Introduction
Dietary protein is the most important nutrient for increasing the milk nitrogen (N) yield in dairy cows (Huhtanen and Hristov, Reference Huhtanen and Hristov2009). However, over-feeding of protein increases the cost of production and excretion of N to the environment (NRC, 2001; Lee et al., Reference Lee, Hristov, Cassidy, Heyler, Lapierre, Varga, de Veth, Patton and Parys2012a). For instance, it has been reported that increasing dietary crude protein (CP) content from 148 to 167 (Lee et al., Reference Lee, Hristov, Dell, Feyereisen, Kaye and Beegle2012b) and 162 to 201 g/kg dry matter (DM) (Borucki Castro et al., Reference Borucki Castro, Phillip, Lapierre, Jardon and Berthiaume2008) increased the excretion of urinary N to almost two-fold. Former studies showed that a dietary CP concentration greater than 167 g/kg DM had no effect on productive performances of lactating dairy cows (Cunningham et al., Reference Cunningham, Cecava, Johnson and Ludden1996; Broderick, Reference Broderick2003; Socha et al., Reference Socha, Schwab, Putnam, Whitehouse, Garthwaite and Ducharme2008). However, it has recently been reported that milk production peaked at CP concentration of 173 (Law et al., Reference Law, Young, Patterson, Kilpatrick, Wylie and Mayne2009) or 176 g/kg DM (Katongole and Yan, Reference Katongole and Yan2020).
Ruminal fermentation and microbial protein synthesis (MPS) occur in an integrated way in the rumen. Consequently, some dietary factors such as amount and types of carbohydrates have substantial impacts on protein requirement of ruminants, as carbohydrate fermentation rate can alter the efficiency of rumen N utilization (Broderick, Reference Broderick2003 ). Rapidly v. moderately fermentable grains (e.g. barley v. maize) can alter rumen degradation patterns and the requirement of dietary chemical composition for an optimal rumen fermentation of dairy cows (Beauchemin and Rode, Reference Beauchemin and Rode1997). Thus, the concentration of CP in the diet could differ due to differences in starch degradability or the rate of availability of energy for the rumen microbiome, a context that has not been considered well for investigating the optimal dietary CP in dairy cows. As a result, the key should be applying efficient nutritional strategies to induce a favourable synchronization of energy and protein for an optimal MPS and rumen characteristics and subsequently minimizing N excretion in urine and maximizing the animal productivity (Kaswari et al., Reference Kaswari, Lebzien, Flachowsky and terMeulen2007).
Although the theory supports the effect of synchronized energy and protein for optimal MPS and rumen characteristics, practical results have been conflicting (Kolver et al., Reference Kolver, Muller, Varga and Cassidy1998; Shabi et al., Reference Shabi, Arieli, Bruckental, Aharoni, Zamwel, Bor and Tagari1998; Kaswari et al., Reference Kaswari, Lebzien, Flachowsky and terMeulen2007). Differences in experimental methods and animals used have made it difficult to determine the effect of energy and protein synchronization from several other factors (Cabrita et al., Reference Cabrita, Dewhurst, Abreu and Fonseca2006). For instance, changes in feed ingredients or dry matter intake (DMI) alter the supplied amount of organic matter (OM), N and their fermentation rate in the rumen, which affect the efficiency of synchronized energy and N in the rumen (Yang et al., Reference Yang, Beauchemin and Rode2001). Moreover, these studies used different ruminant species in various physiological stages, where the conversion efficiency of dietary N into animal product N is greatly diverse. Thus, it is difficult to generalize the effect of synchronization of energy and N on animal performance across all ruminants with different physiological needs. Primiparous cows have not been widely evaluated in terms of energy-N synchronization. It has been recommended to contain greater concentrations of CP in the diets for primiparous cows than for their multiparous counterparts, as they are in growing stage, their mammary gland is developing and they have lower DMI compared with multiparous cows (NRC, 2001). Consequently, they might benefit more from the amount and type of dietary protein and energy sources, when nutrients partition into the two main processes (i.e. growth and milk synthesis). Therefore, more studies are required to identify the effects of protein and energy availability interaction on ruminal fermentation characteristics and animal performance in a particular physiological condition.
Thus, it was hypothesized that feeding ground barley grain instead of combined ground barley and maize grains with high concentration of CP in diets may lead to a decrease in urinary N excretion, an increase in N utilization efficiency and an improvement of productive performance as a result of improved MPS. This study was, therefore, designed to evaluate the interaction of two dietary protein concentrations (high v. low) and two grain sources with different degradability (barley or barley + maize) on rumen fermentation characteristics, N efficiency and milk production in primiparous Holstein cows.
