Brazil is the world’s largest producer of forage seeds and, as such, generates a large volume of residue that causes problems for the subsequent occupation of the area. The permanence of straw in seed production fields forms windrows that inhibit regrowth and make harvesting under the windrows difficult. Tropical-forage-grass seed production fields occupy an area equivalent to 140,000 ha per year, generating an average of 20 t of straw per hectare. It is thus estimated that 2.8 million tons of this lignocellulosic material are discarded each year in Brazil (Catuchi et al., 2017).

Among the alternatives to optimize production, the use of alternative foods from agricultural residues can be considered. This is possible due to the ability of ruminants to transform plant residues into nutrients (Van Soest, 1994). As it comes from a mature forage, the residue from the production of tropical seeds is characterized by having low nutritional value, with high levels of fibrous carbohydrates and low levels of crude protein (CP), non-fiber carbohydrates, and minerals. An alternative to solve this problem is the compaction of straw through a process called briquetting (Souza and Cardoso, 2003). Processing takes place by means of temperature and pressure, which can result in some destabilization of the lignocellulosic matrix of the original material, causing an increase in the rate of ruminal degradation of the fiber.

Diets with a high concentration of cell wall restrict the access of microorganisms to the food particle, with a consequent decrease in digestibility (Akin, 1979). This limitation is mainly due to the lower rate of degradation and passage of the fibrous fraction through the rumen. For this type of feed, besides its composition, information on its transit and degradation kinetics is essential, so it can be strategically used in ruminant diets.

The total CP present in low quality fibrous foods is little used by rumen microorganisms and does not meet the requirements to maintain adequate fermentation (Oliveira, 2007). The low protein content of these straws can be overcome with the use of protein supplements. Ammonia is essential for microbial protein synthesis in the rumen and can be supplied through protein and non-protein nitrogen (NPN) sources such as urea (Berger et al., 1994).

Nonetheless, conventional urea has a fast degradation in the rumen, which can oftentimes reduce the efficiency of its utilization due to the low synchronization with fermentable carbohydrates in tropical diets. This has fostered the development of new sources of NPN, which provide greater synchronization and rumen efficiency. Slow-release urea degrade less rapidly in the rumen, with potential claims of improved synchronization of ruminal ammonia with energy digestion (Salami et al., 2020, 2021).

Therefore, the hypothesis is that slow-release urea and extruded urea provide greater synchronization with the fermentable carbohydrates and, therefore, improve the degradation dynamics and the transit kinetics of the fiber from the residue of the processed forage harvest. The present study was thus carried out to investigate the dynamics of degradation and transit kinetics of fiber from processed forage-seed harvest residue in response to the use of alternatives sources of NPN to urea in supplements formulated for a low intake.

Research on animals was conducted according to the institutional committee on animal use (CEUA EPAMIG01/2019). The experiment was carried out in São João del-Rei, Minas Gerais, Brazil (Latitude: 21°8'11" South; Longitude: 44°15'43" West), from June to August 2020.

Four rumen-cannulated, castrated Holstein-Gir crossbred cattle, weighing an average of 380±22.64 kg, were distributed into a 4 × 4 Latin square design. Throughout the experiment, the animals remained stabled in individual stalls with concrete floors and partially covered with clay tiles. Treatments were as follows: supplement containing conventional urea (CUS), supplement containing slow-release urea (SRUS), supplement containing extruded urea (EUS), and supplement containing monoammonium phosphate (MAPS).

A basal diet was used to meet the nutritional requirements of cattle with a body weight of 400 kg according to the NASEM (2016) (Tables 1 and 2). Diets were supplied twice daily to the animals, which had ad libitum access to water. The four diets varied only in the source of NPN in the low-intake supplement, which characterized each treatment. Orts were not sampled since the objective was to study fiber transit and degradation kinetics.

