Effect of Supplementing Grass Silage-Based Diets with Concentrate Carbohydrate Sources with Different Fermentation Profiles on N Metabolism of Beef Heifers Fed to Maintenance

The synchronous supply of energy and nitrogen (N) substrates to the rumen microbes on grass silage (GS)-based diets can potentially lead to reduced levels of N excreted in the urine. The objective of this study was to evaluate the effect of supplementing GS-based diet with carbohydrate sources differing in rumen fermentation profile on N metabolism of beef heifers. Six Belgian Blue × Holstein Friesian cross beef heifers (487 ± 29 kg BW) were used in a 3 × 3 Latin Square design (n = 6). Dietary treatments were: (RB) GS supplemented with rolled barley; (MM) GS supplemented with maize meal and; (SH) GS supplemented with soya hulls offered at 40:60 forage to concentrate ratio on a dry matter (DM) basis, at maintenance feeding (40 g DM/kg BW0.75). Carbohydrate source had no effect on DM, organic matter, or N intake or total N excretion and the amount of N excreted in the urine (p > 0.05). Animals offered MM excreted a higher percentage of N in the faeces and a lower percentage of N in the urine compared to animals offered RB (p < 0.05). There was a time by interaction for ruminal ammonia (NH3) concentrations (p < 0.01). Ruminal NH3 concentrations peaked at 2 h post-feeding for all treatments. At 3 h post-feeding, ruminal NH3 concentrations for the RB treatment remained higher compared to MM and SH treatments. Molar proportions and total ruminal volatile fatty acids were similar among dietary treatments (p > 0.05). Supplementing GS-based diets with different carbohydrate sources had no impact on the total level of N excreted or the amount of N excreted in the urine. However, there was a higher percentage of N excreted in the faeces and a lower percentage of N excreted in the urine when animals were offered MM compared to those offered RB (p < 0.05).


Introduction
Globally, there are growing concerns as levels of ammonia (NH 3 ) in the atmosphere continue to rise [1]. Excess nitrogen (N) excreted from agriculture contribute to atmospheric NH 3 [2,3] responsible for 93% of total NH 3 emissions within the European Union (EU) [4]. When redeposited, ammonia increases acidification and the eutrophication of terrestrial and aquatic ecosystems [5]. Furthermore, NH 3 reacts with atmospheric acids to form secondary particles (particulate matter, PM 2.5 ), which contribute to air pollution, estimated to be responsible for 4.2 million premature deaths worldwide in 2016 [6]. In Ireland, agriculture accounts for 99% of total NH 3 emissions, with the cattle sector responsible for 90% of this total, owing to animal housing/storage and land spreading of manures accounting for 47.1%, and deposition at grazing accounting for 12.3% [7].
Beef cattle are inefficient in utilising N, only retaining 10-20% of N consumed [8], resulting in large amounts of ingested N being excreted in urine and faeces. Reducing (DM) basis. All diets were formulated to be isonitrogenous and balanced with soya bean meal (Table 1). Diets were offered at maintenance (40 g DM/kg BW 0.75 ) [26] twice daily as a total mixed ration (TMR) at 08:00 and 16:00 h using a Calan Data Ranger (American Calan, Northwood, New Hampshire, USA). The GS used during this experiment consisted of predominantly perennial ryegrass (Lolium perenne L.). The crop was felled during the early booth stage of vegetation (growth stage 410; [27], wilted for 16 h, baled, and wrapped using a McHale Fusion 3 Integrated baler/wrapper (McHale, Ballinrobe, Co. Mayo, Ireland). The crop was ensiled without the use of an additive. Each experimental period consisted of a 14 d dietary adjustment period, where the animals were fed their respective diets using a Calan Broadbent controlled feeding system (American Calan, Northwood, New Hampshire, USA), followed by an 11 d experimental period, where the animals were housed in metabolism stalls (1.4 × 1.8 m). During this period in the metabolism stalls, animals were allocated the first 3 d for acclimatisation, followed by 8 d to facilitate a N-balance study, rumen sample collection and in sacco DM degradability determination. While in the metabolism house, each animal was assigned to their own individual stall for the duration of the experiment with ad libitum access to water.

