Effects of Supplementing Rumen-Protected Methionine on Production Performance, Apparent Digestibility, Blood Parameters, Ruminal Metabolites and Economic Effectiveness in Lactating Holstein Dairy Cows


 Background: Because of disadvantages of excessive dietary crude protein (CP), decreasing dietary CP of dairy cows has attracted the worldwide attention. Rumen protected methionine (RPM) supplementation can allow lower CP diets and is beneficial to milk production performance, N efficiency of cows and environment. The aim of this study was to evaluate the effects of reducing dietary CP and supplementing RPM on production, digestibility of nutrients, blood parameters, ruminal metabolites and economic effectiveness in lactating Holstein dairy cows. Results: A total of 96 lactating cows (63 ± 25 d in milk; 34.4 ± 5.74 kg/d of milk production; mean ± SD) were randomly assigned to 1 of 2 treatments: diet containing 17.3% CP without RPM (control group; CON; n = 49); diet containing 16.4% CP with supplementing 15.0 g/d of RPM (treatment group; RPM; n = 47). No effect was observed of reducing dietary CP on milk yield and milk composition. The apparent digestibility of nutrients was similar between treatments. The results related to blood showed that cows in RPM group exhibited lower concentration of blood urea nitrogen than that in CON group (P < 0.001). Moreover, there were no differences between treatments on concentrations of aspartate transaminase, alanine transaminase, alkaline phosphatase, globulin and albumin. In ruminal metabolites, microbial crude protein (MCP) of dairy cows in RPM group was higher compared with CON group (P = 0.006). Ruminal volatile fatty acid (VFA) contents were not changed by treatments except that the concentrations of butyrate and isovalerate of RPM group were higher than that of CON group at 2 h after feeding (P < 0.05). In addition, supplying the diet of 16.4% CP with RPM supplementation to cows could reduce feeding cost by 0.5 $/d per cow and boost net profits. Conclusions: Lower dietary CP with RPM supplementation did not limit milk yield, milk composition and apparent digestibility of nutrients, and could improve nitrogen utilization of dairy cows and synthesis of MCP in rumen, change VFA production at 2 h after feeding, as well as boost the economic benefits of the dairy farms.


Background
To satisfy the protein requirement, cows sometimes are received excessive dietary crude protein (CP) and then excreted in the form of urea, which would be emitted into the atmosphere in the form of NO 2 [1], or be hydrolyzed to NH 3 and volatilized [2], ultimately harmful to the environment. In addition, some researchers noted high dietary CP has a negative impact on N utilization for dairy cows [3]. And there is no improvement in milk production and milk composition when dietary CP is increased up to 18% [2] or 18.8% [4]. Furthermore, 40% of feeding cost can be attributed to dietary protein [5], and excess protein in the diets will lead to high cost and reduce pro t margins. In consideration of above-mentioned factors, decreasing dietary CP has attracted the attention from worldwide researchers. In fact, previous study has shown that ensuring ideal balance for AA, especially methionine (Met) and lysine (Lys) [2], is the key to formulate cow rations rather than for dietary protein [6].
Given that rumen undegradable protein (RUP) is signi cant to meet daily protein needs of cows especially during the lactation stage, an alternative strategy of feeding rumen-protected AA has been taken into consideration. Recently years, there have been some research that explored the effects of rumenprotected methionine (RPM) supplementation on dairy cows. Several studies indicated that RPM supplementation and decreasing dietary CP could increase milk production, milk protein, fat as well as lactose contents [7][8][9][10]. Recently research has shown that an increase in overall dry matter intake (DMI) could be found after parturition when cows fed lower dietary CP and RPM [11]. In addition, supplementing RPM may allow lower CP diets to improve N e ciency and reduce urinary N excretion [8,12] and production of total gas and methane volume [13]. Meanwhile, a previous meta-analysis of dietary protein content noted that not overfeeding dietary CP is the most e cient means to increase e ciency of N utilization [14]. In general, reducing dietary CP levels with RPM supplementation would be bene cial to milk production performance and N e ciency of dairy cows, as well as environment. Other effects of RPM, especially in aspects of digestibility of nutrients, health, rumen function and farmer's pro ts need to be further explored.
Thus, the objective of the present study was to investigate the effects of reducing dietary CP levels and supplementing RPM on production performance, apparent digestibility of nutrients, blood parameters, ruminal metabolites and economic effectiveness in lactating Holstein dairy cows. We hypothesized that reducing dietary CP levels and supplementing RPM would not limit milk yield and composition, and could change rumen fermentation and farmer's pro ts.  On d 0, d 30, d 60 of the experimental period, blood samples were randomly collected from the 15 cows of each group from tail vein of cows using 10 mL blood collection heparin-coated tubes (Vacutainer; aosaite Medical Instrument; Shandong) before morning feeding. Blood samples were immediately centrifuged at 3,500×g at 4℃ for 15 min to obtain plasma samples, which stored at − 20°C for further analysis. where Ad = AIA in the diets (g/kg); Af = AIA in the feces (g/kg); Nd = the concentration of a nutrient in the diet (g/kg); Nf = the concentration of a nutrient in the feces (g/kg).