Materials and methods
Cows, treatments and management
This experiment was conducted at the Dairy Facilities of the University of Zanjan (Zanjan, Iran). The Animal Care Committee of University of Zanjan (ID 1353) approved all procedures involving animal care and management.
Eight primiparous Holstein cows (560 ± 65 kg of body weight; 50 ± 15 days in milk; DIM) were used in a replicated 4 × 4 Latin square design with four 21-day periods. Cows were allocated within the square based on DIM (≤50 and >50 days). Each experimental period had 14 days of adaptation followed by 7 days of sampling and data collection. Cows were housed in individual free boxes (4 × 4 m) bedded with wheat straw and were allowed to 1-h daily exercise. Cows were fed with one of the four dietary treatments (Table 1) consisted of: (1) high-protein concentration (195 g/kg of DM) with barley as the only grain source, HP-B; (2) high-protein concentration (193 g/kg of DM) with an equal mix of barley and maize grains, HP-BM; (3) low-protein concentration (159 g/kg of DM) with barley as the only grain source, LP-B and (4) low-protein concentration (156 g/kg of DM) with an equal mix of barley and maize grains, LP-BM. Maize (Zea mays L. ssp.) and barley (Hordeum spp.) grains contained 700 and 580 g/kg of starch; 85 and 110 g/kg of CP; 110 and 200 g/kg of neutral detergent fibre (NDF); 32 and 70 g/kg of acid detergent fibre; 40 and 22 g/kg of ether extract, respectively (all in DM basis). Whole maize and barley grain densities were 540 and 500 g/l, respectively. Both grains were ground by using a commercial hammer mill (sieve size of 2 mm) most commonly used in dairy farms in Iran. In each period, diets were provided as total mixed rations (TMRs) and were formulated using the Cornell-Penn-Miner Dairy model (CPM-Dairy 3.0). Cows were fed ad-libitum twice daily at 09.00 and 16.00 h, and fresh water was available at all times.
Ingredients and chemical composition of the experimental diets
HP, high protein; LP, low protein; DM, dry matter; NEL, net energy for lactation; CP, crude protein; RDP, rumen degradable protein; RUP, rumen undegradable protein; NDF, neutral detergent fibre; peNDF, physically effective NDF; NFC, non-fibre carbohydrate.
a Supplemental minerals and vitamins per kg contained 196 g calcium, 96 g phosphorus, 19 g magnesium, 46 g sodium, 3000 mg iron, 300 mg of copper, 2000 mg manganese, 100 mg cobalt, 3000 mg zinc, 100 mg iodine, 1 mg selenium, 400 mg antioxidants, 500 000 IU vitamin A, 100 000 IU vitamin D3 and 100 mg vitamin E.
b Calculated from CPM Dairy (V. 3.0).
Sample collection and chemical analysis
Samples of TMRs and orts were collected during days 15–21 of each period and stored at −20°C until analysis. Samples were composited by period, dried at 55°C for 48 h in an oven and ground through a 1-mm screen using a Wiley mill (Wiley Company pulverizer Ogaw Seiki, Ltd., Tokyo, Japan). OM concentration was determined after subtraction of ash content that was determined after burning the samples at 600°C for 2 h. CP concentration was analysed using the Kjeldahl method (Tecator, Hoganas, Sweden). NDF content was determined with sodium sulphite and alpha-amylase according to Van Soest et al. (Reference Van Soest, Robertson and Lewis1991). The acid insoluble ash (AIA) concentration of the diets and faecal samples was determined according to Van Keulen and Young (Reference Van Keulen and Young1977). The concentration of AIA was used as an internal marker to calculate total tract apparent digestibility of dietary components. In this regard, faecal samples were collected at 4-h intervals for 24 h on day 15. On the last day of each period at 11.30 a.m., rumen fluid sample was collected using a stomach tube. The initial 100 ml of aspirated ruminal fluid was discarded to minimize saliva contamination. The pH of the rumen fluid was measured using a mobile pH meter (HI8314, Hanna Instruments, Italy). An 8-ml aliquot of strained ruminal fluid sample was mixed with 2 ml of 25% (wt/vol) metaphosphoric acid solution containing 2 g/l of 2-ethyl butyric acid (an internal standard, Sigma). The content was allowed to settle for 15 min. Rumen fluid samples were centrifuged at 3000 × g for 20 min at 4°C (Mikro 220R, Hettich, Germany) and the supernatant was kept at −20°C for subsequent volatile fatty acid (VFA) analysis. For VFA analysis, a 5-ml supernatant was transferred into a 15-ml test tube. The tube was centrifuged at 10 000 × g and 4°C for 15 min. A 1-μl aliquot of the upper phase was injected into a Varian 3400 gas chromatograph (Varian Inc., Walnut Creek, CA, USA) equipped with an injector at 170°C, a flame-ionization detector at 175°C and a packed column (2 m × 2 mm i.d. glass column containing 1–1965 10% SP-1200/1% H3PO4 on 80/100 Chromosorb W). Another 8-ml aliquot of rumen fluid was preserved with 2 ml of 1% (wt/vol) sulphuric acid and stored at −20°C for ammonia-N analyses. . Urine samples were collected before the morning milking in 50-ml tubes on day 21. Fifteen millilitres of samples was acidified immediately by diluting with 60 ml of 0.072 N H2SO4. The representative sample was stored at −20°C until analysis for total N by the Kjeldahl method. Daily milk yield was recorded for all cows for 84 days in the four experimental periods. All cows were milked three times daily at 06.00, 14.00 and 20.00 h. To determine milk composition, milk samples were collected for two consecutive days per week (on days 18 and 19 of each period) and then analysed for milk fat, protein and urea nitrogen concentrations (MUN; Milcoscan TM S 50, Denmark).
