Balchem Research Summary

In Vitro Release of Ammonia Nitrogen from Various Nitrogen Sources in Batch Culture

A summary of a study conducted by N. DiLorenzo and A. DiCostanzo. “In vitro release of ammonia nitrogen from various nitrogen sources in batch culture.”

Background

Meeting rumen degradable protein (RDP) requirements of cattle is necessary for optimized carbohydrate digestion in the rumen, and to maximize bacterial crude protein (CP) synthesis. Not balancing available nitrogen with available energy may cause temporary nutrient deficiencies for bacteria (Newbold and Rust, 1992). Degradation kinetics of protein sources differ greatly, even amongst NPN sources, are highly variable and are influenced by DMI. In vivo determination of protein degradation kinetics can be expensive, time-consuming and highly variable. A simple in vitro procedure to determine NH3-N release was developed (modified from Kung et al., 2000). In spite of being a static system, relative differences amongst protein sources can provide an indication of NH3-N release in vivo. This study was conducted to determine the relative rates of release of several NPN sources, including NitroShure™ Precision Release Nitrogen, compared to SBM.

Materials and Methods

Batch culture incubations were conducted using 250-mL flasks fitted with a one-way rubber stopper gas release valve. Each flask was inoculated with 200 ml of a nutrient buffer: rumen fluid solution (4:1 ratio). Rumen fluid came from a cannulated steer fed a 95% corn silage 5% protein supplement diet. The nutrient buffer solution contained a mixture of macro and micro minerals, buffer and cysteine hydrochloride as a reducing agent.

Nitrogen sources tested included: 1. NitroShureTM (NTS), 2. Feed grade urea (URE), 3. Biuret (BIU), 4. Fermenten^® (FER; Church & Dwight Co., Inc. Princeton, NJ), 5. Soybean meal (SBM). Flasks containing no added nitrogen sources were also incubated to serve as blanks (BLK). Four flasks per treatment were incubated per period and there were two periods in the study. All flasks received equal amounts of nitrogen from each source (155 mg; equal to 2 g of SBM) based on their nitrogen content.

During the fermentation, 10 ml samples were taken from each flask at 0, 0.5, 2, 4, 6, 8, 12 and 24 h of incubation. Each sample was analyzed for NH3-N by Kjeldahl (Kjeltec 2300 Anayzer Tecator™, Herndon, VA).

The proportion of NH3-N released from the total incubated N (N provided by the N source + N in the nutritive solution) was calculated as follows:

Proportion of NH3-N released from incubated
N(%) = [(TRTmM-BLKmM/mM Media] x 100

Where: TRTmM was the NH3-N value observed for each treatment at a given incubation time, BLKmM was the NH3-N value observed for BLK at a given incubation time and mM Media was the total mM of N provided by the N source and the N in the nutritive solution.

Results and Discussion

Figure 1 shows the results of NH3-N concentration changes over time of incubation. There were significant (p<0.01) treatment x time interactions. At 6 h, deamination from SBM protein began to be significant as seen by NH3-N accumulation relative to the BLK (p<0.02). This, in part, reflects a lag time for hydration, initiation of protein hydrolysis to peptides and amino acids by rumen bacteria, uptake of peptides and amino acids and the subsequent deamination of amino acids by bacteria to form volatile fatty acids and ammonia. The lag may also reflect incorporation of absorbed peptides and amino acids directly into microbial protein rather than deamination. Ammonia accumulation associated with SBM became significantly different from the BLK at six hours (p<0.02).