Data and Sample Collection
During the N-balance study, all animals were fitted with a specially constructed harness system to facilitate the separate collection of urine and faeces as previously described in Kirwan et al. [28].
Samples of concentrates (rolled barley, maize meal, soya hulls and soya bean meal) were collected weekly, while GS and TMR samples were collected daily, later pooled per treatment and per animal for each experimental period. Samples were dried at 55 • C for 48 h for chemical analysis with additional samples frozen and stored at −20 • C for later total N analysis. Faecal and urine samples collected during the N-balance study were prepared as previously described in Whelan et al. [29].
On d 1 and 5 of each N-balance period, blood samples were collected by jugular venepuncture at 1600 h prior to pm feeding into blood collection tubes containing Lithium Heparin (REF: 367526, BD-Plymouth, UK), prepared as described in Kirwan et al. [28] and then stored at −20 • C pending analysis for plasma urea N, total protein, and creatinine concentrations.
In sacco DM degradability determinations were conducted on d 8 and d 9 in each experimental period to determine the extent of rumen digestion of each of the three carbohydrate sources offered (rolled barley, maize meal, and soya hulls) over a 48 h period.
In situ filter bags (5 × 10 cm; 50 µm pore size) (Ankom Technology, Macedon, New York, USA) containing approximately 5 g DM feed were placed inside large mesh nylon bags and inserted into the ventral sac of the rumen and secured with a metal weight. The in situ bags were inserted at 1700 h on d 8 of each experimental period and incubated for 0, 2, 4, 6, 8, 12, 24, 48 h in reverse order. All feed samples were previously ground using a Norris hammer mill fitted with a 2 mm screen (Lab Mill Christy Turner, Suffolk, UK). After removal from the rumen, all bags were immediately submerged in ice cold water, thoroughly washed and frozen at −20 • C. Upon thawing, in situ bags were rinsed in a domestic washing machine for 30 min using the cold rinse cycle in the absence of detergent, then dried at 55 • C for 48 h. Degradability constants a, b, and c were estimated according to the non-linear model: [30], where 'a' represents the soluble degradable fraction, 'b' represents the slowly degraded fraction within the rumen and 'c' is the constant rate of degradation per hour of the 'b' fraction with time 't'. Effective degradability (ED) was calculated using the equation a + [bc/(c + k)] [30], where k is the fixed rumen outflow rate 0.03 h −1 [31].
Rumen fluid samples were collected at 1, 2, 4, 6, 8 h post-feeding on d 10 and 11 while in the metabolism house via the cannula for pH, NH 3 and volatile fatty acids (VFA) determination as described previously in Kirwan et al. [28] and analysed for NH 3 concentrations using the phenol hypochlorite method of Weatherburn [32].

Chemical Analysis
Samples of TMR, concentrates, GS and faeces were prepared and analysed for DM, NDF, acid detergent fibre (ADF), starch, ash, N, gross energy, ether extract, and N in urine as described in Kirwan et al. [28]. The apparent digestibility (%) of nutrients [DM, organic matter (OM), crude protein (CP), NDF and starch] was calculated according to the following equation [33] (intake and output of nutrients in kilograms): Apparent nutrient digestibility = (1 − (faecal nutrient/total nutrient intake)) × 100. Data were analysed as a replicated 3 × 3 Latin Square design using the PROC MIXED procedure of Statistical Analysis Software (SAS v9.4, Inst. Inc., Cary NC, USA) [34]. Normal distribution and homogeneity of variance were analysed using the UNIVARIATE procedure. Animal within period was the experimental unit. Model consisted of animal, period, and dietary treatment. Animal within period was a random effect. Ruminal data collected at different times after feeding were analysed using the PROC MIXED procedure for repeated measures. The model contained the same fixed effects as before, except that time after feeding and its interaction with the main effects were included. Effects were considered significant at p < 0.05, with a tendency towards significant p < 0.10. When significant differences were detected, difference among treatment means and treatment by time point interaction were tested using Tukey's multiple comparison test.

