Effect of Slow-Release Non-Protein Nitrogen Produced from Agro-Industrial Byproducts on Bio Gas Production, Feed Digestibility and Inoculum Parameters

In the recent decades, air and surface water pollution by nitrogen from agro-industrial discards has become a global environmental concern. Generally, these byproducts and or discards are nutrient rich and could inexpensively be utilized for various purposes marginally helping with mitigation strategies. In this context, our study was conducted in two phases: producing lactosylurea from agro-industrial by-products and subsequently evaluating the possibility of using lactosylurea as a non-protein nitrogen source in the ruminant’s diet and its effect on feed digestibility as well as ruminal parameters. Gas production test and in-vitro disappearance method were used to describe the kinetics of digestion in both dry matter (DM) and crude protein (CP) of the four experimental treatments. Protozoa count and total volatile fatty acids concentration were utilized to evaluate the ruminal parameters. The treatments were 1) basal diet + urea (BDU), 2) basal diet+ lactosylurea (BDL), 3) basal diet+ concentrated lactosylurea (BDCL), 4) basal diet+ Optigen (slow release NPN) (BDO). According to our ndings, produced gas, DM, and CP disappearance in were signicantly higher in concentrated lactosylurea and positive control groups than the other treatments (P<0.05). Moreover, estimated metabolizable energy, digestible organic matter and short chain fatty Acid were signicantly higher for the same treatments (P<0.05). The values for protozoa count (2.66×106 organism/ml) and total volatile fatty acids concentration (30.96 mmol/L) were signicantly lower and for urea treatment compared with others (P<0.05). In conclusion, lactosylurea as agro-industrial by-products can be a good alternative for urea or Optigen to reduce environmental contamination.


Introduction
In the recent years, nitrogen utilization e ciency has been one of the challenging environmental issues.
The agricultural sector contributes to the issue by adding nitrogen to the surface waters and the atmosphere (Pavlidis and Tsihrintzis, 2018;Wanapat, 2009). The ruminant's production system should be developed to protect nature and reduce environmental pollution (Hermansen, 2003). The use of food processing by-products and discards has played a major role in the sustainability of livestock industry yet fermentation properties of these fermentable materials must be carefully investigated for optimum e ciency (VandeHaar and St-Pierre, 2006). However, it requires enough of the (Fessenden and Van Amburgh, 2016). Of all the process discards, lactose and protein rich whey is considered one of the major ones in the cheese production (Bacenetti et al., 2018). Nonetheless, not only that whey powder production is highly expensive, but the process could also pollute the environment. Whey can be used in livestock feeding as an accessible source of carbohydrates of which lactose is the most abundant one ( . In this context, the main objective of this study was to investigate the use of whey-derived lactosylurea and urea as agro-industrial by-products on feed digestibility and ruminal parameters. The speci c objective of our study was to reduce the environmental contamination by secondary by-products of human-food producing industry.

Samples preparation
Lactosylurea samples were prepared in two different conditions as rst and second methods with slight differences according to the methods proposed by Merry et al. (1982) and Torkashvand and Nezamedost (2009). In the rst method, 200ml of whey (provided by Pegah Factory of Tabriz, East Azarbaijan, Iran.) was mixed with 0.11 ml of sulfuric acid and 0.125 g of urea and incubated in 55°C for 72 h. Neutralization was performed with sodium hydroxide and centrifuged for 10 min at 1300 rpm. The residue was separated, and the remaining liquid was transferred to the refrigerator. After the formation of white crystals, they were separated from the initial liquor and stored at 37°C for 24 h in a vacuum drying oven. In the second method, 200 ml of concentrated whey (provided by Pegah Factory of Tabriz, East Azarbaijan, Iran) was mixed with 0.22 ml of sulfuric acid and 0.25 g of urea and incubated at 55°C for 48 h. Then, the solution was neutralized with sodium hydroxide and centrifuged (10 min, 1300 rpm). The supernatant was transferred to the refrigerator. Then, the formed crystals were washed twice with distilled water and placed in an oven under vacuum for 24 h at 37° C.