Materials And
On the last day of the experiment, 15 cows from each group were selected randomly and were collected samples of rumen uid. Samples were collected before morning feeding and 2, 4, 6, 8, 12 h after morning feeding by oral sampler. Rumen uid samples was ltered with four layers of gauze, centrifuged at 4000 r/min for 15min and took 1mL supernatant, which was acidi ed with 4.5mL 0.2mol/L HCL for later analysis of ammonium nitrogen. Meanwhile, 4mL supernatant was added l mL 25% metaphosphate acid and were stored at − 20°C for later analysis of microbial crude protein (MCP), Ammonia N (NH 3 -N) and volatile fatty acid (VFA) including acetate, propionate, butyrate, valerate, isobutyrate and isovalerate (using Shimadzu GC-7A gas chromatograph, Japan).

Statistical Analysis
Dairy cow was used as the experimental unit, and all data were analyzed using SAS (SAS version 9.2, SAS Institute Inc., Cary, NC, USA). The milk yield, milk composition, apparent digestibility of nutrients, blood parameters, and ruminal metabolites were analyzed using PROC MIXED procedure of SAS. The milk yield, milk composition and blood parameters were calculated by averaging the three samples collected in d 0, d 30 and d 60 and then analyzed. The model of ruminal metabolites included the xed effects of treatment, time, and time × treatment interaction, and cow within treatment as a random effect. Degrees of freedom were counted using the Kenward-Roger approximation option of the MIXED procedure. In order to explicate the repeated measures within-subject, the covariance structures were executed for each repeated variable on the basis of best t determined by the Akaike information criterion. A signi cant difference between treatment group and control group was declared at P < 0.05, tendencies were considered when 0.05 ≤ P < 0.10, a highly signi cant difference indicated at P ≤ 0.01.

Milk Yield, and Milk Composition
The results of the milk yield and milk composition are shown in Table 2. Milk yield and milk composition were not limited by the diet of lower protein with RPM supplementation. Meanwhile, SCC and MUN values for RPM diet decreased numerically and that of milk fat increased numerically. There was a trend for lower SNF with decreased CP feeding and RPM supplementation (P = 0.09).  [10,20] and milk protein yield [21] increased. In contrast, a study reported that milk yield or milk composition like fat, lactose and SNF decreased signi cantly with diet of 11% CP, which may due to 11% CP content was not enough to meet the milk production needs of dairy cows [9]. Furthermore, a report noted that compared with the diet of 17.3% CP, SNF yield in milk was lower in that of 16.1% CP[8], which was accorded with another study where reported that reduced dietary CP (from 17.1-15.8%) could decrease milk SNF yield [22]. These studies indicated that lower dietary CP could result in less yield of milk SNF, which may account for the result of the current study.
In addition, no signi cant treatment effect was observed on MUN, while decreased with RPM group numerically in the current study. A previous study found that MUN concentration was lower when feeding the low-protein diet and supplementing rumen-protected essential amino acids [23]. A report elsewhere observed that compared with 18% CP diet, the MUN values decreased with 16.4% and 15.6% CP diets supplementing RPM [2]. However, some researchers noted that there was no signi cant change in MUN value if the dietary CP content changed by 1% [24]. Therefore, the similar content of MUN in our study could be explained by that the difference in dietary CP between the two experimental groups was 0.9% (< 1%). Hence, reduced MUN in RPM group may bene t to conception rates of dairy cows [25]. SCC level, an indicator of animal mammary disease, often increases when an infection of the mammary gland occurs [26,27]. In the current study, SCC values did not change and was numerically lower in RPM group. And in these two groups the SCC values were both within the normal levels (100,000 ~ 314,000 cells/mL) [28], which indicated the health condition of dairy cows may not be affected by treatments.