Table 1

Ingredient	%
Straw briquette - straw from forage-seed harvest, processed1	85
Potato starch	13.5
Mineral supplements containing different sources of NPN	1.5
Total	100

NPN - non-protein nitrogen.

Table 2

Feedstuff	DM	CP	CF	aNDF	ADF	Lignin	MM	Starch
Straw briquette	918.2	51.2	ND	736.6	439.4	35.2	54.5	ND
Potato starch	957.5	3.3	ND	40.8	29.5	ND	4.3	801.4
Mineral supplement	925.0	312.5	ND	ND	ND	ND	1000	ND

DM - dry matter; CP - crude protein; CF - crude fat; aNDF - neutral detergent fiber; ADF - acid detergent fiber; MM - mineral matter; ND - not detectable.

Straw briquette, used to compose the diet, is a product obtained from the industrial processing of residues from the production of tropical forage seeds (Urochloa brizantha, cv. MG4), composed of stems, leaves, and remaining inflorescences. This processing consists of grinding and subsequently compacting the material and cutting the briquettes formed. The potato starch, originated from the processing for the production of straw potatoes (Croques company, São João del-Rei, MG, Brazil), was chosen to be used in the diet because of their low protein content. Straw briquette was hydrated at the ratio of 2:1 (2 L of water to 1 kg of straw briquette) and supplied to the animals together with starch and mineral supplement. To allow an equal concentration of NPN between the sources, its content was fixed by its concentration in monoammonium phosphate. Mineral supplements containing the test sources (Table 3) were inserted directly into the rumen once a day via cannula, throughout the trial period. This procedure was adopted to ensure the complete supply of NPN for each treatment.

Table 3

Warranty level
Calcium (g/kg)	160
Phosphorus (mg/kg)	90
Sodium (mg/kg)	65.35
Sulfur (mg/kg)	5
Magnesium (g/kg)	5
Cobalt (mg/kg)	24
Copper (g/kg)	1600
Iron (mg/kg)	600
Iodine (mg/kg)	60
Manganese (g/kg)	3600
Selenium (mg/kg)	12
Zinc (mg/kg)	4800
CP (g/kg)	312.46
NPN Eq CP1 (g/kg)	312.46

1 Crude protein equivalent from non-protein nitrogen refers to the sources of non-protein nitrogen in the low-intake supplement, which characterized each treatment.

In the basal diet, the protein and fiber contents were analyzed. Total nitrogen was determined by method 981.10 of AOAC (2012). The CP content was calculated by multiplying the percentage of N by 6.25. Neutral detergent fiber (aNDF) was determined according to Mertens (2002), without the addition of sodium sulfide and with the addition of thermostable alpha-amylase. The aNDF content was corrected for protein and ash for all samples (Licitra et al., 1996).

The experiment lasted 92 days, which were distributed into four experimental periods of 23 days each. Of these, the first 14 were used for the animals to adapt to the diets, and the subsequent nine for sample collection.

The following models were fitted to the degradation profiles:

Equations 4, 5, and 6 represent the exponential, generalized Michaelis-Mentem (GMM), and generalized compartment (GNG1) models, respectively (López et al., 1999; Vieira et al., 2008), in which A is the potentially degradable fraction. In equations 4 and 6, k is the degradation rate of potentially degradable fractions; in equation 5, c is a scale parameter and K (h) represents the time taken until half of the substrate is consumed (half-life). In equation 6, N is a positive integer representing the time-dependent order, λ_a (1/h) is the asymptote of the rate of preparation for digestion, δ = λ_a/(λ_a − k) is a constant, U is the indigestible fraction, and e_t is the random error. The models were fitted to the degradation profiles using the nlme package procedure of R (Pinheiro and Bates, 2000). The best fit of the model to the profile was evaluated by computing the corrected Akaike information criteria and its derived measures (Akaike, 1974).

The conventional assumption of homoscedasticity was tested using the nlme package of R (Pinheiro and Bates, 2000).