Results
The effect of carbohydrate source on nutrient intake and total tract apparent digestibility of nutrients is presented in Table 2. There was no difference among dietary treatments for dry matter, OM, or CP intake (p > 0.05). Animals offered SH had a higher NDF and lower starch intake compared to animals offered RB and MM (p < 0.001) whereas animals offered MM had a higher starch intake compared to animals offered RB (p < 0.05).
Total tract apparent digestibility of DM, OM, and CP did not differ (p > 0.05) between dietary treatments; however, total tract digestibility of CP tended to be higher for RB compared to those offered MM (p < 0.10). Neutral detergent fibre total tract digestibility was higher for animals offered SH (p < 0.001) with no difference between animals offered RB and MM (p > 0.05). Starch total tract digestibility was higher for animals offered RB compared to animals offered MM (p < 0.05). The in sacco ruminal digestion kinetics and effective degradability of carbohydrates are presented in Table 3. Fraction a (rapidly degradable component) was different for all three carbohydrate sources (p < 0.001), 10% higher for rolled barley compared to maize meal, and 73% lower for soya hulls compared to rolled barley. The slowly degradable component b was 77% and 65% higher for soya hulls compared to rolled barley and maize meal, respectively (p < 0.001), while there was no difference between rolled barley and maize meal (p > 0.05). The factional rate of degradation per h of fraction c was higher for rolled barley (p < 0.01) compared to maize meal and soya hulls. Effective degradability was lower for soya hulls compared to rolled barley and maize meal (p < 0.001) which did not differ (p > 0.05). The effect of carbohydrate type on N balance and blood metabolites is presented in Table 4. Nitrogen intake (g d −1 ) was not affected by dietary treatment (p > 0.05). In addition, dietary treatment had no effect on total N excretion (g d −1 ), the amount of N retained (g d −1 ), and the amount of N excreted in the urine (g d −1 ) (p > 0.05). There was a higher percentage of N excreted in the faeces and a lower percentage of N excreted in the urine when animals were offered MM compared to those offered RB (p < 0.05).
Blood plasma urea concentrations were higher for animals offered RB (p < 0.01), while no differences were observed for plasma creatinine and blood glucose levels between treatments (p > 0.05).  Table 5 shows the effect of carbohydrate source on rumen fermentation parameters. Animals offered SH had a higher ruminal pH than animals offered RB and MM (p < 0.001). Postprandial evolution of ruminal pH did not differ with dietary treatment (p > 0.05; Figure 1). Independent of dietary treatment, ruminal pH decreased, reaching nadir 1 h post-feeding, then gradually increasing to 6 h post-feeding (p < 0.001). The effect of carbohydrate type on N balance and blood metabolites is presented in Table 4. Nitrogen intake (g d −1 ) was not affected by dietary treatment (p > 0.05). In addition, dietary treatment had no effect on total N excretion (g d −1 ), the amount of N retained (g d −1 ), and the amount of N excreted in the urine (g d −1 ) (p > 0.05). There was a higher percentage of N excreted in the faeces and a lower percentage of N excreted in the urine when animals were offered MM compared to those offered RB (p < 0.05).
Blood plasma urea concentrations were higher for animals offered RB (p < 0.01), while no differences were observed for plasma creatinine and blood glucose levels between treatments (p > 0.05). Table 5 shows the effect of carbohydrate source on rumen fermentation parameters. Animals offered SH had a higher ruminal pH than animals offered RB and MM (p < 0.001). Postprandial evolution of ruminal pH did not differ with dietary treatment (p > 0.05; Figure 1). Independent of dietary treatment, ruminal pH decreased, reaching nadir 1 h postfeeding, then gradually increasing to 6 h post-feeding (p < 0.001). There was a treatment × time interaction for rumen NH3 concentrations (p < 0.01) (Figure 2). At 1 h post-feeding, animals offered the MM had higher rumen NH3 concen-  a carbohydrate source that is rapidly degraded within within the rumen and in turn reduce