Alfalfa (Medicago sativa
L.) is commonly used as an excellent high-protein forage for dairy cows, but the availability in China is limited by forage quality and planting scale. Since the quantity of alfalfa can no longer meet the requirements of dairy farms, the contradiction between demand and supply has increased sharply [1
]. Finite access to high-quality forage decreases milk and meat production for livestock industry, undermines health and welfare of animals, and contributes to loss of economic benefits for farmers [2
]. Therefore, exploring locally produced, high yielding, alternative forage resource to ensure satisfactory animal production becomes an urgent problem to be solved.
Paper mulberry (Broussonetia papyrifera
L., RY), which is a deciduous tree of Moraceae family, has been widely cultivated as an emerging fodder tree in recent years, owing to its strong adaptability, fast growth, and high biomass yield. As one of China’s top ten targeted poverty alleviation projects, over 300,000 hectares of RY are cultivated in China [3
]. It is rich in crude protein (CP) and contains highly digestible fiber and minerals [2
]. Multiple biologically active ingredients are also associated with its antioxidant and anti-inflammatory functions in ruminants [3
]. These merits make it attractive to exhibit great potential and applicability as a nutrient-enriched forage resource for local animal production.
Dietary inclusion of RY provides an alternative approach to alleviate feed shortages for livestock production [5
]. However, the growing season of RY is majorly concentrated during the summer rainy season in China, which impairs its sustainable application in dairy rations for all years. Over-production would be discarded in the field, causing squandering of resources [6
]. Recently, the conservation of RY as silage has proven to be effective for overcoming the gap between annual animal production and seasonal imbalance [2
]. Nevertheless, similar to other high-protein forages (e.g., legume forages), the fermentation quality of ensiled RY is usually unsatisfactory, as indicated by extensive proteolysis, high butyric acid content, and unfavorable odor [6
]. During the protein degradation process, non-protein nitrogen (NPN), including mainly peptides, free amino acids (FAA), and ammonia nitrogen (NH3
-N), is largely produced and accompanied by the change in true protein fraction and digestibility of the remaining protein [8
]. Extensive proteolysis leads to the low nitrogen utilization by ruminants, thus, resulting in large economic losses to farmers and adverse environmental issues.
Total mixed ration (TMR) silage has been proposed as an ideal method that does not only help develop new feed resources and balance the moisture content of moist crops, but also improves palatability by altering the odors and flavor of feed resources and preserves nutritional value [9
]. Therefore, the utilization of TMR silage may be a preferred method for efficient utilization of RY. The purpose of this study was to evaluate the effects of substituting different levels of RY for alfalfa on the fermentation quality, chemical composition, protein degradation, and in vitro digestibility of TMR silages.
The chemical composition of RY and alfalfa used in this study was similar to the values reported for RY and alfalfa harvested at the juvenile and squaring stages, respectively, in Northern China [7
]. The CP and WSC contents were much higher in RY of raw material than those of alfalfa, indicating that the RY may be used as a superior high-protein forage resource to supplement ruminant feed. Sole RY is difficult to ensile successfully owing to its high buffer capacity and CP content as well as low DM contents. Thus, there is a need to formulate RY with other ingredients as a TMR silage. The purpose of this study was to determine the effects of replacing alfalfa with different levels of RY on fermentation quality, chemical composition, protein degradation, and in vitro digestibility in TMR silages.
After ensiling for 56 days, all TMR silages irrespective of the substitution of RY were well fermented with dominant lactic acid content, low pH, NH3
-N content, and undetectable butyric acid content. This result is consistent with that in our previous studies [25
], which suggested that high-moisture crops can be well-preserved by formulating TMR silage. This may be related to the sufficient WSC and appropriate moisture contents in all samples to meet the conditions for obtaining satisfactory silage quality. The addition of RY increased the lactic acid bacteria load of pre-ensiled TMR, leading to a higher lactic acid content of silages in RY36 relative to RY0 during ensiling. However, consistent with the elevated lactic acid levels, the pH value increased in response to RY addition. This result was mainly related to their high buffering capacity, which impeded the decline in pH during the ensiling process. In that case, substrate consumption in RY36 could not be effectively suppressed by microbial activity. Therefore, in contrast to other treatments, RY36 accumulated more NH3
-N and reserved less WSC. Tian et al. [27
] reported that high pH and NH3
-N content in TMR silages resulted from high buffering capacity, even with a high lactic acid content.