Variance was modeled as shown below (Pinheiro and Bates, 2000):

in which σ² is the residual homogeneous variance as shown by equation 7. Equation 8 represents the power-scaled (ψ) residual variance as a function of the expected mean, R_t. The correlation between measures was also evaluated using the CAR1 of the nlme package of R (Pinheiro and Bates, 2000).

The models were fitted to the profiles of in situ fiber degradation and marker excretion using the nlme package of R software (Pinheiro and Bates, 2000). The best fit of the model to the profile was evaluated by computing the information criteria derived from the corrected Akaike criterion (Akaike, 1974; Sugiura, 1978; Burnham and Anderson, 2004). Additionally, the decision on the best model to describe the profile was made based on the recommendations of Vieira et al. (2012).

Additional estimates were obtained by the NLMIXED package of SAS (Statistical Analysis System, University Edition), using the estimates from the nlme package of R as input.

As aNDF has no soluble component in the rumen fluid phase, the estimated parameter coefficients were normalized by assuming the correction proposed by Waldo et al. (1972), as follows:

The effective degradability coefficient (ED), mean residence time in the rumen-reticulum (MRTR), and mean total residence time (MTRT) were calculated following the equations:

The estimated parameters and additional estimates were compared by contrasts against the control treatment, which is conventional urea (CUS). Contrasts were considered significant when P<0.05.

Because it is a mature material, straw from seed production has low nutritional value (i.e., high fiber content, low protein content). This translates into poor degradation of this fiber, as evidenced by the indigestible fraction (Un) values (Table 5). The high levels of lignin and the incrustation of this lignin onto the other components of the cell wall (cellulose and hemicellulose) found in plants at an advanced stage of maturity make it difficult for microorganisms to access the feed particle, reducing its digestibility (Akin, 1979). In Cynodon nlemfuensis pastures, Oliveira et al. (2013) found that the potential degradability of aNDF was inversely proportional to the age of the plants (83.2, 80.3, 76.8, and 75.3%, respectively for 28, 48, 63, and 79 days of regrowth). Moreover, rumen retention time increases and voluntary intake decreases, ultimately compromising animal performance (Lazzarini et al., 2009).

The size of the potentially degradable and indigestible fractions is characteristic of the substrate, and changes in the ruminal environment cause variations in the rate of degradation by microorganisms (Ørskov, 2000). These effects have a greater impact in tropical conditions relative to aNDF, given its role as the main energy substrate for growth and as a determining factor in the rumen-fill process, which increases as forage quality decreases (Vieira et al., 1997).

The incorporation of NPN sources into strategic low-intake supplementation could improve the degradation and transit parameters of fiber, even if it has a relatively low quality (Galo et al., 2003; Pires et al., 2004; Santos and Pedroso, 2011). The increase in the aNDF degradation rate through the addition of nitrogenous compounds reiterates their priority nature in the supplementation of animals kept on pastures during the dry season, a situation in which energy extraction from fibrous carbohydrates becomes limited due to deficiency of nitrogenous compounds for the synthesis of enzymatic systems of ruminal microorganisms (Costa et al., 2008; Detmann et al., 2009; Lazzarini et al., 2009; Sampaio et al., 2009).

Positive alterations in the escape of particles from the ruminal environment are associated with the use of nitrogen supplements in low-quality forages and are strongly related to an increase in total intake by the animal (McCollum and Galyean, 1985). Experiments carried out in several places in the United States showed an average increase of 22% in the intake of ammoniated forages (Berger et al., 1994). In nine experiments with growing cattle, the animals that received forage treated with ammonia gained 163 g/day more than those that received untreated straw. In eight tests with pregnant cows, ammonia-treated hay and straw provided a 313-g higher daily gain compared with that of animals that received untreated forages (Kunkle, 1998).