N excretion was study from the in sacco degradability of the three feed nce in ruminal DM degradation of each carbohydrate enerally represents a large proportion of the feed DM, n the ED of DM and ED of starch [35]. The high values ly degradable component) with rolled barley and maize ch was immediately washed out upon immersion of the the high solubility rate obtained with these ingredients e to mechanical particle loss [36] or the smaller particle d to that of soya hulls [37][38][39]. The animals used herein account for the underestimation in the digestibility of nover rates, the rumen outflow rate was fixed at 0.03 h −1 vered in the urine was similar across all treatments at covered in the faeces tended to be higher for animals ffered RB (32% vs. 28%, respectively). 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The animals used herein and to account for the underestimation in the digestibility of men turnover rates, the rumen outflow rate was fixed at 0.03 h −1 , N recovered in the urine was similar across all treatments at reas N recovered in the faeces tended to be higher for animals those offered RB (32% vs. 28%, respectively). The partitioning and faeces is largely dependent on diet, with up to 75% of N igh protein, high concentrates diets are fed [41,42]; but can be N in urine when diets are formulated to NRC recommended CP larly, Colmenero and Broderick, [44] observed that dairy cows P and RDP had higher ruminal NH3 concentrations, resulting in d in the urine. is an environmental concern as it is a major contributor to NH3 the urine is rapidly hydrolysed to NH3 due to the prevalence of Ammonia is the principle source of urea that is produced in the excess or an insufficient energy supply to rumen microbes, meliver and excreted in the urine [44]. It was hypothesised in this d barley, which has a more rapid rate of ruminal fermentation hydrate source that is rapidly degraded within the rumen and in turn reduce N excretion was om the in sacco degradability of the three feed uminal DM degradation of each carbohydrate represents a large proportion of the feed DM, of DM and ED of starch [35]. The high values dable component) with rolled barley and maize mmediately washed out upon immersion of the solubility rate obtained with these ingredients hanical particle loss [36] or the smaller particle t of soya hulls [37][38][39]. The animals used herein for the underestimation in the digestibility of tes, the rumen outflow rate was fixed at 0.03 h −1 the urine was similar across all treatments at in the faeces tended to be higher for animals B (32% vs. 28%, respectively). The partitioning rgely dependent on diet, with up to 75% of N h concentrates diets are fed [41,42]; but can be diets are formulated to NRC recommended CP and Broderick, [44] observed that dairy cows igher ruminal NH3 concentrations, resulting in ntal concern as it is a major contributor to NH3 dly hydrolysed to NH3 due to the prevalence of principle source of urea that is produced in the fficient energy supply to rumen microbes, med in the urine [44]. It was hypothesised in this as a more rapid rate of ruminal fermentation 9.94 9.68 ntrate carbohydrate source on rumen fermentation parameters in beef d diets. offering a carbohydrate source that is rapidly degraded within more N within the rumen and in turn reduce N excretion was in this study from the in sacco degradability of the three feed e difference in ruminal DM degradation of each carbohydrate starch generally represents a large proportion of the feed DM, n between the ED of DM and ED of starch [35]. The high values he rapidly degradable component) with rolled barley and maize f the starch was immediately washed out upon immersion of the owever, the high solubility rate obtained with these ingredients ated due to mechanical particle loss [36] or the smaller particle compared to that of soya hulls [37][38][39]. The animals used herein and to account for the underestimation in the digestibility of men turnover rates, the rumen outflow rate was fixed at 0.03 h −1 , N recovered in the urine was similar across all treatments at eas N recovered in the faeces tended to be higher for animals those offered RB (32% vs. 28%, respectively). The partitioning