Statistical analysis
Data were analysed using the Proc MIXED in SAS. The REML method was set to estimate least squares means, and Kenward–Roger method was used to calculate denominator's degrees of freedom. The model for data analysing was as follows:
where Yijkl is the variable of interest, μ is the overall mean, Tj is the fixed effect of period, Ck is the random effect of cow, Gl is the fixed effect of the grain source, Pm is the fixed effect of protein level, (Gk × Pl) is the interaction of grain source and protein level and eijkl is the residual error.
The other possible interactions except for the effect of grain source × protein level as well as square and its interaction with period, grain source and protein level were tested earlier and they were excluded from final models due to non-significance. Normality of distribution and homogeneity of variance for residuals were tested and ensured using Proc Univariate. Post hoc comparisons with evaluate the differences among means was tested by Tukey test. The P values <0.05 were declared as significant.
Results
Diet characteristics
The nutrient composition of the ingredients and their inclusion rates in the diet are shown in Table 1. Across diets (on DM basis), the NDF content ranged from 319 to 335, and NFC from 367 to 424 g/kg. The net energy for lactation contents in HP-B, LP-B, HP-BM and LP-BM were 7.21, 6.98, 7.35 and 7.23 MJ/kg of DM, respectively. The NFC and starch contents of high-protein diets (g/kg of DM) were lower than those of low-protein diets. Therefore, low-protein diets provided more carbohydrate compared with high-protein diets.
Dry matter intake, body condition score and body weight, and nutrient digestibility
No significant differences in dry matter intake, body weight and body condition score were observed among dietary treatments (Table 3). Coefficients of total tract digestibility of dietary components are presented in Table 2. The interaction of grain source and protein level was significant for apparent digestibility of all dietary components (DM, OM, NDF and CP). Our results showed that LP-BM had the greatest coefficient of DM, OM and NDF digestibility compared with other treatments (P < 0.05). CP digestibility was greater for high-protein diets than that for low-protein diets (P < 0.001) and the greatest CP digestibility was observed for cows fed with HP-B diet.
Coefficients of total tract apparent digestibility of dietary components in dairy cows fed with four diets with different protein concentrations and grain sources
HP, high protein; LP, low protein; P × G, protein and grain interaction; DM, dry matter; OM, organic matter; NDF, neutral detergent fibre; CP, crude protein.
Milk yield, milk composition and blood urea nitrogen (BUN)
Regardless of the level of CP, an equal mix of barley and maize grains resulted in higher milk fat yield (kg/day). The effect of the grain source was observed for ECM, as HP-BM cows had the greatest ECM compared with other dietary treatments (P < 0.01; Table 3). Concentration of MUN was influenced by level of protein, as cows fed with high-protein diets had greater MUN compared with cows fed with low-protein rations. Other measurements of milk yield and composition were not affected by protein concentration, grain type or their interaction (Table 3). Similar to MUN, concentration of BUN in cows fed with high-protein diets was greater (P < 0.01) than in cows fed with low-protein rations.
DMI, body condition score, blood urinary nitrogen, milk yield and composition of dairy cows fed with four diets with different protein concentrations and grain sources
HP, high protein; LP, low protein; P × G, protein and grain interaction.
a Energy-corrected milk; calculated as (milk production × (0.383 × % fat + 0.242 × % protein + 0.7832)/3.1138) (Østergaard et al., Reference Østergaard, Sorensen and Houe2003).