Blood Parameters
The effects of RPM supplementation on the blood parameters contents are shown in Table 3. The BUN value decreased (P < 0.001) with RPM supplementation. GLB was numerically higher in cows consuming lower protein diet supplemented RPM; however, this difference was not statistically signi cant. Other blood parameters did not exhibit any difference by treatment. The signi cant decline of BUN in our ndings accords with several earlier research. Bahrami-Yekdangi et al. conducted research in 2013, 2016 respectively. They found plasma urea concentrations were lower in cows consuming low-protein diets with RPM supplementation in 2013 [2]. In another study, they also observed that BUN concentrations had a trend of a linear decline (P = 0.06) with reduced dietary CP and rumen degradable protein (RDP) [29]. These results may be explained by the fact that low-protein diets and ameliorating the balance of dietary AA, especially the ratio of Lys to Met, could enhance the protein utilization of dairy cows [23]. Moreover, it's worth to mention that some researchers indicated higher BUN may lead to saturation of transfer process because of the inhibitory effect of increased NH 3 -N contents in the rumen or a limitation of urease activity [30]. Reduced BUN concentrations might stimulate rumen urea transfer and increase urea transport rates [31]. Several research also noted the negative effect of high MUN or BUN value on reproductive performance of cows [32,33]. Therefore, these evidences indicate that reasonable BUN decline has a positive impact on the nitrogen utilization of dairy cows. A previous study supported that RPM supplementation could decrease in ammation of dairy cows, which may attribute to there is an association between RPM and galectin expression and secretion [34]. However, the ndings from the abovementioned study differed from our results in that there was no difference in GLB contents, an indicator of immune system [35], between the two groups. We have no explanation for this result, and further experimentation may be required.

Apparent digestibility of nutrients
The results of the apparent digestibility of nutrients of dairy cows are shown in Table 4. The total apparent digestibility of DM, CP, NDF, ADF, ash, calcium and phosphorus were not affected by treatment diet, despite an overall reduction in lower dietary CP of 16.4%. Furthermore, apparent minerals digestibility, calcium and phosphorus, were numerically higher in cows consuming lower protein diets; however, these differences were not statistically signi cant.

Ruminal fermentation
Effects of experimental diets on ruminal metabolites concentrations are shown in Table 5. The results showed that MCP in the RPM group was higher than that in CON group (P = 0.006). All ruminal metabolites contents were affected by time signi cantly (P < 0.05), but no differences were found between treatments. Treatment × time interaction effects (P < 0.05) were observed for ruminal butyrate and isovalerate concentrations, with cows fed RPM having higher butyrate and isovalerate contents at 2h after feeding, as shown in Fig. 1. It is worth to mention that the mean content of NH 3 -N was numerically higher in treatment diet; however, the effect was not statistically signi cant. A previous study noted that with the development of RPM technology, most of protected Met is able to escape ruminal degradation, while a small proportion of Met is still released into the rumen and may alter the community composition of microbiota in rumen and their metabolism [11]. Several researchers have shown that unprotected methionine promoted ruminal bacteria growth rate and protein synthesis in vitro [41,42]. And the study also found that rumen degradable methionine could increase the abundance of Selenomonas ruminantium [11], which is Gram-negative bacteria that could account for up to 51% of the ruminal total viable bacterial counts [43]. Some earlier scholars noted this strain was a typical ureolytic gastrointestinal tract anaerobe [44][45][46]. In the current study, we found that ruminal MCP content increased signi cantly in the PRM group (P < 0.01), which agreed with the increase of ruminal NH 3 -N concentration numerically, though. Based on the above-mentioned research, a possible explanation for our results might be that a small fraction of methionine RPM contained could be degraded in the rumen and then increase the bacteria populations in the rumen, of course. Therefore, more peptides and AA in the rumen were degraded to NH 3 -N via microbial deamination [41,47] and then synthesize MCP better.
The similar results were also observed in another study [13].
Although no effects were found in VFA concentration during the whole period, cows fed RPM had higher ruminal butyrate and isovalerate contents at 2h after feeding, which is consistent with other studies [13,48]. A previous research described that ruminal VFA production depended on substrates, bacterial populations and ber degradation [49]. Some researchers noted that bacterial populations changed in fermenter uid if rumen inoculum accepted different dietary CP with or without RPM in vitro, and abundance of Ruminococcus albus was the highest as dietary low protein with high RPM level [13]. In addition, another study started the diets which had well-balanced essential AA pro les especially RPM evaluated the abundance of ruminal brinolytic bacterial (R. avefaciens, F. succinogenes, and R. albus) [50]. Thus, we speculated the increase of ruminal butyrate and isovalerate contents at 2h after feeding was because abundance of cellulolytic species changed in rumen when cows fed RPM and subsequently produced more VFA. However, a limitation of the current study is analysis of ruminal speci c bacteria species was not included in the design.

Feeding cost
The price of TMR ingredients and the cost of feeding formulations are shown in  concentrations of butyrate and isovalerate in the rumen of RPM group were higher than that of CON group at 2h after feeding. In economic terms, supplying the diet of 16.4% CP with RPM supplementation to cows could reduce feeding cost by 0.5 $/d per cow and boost net pro ts. In general, RPM may make a positive effect on dairy cows and several research programs are still needed to explore RPM more to discover its other vital roles in dairy cow life. Ruminal butyrate and isovalerate concentrations in Holstein cows fed a control diet (CON) containing 17.3% CP or treatment diet containing 16.4% CP with RPM supplementation (RPM) at 0h, 2h, 4h and 8h after feeding. a,bLeast squares means within a row with different superscripts differ (P < 0.05). Bars indicate standard error of the means.