and faeces is largely dependent on diet, with up to 75% of N igh protein, high concentrates diets are fed [41,42]; but can be N in urine when diets are formulated to NRC recommended CP larly, Colmenero and Broderick, [44] observed that dairy cows P and RDP had higher ruminal NH3 concentrations, resulting in d in the urine. is an environmental concern as it is a major contributor to NH3 the urine is rapidly hydrolysed to NH3 due to the prevalence of Ammonia is the principle source of urea that is produced in the xcess or an insufficient energy supply to rumen microbes, meiver and excreted in the urine [44]. It was hypothesised in this barley, which has a more rapid rate of ruminal fermentation cript letter differ (p < 0.05). ⸸ Tendency towards sigplemented with either RB rolled barley, MM maize c acid to propionic acid (acetic:propionic). 3 TVFA = ydrate source that is rapidly degraded within the rumen and in turn reduce N excretion was m the in sacco degradability of the three feed minal DM degradation of each carbohydrate represents a large proportion of the feed DM, of DM and ED of starch [35]. The high values able component) with rolled barley and maize mediately washed out upon immersion of the solubility rate obtained with these ingredients hanical particle loss [36] or the smaller particle of soya hulls [37][38][39]. The animals used herein for the underestimation in the digestibility of tes, the rumen outflow rate was fixed at 0.03 h −1 the urine was similar across all treatments at in the faeces tended to be higher for animals B (32% vs. 28%, respectively). The partitioning gely dependent on diet, with up to 75% of N concentrates diets are fed [41,42]; but can be diets are formulated to NRC recommended CP and Broderick, [44] observed that dairy cows igher ruminal NH3 concentrations, resulting in ntal concern as it is a major contributor to NH3 dly hydrolysed to NH3 due to the prevalence of rinciple source of urea that is produced in the fficient energy supply to rumen microbes, mein the urine [44]. It was hypothesised in this as a more rapid rate of ruminal fermentation 8. 27 7.59 ntrate carbohydrate source on rumen fermentation parameters in beef d diets. offering a carbohydrate source that is rapidly degraded within more N within the rumen and in turn reduce N excretion was in this study from the in sacco degradability of the three feed e difference in ruminal DM degradation of each carbohydrate starch generally represents a large proportion of the feed DM, n between the ED of DM and ED of starch [35]. The high values he rapidly degradable component) with rolled barley and maize f the starch was immediately washed out upon immersion of the owever, the high solubility rate obtained with these ingredients ated due to mechanical particle loss [36] or the smaller particle compared to that of soya hulls [37][38][39]. The animals used herein and to account for the underestimation in the digestibility of men turnover rates, the rumen outflow rate was fixed at 0.03 h −1 , N recovered in the urine was similar across all treatments at eas N recovered in the faeces tended to be higher for animals those offered RB (32% vs. 28%, respectively). The partitioning and faeces is largely dependent on diet, with up to 75% of N igh protein, high concentrates diets are fed [41,42]; but can be N in urine when diets are formulated to NRC recommended CP larly, Colmenero and Broderick, [44] observed that dairy cows P and RDP had higher ruminal NH3 concentrations, resulting in d in the urine. is an environmental concern as it is a major contributor to NH3 the urine is rapidly hydrolysed to NH3 due to the prevalence of Ammonia is the principle source of urea that is produced in the xcess or an insufficient energy supply to rumen microbes, meiver and excreted in the urine [44]. It was hypothesised in this barley, which has a more rapid rate of ruminal fermentation  Within a row, means with a different superscript letter differ (p < 0.05). ⸸ Tendency towards significant (p < 0.10). 1 Grass silage-based diets supplemented with either RB rolled barley, MM maize meal, or SH soya hulls. 2 Ac: Pr = ratio of acetic acid to propionic acid (acetic:propionic). 3 TVFA = total volatile fatty acids.