Although urea positively affects ruminal fermentation parameters and microbial growth in ruminants, it is rapidly degraded, making it difficult for microorganisms to readily utilize it and leading to waste of nitrogen sources. Greater synchrony of urea hydrolysis with carbohydrate degradation could improve the efficiency of NPN incorporation into microbial protein (Firkins, 1996). It is believed that the main effect of slow-release urea compared to conventional urea is to balance ammonia concentrations throughout the day.

In the present study, conventional urea showed a lower rate of degradation of the potentially degradable fraction of the fiber and effective degradability than the other sources in the NPN test, slow-release urea, extruded urea, and monoammonium phosphate. Considering that conventional urea is the most readily soluble source of the four NPN sources under study, this may have caused an excess of ammoniacal nitrogen in the rumen, which is not used efficiently by ruminal microorganisms for microbial protein synthesis, and therefore, is absorbed by the rumen wall and metabolized in the liver. Its excess in the liver increases the energy costs associated with nitrogen metabolism, the “urea cycle”, to finally be able to excrete urea via urine. With this, the animal starts directing the ingested energy to eliminate urea (Verbic, 2002).

Extruded urea showed improvement in all parameters of fiber degradation kinetics and effective degradability when compared with conventional urea. The solubility and concentration of ammoniacal nitrogen of extruded urea is lower than the value observed for conventional urea (Ítavo et al., 2016). This fact indicates that by reducing solubility, urea extrusion allows a slower release of nitrogen in the ruminal environment when compared with conventional urea. Ítavo et al. (2016) worked with different combinations of NPN sources to obtain different NPN solubilization rates, urea + extruded urea + coated urea, urea + coated urea, urea + extruded urea, and extruded urea. Although the animals in the extruded urea treatment showed lower intake compared with the animals supplemented with the other sources, a higher weight gain was observed, which indicates that extruded urea, as it is a source of medium solubility nitrogen release, may have been used with greater efficiency by ruminal microorganisms and enabled adequate synchrony between the digestion of forage fibrous carbohydrate and the release of nitrogen from the source for microbial protein synthesis.

Ferreira et al. (2005) observed satisfactory results when comparing conventional urea with encapsulated urea, with the former showing more constant ammonia levels throughout the day, which resulted in greater fiber degradability. Dietary urea or slow-release urea supplementation improves the efficiency of microbial protein synthesis in Nellore steers (Corte et al., 2018). The synchronization between fermentable energy and degradable nitrogen in the rumen, promoted by using slow-release urea, maximizes microbial growth and promotes microbial protein synthesis. In the rumen, the microbial growth rate is slower than the urea degradation rate, and rapid ammonia release results in inefficient utilization of nitrogen and wastage of nitrogen sources (Xin et al., 2010). The coated urea supplementation improves milk production and fat content in dairy buffaloes through microbial protein turnover and increased fiber degradation (Aquino et al., 2014; Nadeem et al., 2014).

In general, the results regarding particle transit kinetics obtained in this study were similar to those described by Andrade (2018), who evaluated the parameters of corn silage and found an asymptotic rate of passage from the reticulorumen of 0.053, a mean total residence time of 62.1 min, and a mean residence time in the reticulorumen of 56.6 min. The similarity between the parameters, especially for the rate of passage, in the comparison of corn silage and straw briquette, can be said to be due to the particle size of straw briquette, which decreases with processing (grinding).

Disagreeing with these studies, Bourg et al. (2012) found no differences between a lipid-coated urea and urea on diet intake or digestibility in an N balance study using Holstein steers fed a diet based on steam flaked corn. In the study by Azevedo et al. (2008), neither protein supplementation nor urea encapsulation affected the aNDF degradation parameters. Likewise, in an experiment with grazing animals, Hess et al. (1994) did not observe differences in aNDF degradation rates after supplementing them with different protein sources (alfalfa hay, cottonseed meal, and corn gluten).

When comparing parameters of fiber degradation and transit kinetics as a function of different sources of non-protein nitrogen, in low-intake supplements for cattle, it was found that, in general, the worst results are obtained with supplements that contained conventional urea.