Demonstration of constant nitrogen and energy amounts in pig urine under acidic conditions at room temperature and determination of the minimum amount of hydrochloric acid required for nitrogen preservation in pig urine

Objective The objectives were to demonstrate that the nitrogen and energy in pig urine supplemented with hydrochloric acid (HCl) are not volatilized and to determine the minimum amount of HCl required for nitrogen preservation from pig urine. Methods In Exp. 1, urine samples of 3.0 L each with 5 different nitrogen concentrations were divided into 2 groups: 1.5 L of urine added with i) 100 mL of distilled water or ii) 100 mL of 6 N HCl. The urine in open plastic containers was placed on a laboratory table at room temperature for 10 d. The weight, nitrogen concentration, and gross energy concentration of the urine samples were determined every 2 d. In Exp. 2, three urine samples with different nitrogen concentrations were added with different amounts of 6 N HCl to obtain varying pH values. All urine samples were placed on a laboratory table for 5 d followed by nitrogen analysis. Results Nitrogen amounts in urine supplemented with distilled water decreased linearly with time, whereas those supplemented with 6 N HCl remained constant. Based on the linear broken-line analysis, nitrogen was not volatilized at a pH below 5.12 (standard error = 0.71 and p<0.01). In Exp. 3, an equation for determining the amount of 6 N HCl to preserve nitrogen in pig urine was developed: additional 6 N HCl (mL) to 100 mL of urine = 3.83×nitrogen in urine (g/100 mL)+0.71 with R2 = 0.96 and p<0.01. If 62.7 g/d of nitrogen is excreted, at least 240 mL of 6 N HCl should be added to the urine collection container. Conclusion Nitrogen in pig urine is not volatilized at a pH below 5.12 at room temperature and the amount of 6 N HCl required for nitrogen preservation may be up to 240 mL per day for a 110-kg pig depending on urinary nitrogen excretion.


INTRODUCTION
Metabolizable energy (ME) in feeds has been widely employed in swine diet formulations [1] as energy utilization is better reflected in the ME system compared with the gross energy (GE) or digestible energy (DE) system. Feed ME values are determined by subtracting urinary and gaseous energy from ingested DE. In this calculation, gaseous energy is often neglected due to the small quantity in pigs. Thus, an accurate measurement of urinary energy is essential for determining ME values in feeds [25].
Energy in the pig urine consists mainly of urea which can be hydrolyzed to ammonia and evaporated into the air [6]. As the prevention of ammonia volatilization from urine is essential for an accurate ME determination, the addition of acids in the urine collection containers is a general practice to keep the urine acidic [7,8]. However, the amounts of acids used for nitrogen preserva tion vary among experiments [4,911]. To our knowledge, little information is available on the amount of hydrochloric acid (HCl) required for nitrogen preservation in pig urine. Therefore, the objectives of the present experiments were to demonstrate that nitrogen and energy in pig urine are not volatilized under acidic conditions and to determine the amounts of HCl required for nitrogen preservation.

Animal care
The experimental protocol was approved by the Institutional Animal Care and Use Committee at Konkuk University, Re public of Korea (KU17049 and KU19058).

Exp. 1. Nitrogen and energy contents in pig urine under acidic condition
Urine samples were collected from 5 barrows (Landrace× Yorkshire) with a mean body weight (BW) of 68.1±4.0 kg for 24 h with no acid in the urine collection containers and were filtered using cotton cloth (0.5 mm pore size) to remove impurities. The samples were stored in a sealed container at -20°C. Nitrogen concentrations in the urine samples were 0.29%, 0.58%, 0.63%, 0.66%, and 0.68%. Each urine sample (approximately 3.0 L) was divided into 2 groups of 1.5 L which were supplemented with either 100 mL of 6 N HCl to obtain a pH below 2 or 100 mL of distilled water. Each 200 mL urine sample added with HCl, or distilled water was placed in a plastic container. All plastic containers with the urine samples were placed on a laboratory table for 10 d at room temperature of 18°C to 23°C. The weight, nitrogen concentration, and GE concentration of urine were deter mined every 2 d. Urinary nitrogen concentrations were determined using an automatic Kjeldahl analyzer (method 990.03) as described in AOAC [12] and urinary GE con centrations were determined using the procedure described by Kim et al [13].
Experimental data were analyzed using the MIXED pro cedure (SAS Inst. Inc., Cary, NC, USA). The statistical model included day, supplementation of 6 N HCl, and interaction between day and supplementation of 6 N HCl as fixed vari ables, and the day was the repeated term in this model. The values of least squares mean were calculated. Orthogonal polynomial contrasts were used to test the linear and qua dratic effects of day and the interaction between day and supplementation of 6 N HCl. Each plastic container was an experimental unit. Statistical significance and tendency were declared at alpha less than 0.05 and 0.10, respectively.

Exp. 2. A maximum pH for nitrogen preservation in pig urine
Urine samples were collected from 10 barrows ([Landrace× Yorkshire]×Duroc) with a mean BW of 41.2±2.1 kg with no acid in the urine collection containers and were filtered using cotton cloth (0.5 mm pore size) to remove impurities. The samples were stored in a sealed container at -20°C. Three urine samples were selected to contain variable nitrogen con centrations of 0.12, 0.53, and 0.94 g/100 mL. To determine of the maximum pH for nitrogen preservation, six 100mL aliquots from each urine sample were added with 6 N HCl to achieve pH values of 0 A break point of a pH value for nitrogen preservation was estimated by a oneslope brokenline model using the NLIN procedure of SAS (SAS Inst. Inc., USA). A plastic container was an experimental unit and statistical significance was de clared at an alpha less than 0.05.