Discussion
The hypothesis that offering a carbohydrate source that is rapidly degraded within the rumen would capture more N within the rumen and in turn reduce N excretion was rejected.

In Sacco Degradability
The results obtained in this study from the in sacco degradability of the three feed ingredients fed reveal the difference in ruminal DM degradation of each carbohydrate source. In cereal grains, starch generally represents a large proportion of the feed DM, with a positive correlation between the ED of DM and ED of starch [35]. The high values obtained for fraction 'a' (the rapidly degradable component) with rolled barley and maize meal indicate that most of the starch was immediately washed out upon immersion of the bags within the rumen. However, the high solubility rate obtained with these ingredients may have been over estimated due to mechanical particle loss [36] or the smaller particle size of barley and maize compared to that of soya hulls [37][38][39]. The animals used herein were fed at maintenance and to account for the underestimation in the digestibility of nutrients due to higher rumen turnover rates, the rumen outflow rate was fixed at 0.03 h −1 [40].

N-Balance
In the current study, N recovered in the urine was similar across all treatments at 55% of ingested N, whereas N recovered in the faeces tended to be higher for animals offered MM compared to those offered RB (32% vs. 28%, respectively). The partitioning of N excreted into urine and faeces is largely dependent on diet, with up to 75% of N excreted in urine when high protein, high concentrates diets are fed [41,42]; but can be reduced to 52% excreted N in urine when diets are formulated to NRC recommended CP concentrations [43]. Similarly, Colmenero and Broderick, [44] observed that dairy cows fed increasing levels of CP and RDP had higher ruminal NH3 concentrations, resulting in higher levels of N excreted in the urine.
Urinary N excretion is an environmental concern as it is a major contributor to NH3 emissions because urea in the urine is rapidly hydrolysed to NH3 due to the prevalence of urease in the faeces [45]. Ammonia is the principle source of urea that is produced in the rumen from RDP fed to excess or an insufficient energy supply to rumen microbes, metabolised to urea in the liver and excreted in the urine [44]. It was hypothesised in this study that offering rolled barley, which has a more rapid rate of ruminal fermentation Tendency towards significant (p < 0.10). 1 Grass silage-based diets supplemented with either RB rolled barley, MM maize meal, or SH soya hulls. 2 Ac: Pr = ratio of acetic acid to propionic acid (acetic:propionic). 3 TVFA = total volatile fatty acids.
There was a treatment × time interaction for rumen NH 3 concentrations (p < 0.01) (Figure 2). At 1 h post-feeding, animals offered the MM had higher rumen NH 3 concentrations than those offered the RB (p < 0.05), but this response was reversed at 4 and 6 h post-feeding (p < 0.01), while at 6 h post-feeding, NH 3 concentrations for the animals offered RB were higher than those offered the SH (p < 0.05). There were no differences observed between dietary treatments for ruminal NH 3 concentrations (p > 0.05). The animals offered RB had a higher concentration of ruminal valeric acid than those offered SH and MM (p < 0.05). No differences were observed between dietary treatments for ruminal; acetic acid, propionic acid, butyric acid, branched chain fatty acids (isovaleric acid and isobutyric acid) and total rumen VFA concentrations (p > 0.05). However, ruminal acetic acid concentrations tended to be higher for animals offered SH compared to MM (p < 0.10), and animals offered RB tended to have higher ruminal propionic acid and butyric acid concentrations compared to animals offered SH (p < 0.10). Animals offered RB had lower (p < 0.05) Ac:Pr compared to animals offered SH and MM, which did not differ (p > 0.05) among each other. Concentrations of ruminal butyric acid, valeric acid, and isovaleric acid concentrations were highest 2 h after feeding (p < 0.001).

Discussion
The hypothesis that offering a carbohydrate source that is rapidly degraded within the rumen would capture more N within the rumen and in turn reduce N excretion was rejected.

In Sacco Degradability
The results obtained in this study from the in sacco degradability of the three feed ingredients fed reveal the difference in ruminal DM degradation of each carbohydrate source. In cereal grains, starch generally represents a large proportion of the feed DM, with a positive correlation between the ED of DM and ED of starch [35]. The high values obtained for fraction 'a' (the rapidly degradable component) with rolled barley and maize meal indicate that most of the starch was immediately washed out upon immersion of the bags within the rumen. However, the high solubility rate obtained with these ingredients may have been over estimated due to mechanical particle loss [36] or the smaller particle size of barley and maize compared to that of soya hulls [37][38][39]. The animals used herein were fed at maintenance and to account for the underestimation in the digestibility of nutrients due to higher rumen turnover rates, the rumen outflow rate was fixed at 0.03 h −1 [40].

