larger than those from the other enzy mes, as well as those from th e in situ and in vivo methods.We also observed that in several cases, th e amount of N remaining after incubation in SGP, ficin, or BR for a defined length of time closely approximate d in vivo UIP.As a result, we felt that further exploratio n of simple, single time-point assays was justified (see Exp. 2).
Experiment 2. The main focus of this experimen t was to develop a rapid, commercially viable, UIP assay.We used a range of enzyme concentrations and incubation times to see if assay length could be reduced by using higher enzyme concentrations.The highest concentration s of ficin (21.5 units/ml) and BR (50 units/ml) resulted in viscous solutions, causing filtration problems that prevented adequat e washing of the residue from solubilized N. Consequently , results obtained at these high enzyme concentrations, particularly at short incubation times, were unreliable.
The two combinations of enzyme concentration and incubation time that compared best to in vivo values were the 4-hour incubation in SGP at 33 units/ml and the 48-hour incubation in SGP at .33 units/ml (Table 2) .Results with the long incubation, low concentration study concur with research from Cornell University.Although short-ter m incubations in ficin did not yield particularly good predic t ions of UIP across both forages, the 48-hour incubation at 2.15 units/ml yielded values reasonably close t o in vivo values.The BR method yielded reasonable values in some cases for alfalfa but not for prairie hay.
In conclusion, single tim e-point estimates of UIP using SGP and possibly ficin appear to have potential for estim ating forage UIP content in a commercial setting.The p o tential for shortterm, single time-point assays of forage UIP across a wide array of forages and different stages of maturity deserves further evaluation.) and K = degradation rate of the B fraction.
b B and C fractions estimated using a single-pool kinetic model where B = insoluble potentially c degradabl e protein fraction and C = undegradable protein fraction; A = (100% -C -B); undegradable intake protei n (UIP) = B × [ K /(K + K )] + C where K = rate of passage (.

d
In vivo UIP = 44.5 ± 3.5, % of total protein.e b

Table 2 . Effect of Protease Type, Concentration (unite/ml), and Incubation Time on UIP Estimates for Alfalfa and Prairie Hay (Experiment 2) a
Higher enzyme concentrations caused filtration difficulties resulting in unreliable estimates. a