Exp. 3. A minimum amount of HCl required for nitrogen preservation in pig urine
Five urine samples were selected from 10 samples of Exp. 2 to obtain variable nitrogen concentrations of 0.12, 0.26, 0.53, 0.61, and 0.94 g/100 mL. The pH changes of each 100 mL of urine samples were measured every addition of 0.2 mL of 6 N HCl using a pH meter (SevenEasy pH Meter S20; Mettler Toledo, Columbus, OH, USA).
The NLIN procedure (SAS Inst. Inc., USA) was employed to develop exponential equations for estimating urine pH by the volume of added HCl in each urine sample with various nitrogen concentrations. An equation for determining the minimum amount of 6 N HCl for nitrogen preservation in urine was generated by the REG procedure of SAS (SAS Institute, 2012) with 6 N HCl concentrations in urine as a dependent variable and nitrogen concentrations in urine as an independent variable. A plastic container was an ex perimental unit and statistical significance was declared at alpha less than 0.05. Additionally, the amounts of 6 N HCl required for nitrogen preservation were calculated based on actual daily nitrogen excretion data from 9 published experiments and 8 unpublished experiments conducted in our laboratory.

Exp. 1. Nitrogen and energy contents in pig urine under acidic condition
The amount of nitrogen in the urine showed a linear inter action (p<0.001) between acid supplementation and time (Table 1). The amount of nitrogen in the urine supplemented with distilled water decreased linearly with time, whereas that supplemented with 6 N HCl remained constant regardless of the time. The amount of GE in the urine had a tendency for linear interaction (p = 0.053) between acid supplementa tion and time. The amount of GE in the urine supplemented with distilled water tended to decrease linearly with time, whereas that supplemented with 6 N HCl remained con stant regardless of the time. These results indicate that urea was hydrolyzed to ammonia molecules and volatilized un der alkaline conditions, but not under acidic conditions. In the energy metabolism experiments, urine is collected daily basis with acids in the collection containers and subsamples are stored in the freezer [3,4,9,14]. The present results also indicate that urine samples can be stored at room temperature for at least 10 d without nitrogen or energy volatilization if the urine pH is kept below 2. Under acidic conditions, a large quantity of hydrogen ions traps ammonia as ammo nium ions [15,16] that do not dissociate into hydrogen ions and ammonia [17].
As urea is a major energy source in urine [18], the con stant urine energy under acidic conditions regardless of the time is reasonable. The amounts of energy at d 0 were ex pected to be the same between the 2 groups. However, the amount of energy in the distilled wateradded urine was 30.6% less (12.2 vs 17.6 kcal; Table 1) than that in the acidadded urine on d 0. This unexpected result is most likely due to the volatilization of ammonia from distilled wateradded urine during the lyophilization procedure before GE determination. Thus, the addition of acids to pig urine is critical to prevent ammonia volatilization from urine during collection and sample drying processes for an accurate determination of urinary GE.

Exp. 2. A maximum pH for nitrogen preservation in pig urine
Based on the oneslope brokenline analysis (Figure 1), ni trogen in pig urine was not volatilized at pH below 5.12 (R 2 = 0.98, standard error = 0.71, and p<0.01). The present re sults demonstrate that the use of acid in the urine collection container to keep pH below 5 would be sufficient for nitrogen preservation. This result agrees with the previous suggestions that the pH of the urine should be kept below 5 to avoid ni trogen volatilization [19,20]. Although urine pH values are rarely measured in energy metabolism or nitrogen balance experiments, the pH of collected urine may have exceeded 5 due to insufficient addition of acid to the urine containing a large quantity of urea. Thus, the addition of a sufficient amount of acid to make the pH less than 5.12 is critical for nitrogen preservation in pig urine.

Exp. 3. A minimum amount of HCl required for nitrogen preservation in pig urine
Exponential models were developed for each of the 5 urine samples to estimate urine pH changes by adding 6 N HCl (Figure 2). Using these models, the amount of 6 N HCl required for 100 mL of urine to achieve the pH 5.12 was calculated for each of the 5 urine samples with various nitrogen con centrations. Based on these data, an equation was generated using nitrogen concentration in urine (g/100 mL) as an in dependent variable to determine a minimum amount of 6 N HCl required for nitrogen preservation in pig urine ( Figure  3).
Nitrogen concentrations in pig urine are affected by water intake and dietary fiber concentrations due to changes of water absorption to the circulation system of pigs and uri nary water excretion [2,7,21]. However, these 2 factors do not affect absolute amounts of urinary nitrogen excretion. For the calculation of an amount of HCl required for nitro gen preservation in urine, the amount of excreted urinary  nitrogen should be considered. High dietary protein con tents [2224] and an imbalance of dietary amino acids [25,26] cause an elevation of urinary nitrogen excretion in pigs.
Based on actual data for daily nitrogen excretion, the amount of 6 N HCl required for nitrogen preservation was calculated to be 48 to 240 mL per day for the largest daily urinary nitrogen excretion for each BW range (Table 2). In this calculation, the maximum quantity of daily nitrogen ex cretion (g/d) for each BW range was multiplied by the required amount of 6 N HCl for nitrogen preservation per gram of urinary nitrogen (3.83 mL/g) which is the slope in Figure 3. In energy metabolism or nitrogen balance experiments, the amount of urinary nitrogen excretion is largely variable and difficult to predict accurately. Therefore, a sufficient amount of acids should be used for nitrogen preservation based on the calculations provided in this work.      1) Data were from 9 published experiments and 8 unpublished experiments that measured daily urinary nitrogen excretion.
2) The amount of 6 N HCl required for urinary nitrogen preservation was calculated by multiplying the maximum quantity of daily nitrogen excretion for each BW range with the required amount of 6 N HCl for nitrogen preservation per gram of urinary nitrogen (3.83 mL/g) which is the slope in Figure 3.