N-Balance
In the current study, N recovered in the urine was similar across all treatments at 55% of ingested N, whereas N recovered in the faeces tended to be higher for animals offered MM compared to those offered RB (32% vs. 28%, respectively). The partitioning of N excreted into urine and faeces is largely dependent on diet, with up to 75% of N excreted in urine when high protein, high concentrates diets are fed [41,42]; but can be reduced to 52% excreted N in urine when diets are formulated to NRC recommended CP concentrations [43]. Similarly, Colmenero and Broderick, [44] observed that dairy cows fed increasing levels of CP and RDP had higher ruminal NH 3 concentrations , resulting in higher levels of N excreted in the urine.
Urinary N excretion is an environmental concern as it is a major contributor to NH 3 emissions because urea in the urine is rapidly hydrolysed to NH 3 due to the prevalence of urease in the faeces [45]. Ammonia is the principle source of urea that is produced in the rumen from RDP fed to excess or an insufficient energy supply to rumen microbes, metabolised to urea in the liver and excreted in the urine [44]. It was hypothesised in this study that offering rolled barley, which has a more rapid rate of ruminal fermentation than maize meal and soya hulls would capture more NH 3 within the rumen and lead to a reduction in urinary N excretion. However, urine excretion was unaffected by carbohydrate source and was the major route of N excretion across all dietary treatments (79 g d −1 ). Ferreira et al. [46] observed that replacing maize corn with increasing levels of SH in the diets of lambs increased urinary excretion. This increase in urinary excretion can be explained by the increase in DMI intake as the level of SH in the diet increased, while simultaneously increasing the intake of CP in the diet. Similarly, Yan et al. [47] established that the correlation between N intake and DMI is positive. Therefore, in this study, feed intake was restricted to maintenance, to ensure that DMI had no influence on N intake due to difference in energy density between the three feed ingredients [46], in addition to diets formulated to be isonitrogenous (142.6 g d −1 ). The intake level of carbohydrates in the diet can impact the level of N excreted in the urine, as the rate and extent of carbohydrate fermentation within the rumen determines the utilisation of ruminal NH 3 for microbial synthesis [48] and the type of protein therein [49].
Offering maize meal, which is more resistant to rumen degradation compared to rolled barley [35], increases the percentage of total N excreted in the faeces by 39.30 vs. 32.80 %, respectively. Surber and Bowman [50] reported similar findings with beef cattle offered maize meal, where degradation of maize starch within the rumen was lower than those offered rolled barley leading to higher levels of N excreted in the faeces (35 vs. 30 g d −1 ). The site and the extent of carbohydrate fermentation can influence the level of faecal N excretion. Faecal N is primarily of microbial origin with lesser amounts of undegraded feed protein and endogenous secretions [51]. Despite no differences in urinary N excretion observed between treatments in this study (79 g d −1 ), the animals offered MM excreted a higher amount of N in the faeces compared to animals offered RB. Despite the higher level of starch intake with the animals offered MM, the animals offered RB had a higher apparent total tract digestibility of starch in addition to a tendency for a higher apparent total tract digestibility of CP, which would suggest undigested protein in the starch/protein matrix with animals offered MM [52]. Maize starch is more resistant to rumen degradation than other cereal grains, as the starch granules in maize are embedded in the protein matrix, prolamins, which are more resistant to degradation at higher pH [53].