Ruminal fermentation characteristics and nitrogen efficiency
The concentrations of total VFA (mmol/l) was greater for the diets containing mix of maize and barley than that for the diets containing only barley. However, the proportions of different VFAs (mol/100 mol) as well as acetate : propionate ratio were not different among the dietary treatments (Table 4). Ruminal pH (P = 0.02) and NH3-N concentrations (P = 0.004) were affected by protein concentration and grain source interaction. Ruminal pH had lowest in the LP-B group, followed by HP-B and then in LP-BM and HP-BM groups. Ruminal NH3-N concentrations were greatest in the HP-BM group, followed by the HP-B group and lowest in the LP-BM group, and it was greater in high-protein diets than in low-protein diets. Cows fed with high-protein diets had lower (P < 0.001) N utilization efficiency and higher urinary N concentration than cows fed with high-protein diets.
Ruminal fermentation characteristics, urinary nitrogen and nitrogen utilization efficiency of dairy cows fed with four diets with different protein levels and grain sources
HP, high protein; LP, low protein; P × G, protein and grain interaction.
a Total volatile fatty acids.
Discussion
Contrary to our assumption that primiparous cows may gain advantages from high-protein diets, feeding extra protein either with barley as the sole grain source or with an equal mix of barley and maize grains did not improve N utilization efficiency (milk protein/CP intake). Our results are consistent with other researchers who did not find advantages of feeding an increased dietary CP from 160 to 172 g/kg DM on N efficiency (milk protein/CP intake) or BCS of primiparous cows in early and mid-lactation (Mäntysaari et al., Reference Mäntysaari, Huhtanen, Nousiainen and Virkki2004). However, these authors reported an increase in milk and milk protein yield by increasing dietary CP content (Mäntysaari et al., Reference Mäntysaari, Huhtanen, Nousiainen and Virkki2004), which was not in agreement with our result. Although studies on primiparous cows are limited, some studies showed positive effects of feeding diets with CP >160 g/kg DM on milk production of multiparous Holstein lactating cows (Broderick, Reference Broderick2003; Reynal and Broderick, Reference Reynal and Broderick2003). The higher milk yield by additional CP in these studies mostly has been related to an increased DMI due to greater rumen degradable protein (RDP) concentration. Therefore, diets with low levels of RDP may impair nutrient digestibility and thus do not support an efficient MPS, resulting in decreased DMI (NRC, 2001; Broderick, Reference Broderick2003; Law et al., Reference Law, Young, Patterson, Kilpatrick, Wylie and Mayne2009). In the current study, although cows fed with low-protein diets tended to receive a lower amount of RDP (1.70 ± 0.07 v. 1.98 ± 0.03 kg/day for low- v. high-protein diets, respectively), DMI or milk yields were not affected by this difference. Similarly, the greatest NDF digestibility observed for LP-BM may indicate that CP or RDP was sufficient in low-protein rations for proper fibre digestion (Lee et al., Reference Lee, Hristov, Cassidy, Heyler, Lapierre, Varga, de Veth, Patton and Parys2012a, Reference Lee, Hristov, Dell, Feyereisen, Kaye and Beegleb). Therefore, low-protein rations in this study were not likely deficient in RDP fraction. Similarly, it has been reported that inadequate rumen undegradable protein (RUP) also can cause a reduced metabolizable protein flowing to the duodenum (NRC, 2001; Broderick, Reference Broderick2003; Law et al., Reference Law, Young, Patterson, Kilpatrick, Wylie and Mayne2009). Therefore, deficient RUP can limit milk yield and milk protein production as well. In the current study, cows that received high-protein rations consumed 290 g/day more RUP (1.19 ± 0.00 v. 0.90 ± 0.03 kg/day for high- and low-protein rations, respectively). However, either milk protein or milk yield was not affected by this difference. One of the limitations of this study was using only eight cows for investigating the animal productive performances, however, similar to our results, milk protein content and yields were not affected by increasing dietary CP content in other studies (Wang et al., Reference Wang, Liu, Yuan, Wu, Zhai and Ye2007; Cyriac et al., Reference Cyriac, Rius, McGilliard, Pearson, Bequette and Hanigan2008; Law et al., Reference Law, Young, Patterson, Kilpatrick, Wylie and Mayne2009). Thus, diets with CP concentration at 160 g/kg DM with RUP of 54 g/kg DM could be optimum to meet dietary CP requirement of cow yielding around 30 kg/day of milk and feeding extra protein may not result in additional benefits for primiparous cows.