Gas production test and in-vitro digestibility
We loaded 300 mg of the ground test treatments (Wiley Mill, 2mm) into four 50 ml glass phials. Buffer solution (synthetic saliva) was prepared as proposed by McDougall (1948). Ruminal uid of at least three freshly slaughtered beef (Lutakome et al., 2017) were percolated through a 4-layer cheese cloth to a ask which had been warmed at 39C, and promptly taken to the laboratory. Strained ruminal inoculums were mixed thoroughly at 39 • C together with the synthetic saliva (1:2 v/v) to have a homogeneous digestion medium. Glass phials were loaded by 20 ml of homogeneous digestion medium in six replicates for each treatment. Only digestion medium was loaded into blank phials. Phials were placed in shaker adjusted to 39 • C and 120 rpm (Shirmohammadi et al., 2020). Gas production data were recorded at 2, 4

In-vitro disappearance method
In-vitro digestibility of treatments were determined according to Khajehdizaj et al. (2014). Brie y, along with gas production test, ve vials of loaded treatments with digestion medium were incubated for 2, 12, 24 and 48 h, but in order to release the vials gas production during incubation hours syringe needles were tted to the sealed vials cap. After each incubation hour vials were stored at -20 • C till further analyses.
Prior to analyses, vials were thawed at 39 • C and contents were transferred to 50 ml falcon tubes. Subsequently, tubes were centrifuged (3000 g,10 min), supernatant was pipetted out and the pellets were

Measurement of total volatile fatty acids
Total volatile fatty acid content of the samples was measured according to a previously described method Markham (1942). Brie y, total volatile fatty acids of rumen uid that incubated in three replicates for 12 h were measured in two distillation and titration steps. After collection, about 50 ml of the solution in distillation step, it was titrated by addition of a few drops of phenolphthalein reagent with 0.05 N NaOH solution.

Calculations and Statistical model
All documented data were analyzed in a complete randomized design (CRD) utilizing SAS software (version 9.2, the ANOVA procedure, Duncan's multiple range test). Gas production kinetic was calculated by the following model: Where A is the gas production from the immediately soluble and insoluble fraction; c is the rate constant (%/h) of gas production from the insoluble fraction; t is the incubation time (h); and Y is the volume of gas

Results And Discussion
Experimental diets compositions are shown in Table 1. Experimental diets, except for NPN source, had same composition and were assumed to be fed at the dosage of 60 gram per cow per day at the farm level.

Gas production
The obtained data from the gas production (mL/g DM) of the experimental diets were tabulated in Table  2. According to the obtained results, BDCL had the highest gas production volume (290.91 mL/g DM) after 96 h of incubation which was considerably different from BDL diet. The lowest volume of produced gas was related to BDU. We found no signi cant differences between treatments at the initial incubation times (P<0.05), but after 6 h of incubation signi cant differences were observed (P<0.05). BDL and BDCL did not differ signi cantly up to 48 h, which could be due to their same NPN source. BDU showed a signi cant difference with the treatments containing Lactosylurea and Optigen, which might be due to the faster release of ammonia from urea compared with the other NPN sources after 4 h of incubation. BDU had the lowest gas production at all incubation times except for the rst 4 h (P<0.05). in the present study, we estimated higher extent of fermentation (A (ml/g DM)) for BDCL and BDO, however, BDCL had lower rate (c ml/h) of gas production (P<0.05). Ruminal bacteria consortium is able to produce biogas from different sources of feedstuff (Zhang et al., 2016). Cherdthong and Wanapat (2010) reported the highest gas production for urea calcium (product of slow-release urea) along with cassava chips. They reported that an increase in the extent and rate of fermentation was related to the energy source (cassava chips). The cause of high gas production for BDCL was probably related to the presence of lactose as energy source in concentrated lactosylurea. The high cumulative amount of produced gas indicates high metabolic energy, fermentable nitrogen and other nutrients for the activity of microorganisms Menke et al. (1979). Gas production is positively correlated with the dry matter digestibility indicating that gas production is an integral part of food fermentation. Usually, high gas production was achieved from the carbohydrate section of the feed compared to the other nutrients. The amount of gas production in the early times is due to the differences in the level of nonstructural carbohydrates (NSC) such as sugars, pectin, and starches that are rapidly fermenting and producing gas (Menke, 1988), yet, we didn't observe any signi cant differences at early hours (P<0.05).
Calculated gas production parameters including ME, NE l , DOM, SCFA are reported in Table 3. Buchneri to alfalfa silage on in vitro gas production and degradability, they reported that adding fresh whey had increased Calculated gas production parameters.