Rumen pH
Rumen pH is a critical factor in the normal and stable function of the rumen because of its profound effect on microbial populations and fermentation products, and on physiological functions of the rumen, with typical ruminal pH in grain fed beef cattle ranging from 5.5 to 6.2 [54]. As the forage to concentrate ratio of the diet is decreased with high dietary levels of rapidly fermentable carbohydrates such as starch with low levels of effective fibre the probability of acidosis increases. Low ruminal pH may have been anticipated in the current study with the high concentrate to forage ratio offered. However, as a result of animals being fed to maintenance, and for additional rumen fill, all diets were supplemented with 1 kg DM of barley straw. The provision of straw in the diet enhances the level of fibre and physically effective fibre in the rumen, promoting rumination and saliva secretion, helping to buffer the acids from the fermentation of the feed. Additionally, higher pH values obtained in this current study may be associated with the decreased volume of rumen digesta (low DMI) and increased dilution rate of rumen liquid or because of the increased extent of chewing [55].

Rumen NH 3 Concentration
There was a time by treatment interaction in ruminal NH 3 concentrations, where the initial increase in ruminal NH 3 concentration with the animals offered MM is likely as a response to the lower availability of carbohydrate in the MM compared to the other dietary treatments [56]. Additionally, the lower levels of NH 3 associated with RB and SH in the initial 3 h post-feeding suggest that more energy was available to allow for better capture of NH 3 by the rumen microbes [57]. Across all dietary treatments, the highest ruminal NH 3 concentration was detected 2 h after feeding as a response to the rapid degradation of all sources of dietary protein similar to findings of Grigsby et al. [58]. There was no difference in mean ruminal NH 3 concentration between treatments. However, the overall mean ruminal NH 3 concentration was 2.48 mmol L −1 , which was lower than those reported in [59] but similar to [60]. Kang-Meznarich and Broderick, [61] reported 1.94 to 5 mmol L −1 as the optimum level of ruminal NH 3 concentration adequate for microbial synthesis and fibre digestion, suggesting the levels of ruminal NH 3 produced in this study were adequate.

VFA Concentrations
The concentrations of VFA within the rumen are the net result of substrate consumed by the animal and their absorption rate [62], with the rate of absorption increasing as the ruminal pH decreases [63]. The total average rumen VFA concentrations observed in this study (90.43 mmol L −1 ) were lower compared to similar studies (148 mmol L −1 ) involving beef cattle offered carbohydrates differing in rumen degradation rates [60]. However, these diets were offered ad libitum, whereas, in this current study DMI was restricted to maintenance. As the mean ruminal pH in this current study never dropped below 6.0, the lower rumen VFA concentrations were more likely as a result of lower rumen VFA production due to lower DMI consumed [55] rather than greater VFA absorption through the rumen epithelium [64]. While not significant, the higher concentrations of acetic acid observed with the animals offered SH is a result of the higher proportion of NDF within the diet and higher total tract digestibility of NDF in animals offered SH [55] and as a consequence resulted in a higher acetic acid: propionic acid ratio compared to the animals offered RB. The starch contained in barley is more fermentable within the rumen compared with maize starch [35]. However, the similar VFA concentrations observed in this study may be as a result of the different levels of processing associated with each ingredient [65] compared to the dry rolling of barley, maize grain was finely ground which produced large numbers of fine particles, increasing the surface area of the endosperm for utilisation by the rumen micro-organisms [66]. Similar observations were noted when substituting maize meal with rolled barley in beef cattle [67] and with dairy cows [68,69] and substituting maize meal with soya hulls [70].

Conclusions
Offering a carbohydrate source that is rapidly degraded within the rumen such as rolled barley did not alter ruminal NH 3 concentrations, and thus reduce N excretion in beef heifers offered GS-based diets fed to maintenance. Similar ruminal NH 3 concentrations were observed across all treatments, highlighting that protein degradation exceeded carbohydrate fermentation 2 h post-feeding. In conclusion, supplementing grass silagebased diets with concentrate carbohydrate sources with different fermentation profiles had no effect on N metabolism of beef heifers fed to maintenance. However, this approach is unlikely in practice as the animals were fed to maintenance on a diet that contained 60% concentrates.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.