Feeding large amounts of highly fermentable carbohydrate may cause a low rumen pH (i.e. <5.8) (Bramley et al., Reference Bramley, Lean, Fulkerson, Stevenson, Rabiee and Costa2008). In this situation, microbial maintenance requirements increase and MPS rate reduces remarkably (Russell and Wilson, Reference Russell and Wilson1996). In the current study, the lowest ruminal pH was observed in cows fed with the LP-B diet. This group of cows received the greatest barley amount compared with other treatments (362 v. 282, 123 and 168 g/kg DM for LP-B, HP-B, HP-BM and LP-BM, respectively). Although a reduction in rumen pH after feeding highly fermentable carbohydrate (e.g. barley) is expected (Obara et al., Reference Obara, Dellow, Nolan, Tsuda, Sasaki and Kawashima1991), it has not been observed in all studies (Moran, Reference Moran1986; Gozho and Mutsvangwa, Reference Gozho and Mutsvangwa2008). Regardless of the grain type, the differences in the amount of starch consumed by cows should be considered as an important factor on ruminal pH changes, which is likely one of reasons showing no difference in the rumen pH by feeding different types of grains (Moran, Reference Moran1986; Gozho and Mutsvangwa, Reference Gozho and Mutsvangwa2008). Cows in the LP-B group had the lowest digestibility of NDF and CP. Given that the fermentation of dietary components is limited by the rumen pH below 6.0 (Mould et al., Reference Mould, Ørskov and Mann1983; Cerrato-Sánchez et al., Reference Cerrato-Sánchez, Calsamiglia and Ferret2007), the reduced digestibility of NDF and CP for LP-B group may be related to low rumen pH this group. Due to the confounding effects of different feed ingredient levels in the diet (e.g. fish meal, cottonseed meal and soybean meal), the reasons of changes of nutrient digestibility among treatments are not clearly explainable. However, the high DM, OM and NDF digestibility observed in LP-BM might, in part, indicate that high levels of CP may impact negative effects on nutrient digestibility (Mould et al., Reference Mould, Ørskov and Mann1983). A proper rumen pH (approximately >6.0 for modern dairy cows) allows most groups of rumen microbes to grow and ferment dietary components more efficiently, especially fibrous feeds by cellulolytic bacteria (Strobel and Russell, Reference Strobel and Russell1986). Similarly, providing a more synchronized rate of protein and energy release supplied by the combination of barley and maize may result in an improved ruminal digestibility of feed fractions (Firkins, Reference Firkins1996).
Many factors determine the efficient utilization of NH3-N by ruminal microbes, but Theurer et al. (Reference Theurer, Huber, Delgado-Elorduy and Wanderley1999) noted that total starch intake should be considered as the main factor. The greater concentrations of NH3-N observed for cows fed with high-protein rations indicate that extra N provided by these diets could not be efficiently used towards MPS. Our results are supported by others, who reported that synchronization between N and carbohydrate release in the rumen improved N utilization by the rumen microorganisms (Chamberlain and Choung, Reference Chamberlain, Choung, Garnsworthy and Cole1995; Seo et al., Reference Seo, Yang, Kim, Upadhaya, Cho and Ha2010). Consequently, the greater concentrations of CP increased the concentrations of urinary N, BUN and MUN in cows fed with the high-protein rations. Accumulated NH3-N is absorbed across the ruminal wall and after conversion to urea causes an increase in BUN concentration. Because urea is a small molecule, it can diffuse freely from the blood stream into tissues such as udder. In agreement with our results, other researchers also reported that urinary N, MUN and BUN concentrations were associated with the dietary CP level (Broderick, Reference Broderick2003; Hynes et al., Reference Hynes, Stergiadis, Gordon and Yan2016).
Conclusion
Considering the low number of cows for a productive performance study, the current study showed that feeding high levels of dietary protein either with the barley diet or with a combination of barley plus maize did not result in an improvement in milk production performance or ruminal fermentation characteristics in early lactating primiparous Holstein cows under our experimental conditions. However, this study showed that diets containing an equal mix of barley and maize instead of diets with barley only may increase ECM and fat yield, which may, in part, be attributed to better ruminal microbial fermentation and health as evident from greater VFA concentration and ruminal pH. However, confounding effects of some dietary ingredients which led to different RDP : RUP ratio in high- or low-protein diets, made it difficult to conclude that synchronization of energy and protein release caused these results.
Acknowledgements
The authors would like to thank Dr Amlan Patra for proofreading of the manuscript. We cordially thank the employees of the Dairy Research Farm of the University of Zanjan for diligent help with animal care.
Financial support
The Ministry of Science, Research and Technology, and University of Zanjan, Iran, are acknowledged for financial support.
Conflict of interest
The authors declare no conflicts of interest.
Ethical standards
Not applicable.