The apparent degradability of dry matter
The mean data for dry matter disappearance are presented in Table 4. According to the results, BDCL and BDO showed the highest degradabilities after 12 h of incubation (P<0.05). Less than 12 h incubation, treatments had no signi cant difference (P<0.05). After 24 h there were signi cant differences not only with BDU but also with BDL. The later difference may be caused by the amount of nutrients presented in the whey as the primary substance in lactosylurea production. Chamebon et al. (2017) observed that dry matter degradability increased by adding urea to the treatments (to orange pomace treatment with 38.5% of wheat straw and 1.5% urea and orange pomace treatment with 37% of wheat straw and 3% urea) compared with non-urea treatment. Mahmoudi-Abyane et al. (2018) studied the effect of utilizing different sources of nitrogen on digestibility and nitrogen balance in Mehraban lambs. They reported the lowest and highest digestibility of NDF and ADF for diet containing soybean meal and slow-release urea, respectively. These results suggested that diets with a contentious source of nitrogen can meet the requirements of the cellulolytic bacteria, the major ammonia consumers in the rumen, consequently improving ber digestion and the activity of rumen microorganisms (Castro et al., 1999). Ruminal bacteria can receive 40 to 95% of their nitrogen from ammonia depending on the diet, and using these sources can create a balance of peptides and amino acids (Nolan et al., 1993). In a previous study, digestibility of the bers increased when Optigen was replaced with soybean meal and rapeseed meal, although ammonia nitrogen was high in both treatments (Sinclair et al., 2012). It has been reported that adding 1.8 kg slow-release urea supplementation to the beef diet containing sugarcane, cane molasses and maize signi cantly improved the digestibility of dry matter and NDF (Galina et al., 2003).

The apparent degradability of crude protein
The mean data of crude protein degradability of the experimental treatments are presented in Table 4. According to the obtained results, the highest degradability was observed in BDCL and BDO. The lowest degradability was reported in BDU. Treatments showed no signi cant differences at 2 h but differences were signi cant after 12 h (P<0.05). This difference may be due to the presence of lactose in BDCL and BDO, which showed nitrogen release compared with other two treatments and Optigen as a commercial slow-release urea product had the kinetics just like BDCL. A recent report by Sevim and Önol (2019) that worked on the effect of supplemental slow-release urea on some feed digestibility and rumen parameters showed that using slow release urea had increased feed protein digestibility which is in line with ours.

Protozoa count
The results of the number of protozoa are presented in Table 5. The number of protozoa in the BDCL and BDL treatments were signi cantly higher than the other treatments (P<0.05). The absence of protozoa reduces the predation of bacteria (Takahashi et al., 2005) resulting reduced end products from ruminal bacteria degradation while increasing the ow of microbial protein into the lower gastro intestinal tract (Hess et al., 2004). The protozoa can use up starch granules, thereby creating a balance in the rumen environment and better cellulose digestion (Orpin, 1984). Eugène et al. (2004) found that use of high levels of concentrate can reduce the protozoa population due to a reduction in ruminal pH. In the present study, diets contained equal concentrate: forage ratio and the only source of difference was related to NPN source, therefore the difference in the number of protozoa cannot be related to the mentioned fact.
Protozoa use cellulose and starch as a source of energy, and ruminal bacteria and insoluble proteins as a source of nitrogen (Coleman, 1986;Jouany, 1996). Diets with high concentrate provide digestible energy sources for the protozoa boosting their growth yet allowing protozoa to better compete with ruminal bacteria (Yuste et al., 2019). According to the results of this study, it can be expressed that the diets containing lactosylurea and Optigen can provide nitrogen source easily available for both of ruminal bacteria and protozoa.

Ruminal total volatile fatty acids
The results of ruminal total VFA (mmol/ml of rumen uid) after 12 h of incubation are presented in Table  6. The lower concentration of volatile fatty acids was observed in the BDU and the higher was recorded for the others (P<0.05). It can be explained that in the treatments containing slow-release urea source, the levels of ruminal VFA signi cantly were high in comparison with the other NPN sources (P<0.05). This case might be related to the nal products of ruminal microbial protein in the rumen uid. Therefore, more microbial protein synthesis that probably occurred in three treatments containing of slow-release urea sources in comparison with diet containing of unprocessed urea (BDU). The ruminal total VFA concentration can varied widely depending on the diet variance and elapsed time from the previous meal

Conclusion
According to our observations, lactosylurea, synthesized using urea and whey, can be a suitable alternative for urea or Optigen since in-vitro dry matter and crude protein disappearance values as well as gas production was improved in the lactosylurea containing diets. Additionally, it could signi cantly improve selected rumen parameters inclusive of ruminal protozoa count and total VFA concentrations.
Therefor it is suitable to make use of it to proceed sustainable dairy farming beside preserving environment.   Within a raw, means without a common superscript letter differ (P<0.05).