Treatment differences in the chemical composition of the TMR silages generally reflected compositional variations in the forage materials and their mixing proportions before ensiling. In this study, the higher CP and lower NDF in RY27 and RY36 silages relative to RY0 after ensiling was mainly attributed to the high CP and low NDF in RY materials. Previous research found that forage chemical composition can affect animal feeding behavior, DM intake, metabolism, and performance [28
]. Feed fiber is conducive to rumination and rumen pH. Consequently, it is negatively correlated with DM intake and digestibility [30
]. With the higher CP and lower NDF, an addition level of RY in TMR silages possessed a higher nutritive value theoretically, which could be preferentially recommended to feed the high-lactation dairy cows. However, the higher condensed tannin and hydrolysis tannin in TMR silages with an increasing level of RY were also detected in this study. Tannin in diets generally has harmful effects on nutrient availability in ruminants [31
]. Thus, a digestion trial is needed to further evaluate the feed value of these TMR silages. The ensiling process apparently changed the chemical composition of all the TMR silages. In comparison to the pre-ensiled TMR, the DM content in all TMR silages constantly decreased with ensiling. The readily degradable components (e.g., WSC) of silages were transferred into organic acids, ethanol, and carbon dioxide by microorganisms when ensiling fermentation. The CP content increased throughout the experiment after ensiling, indicating efficient fermentation and nutritional preservation of all silages. Similarly, Chen et al. [32
] observed an increase in CP content in TMR after ensiling, which was attributed to a concentration effect and microbial crude protein. Compared with pre-ensiled TMR, the ether extract, ADF, and ADL contents increased after ensiling likely as a result of being represented on the basis of DM. In contrast, NDF content decreased slightly in all silages after fermentation in this study. Yin et al. [33
] ascribed this increase to acid hydrolysis of cell wall fractions during silage fermentation. Overall, substituting RY with alfalfa in TMR silages did not have adverse effects on ether extract content, but effectively increased CP content and decreased NDF content over the control silages, suggesting an improved nutritive composition of TMR silages by introducing RY.
In terms of forage crops, especially for high-protein herbages, it is inevitable that large quantities of true protein are converted into NPN during ensiling, resulting in poor nitrogen utilization for ruminants. In the present study, NPN content in all silages increased rapidly during the first 7 days of ensiling, as supported by Hao et al. [34
] in TMR silages. However, higher NPN content with lower silage pH in RY36 compared with RY0 is in agreement with Sousa et al. [35
], who reported that protein breakdown during ensiling can be reduced by a rapid decrease in silage pH. Earlier studies have shown that the protein degradation process during ensiling of tannin-containing species, such as purple prairie clover (Dalea purpurea
) and sainfoin (Onobrychis viciifolia
), is suppressed in comparison to that of non-tannin containing forages, such as alfalfa [36
]. Thus, the inhibition of protein degradation in RY36 during silage fermentation in this study is likely attributed to its highly condensed tannin content. Protein degradation in ensiled forages proceeds through two major pathways. True protein is hydrolyzed to peptides and FAA is hydrolyzed mainly by plant proteases under aerobic conditions during the initial ensiling process, and mainly by microbial enzymes when the FAA is deaminated to produce ammonia during anaerobic fermentation [35
]. In this study, the initial increases in peptide-N content in all TMR silages during the first 14 days indicated that protein hydrolysis initially exceeded deamination by microbes while the contribution of microbes became greater than plant protease during the later stage of ensiling. This explains the gradually decreased peptide-N content after 14 days. Free amino acid nitrogen content is closely related to the extent of proteolysis [37
]. The lower FAA-N content in RY27 and RY36 silages showed that the hydrolysis of true protein and peptides was weaker in RY27 and RY36 silages than control silages. Li et al. [8
] reported that peptides can be protected from degradation by bonding to tannin, thus, inhibiting the production of FAA. Notably, increasing the level of RY did not reduce deamination of amino acids in the present study. Instead, the NH3
-N content in RY36 was even higher than that in RY0 at every phase of ensiling. Guo et al. [37
] found that tannin additives can lower the formation of NH3
-N only when its application rate exceeds 35 g/kg DM. This means that only high levels of tannin may have caused the inhibition of deamination. Balanced amino acid composition of the diet is essential for maximizing nutrient utilization and animal productivity. Lysine and methionine play an important role in the metabolism in organisms and are the first two limiting amino acids necessary for dairy cows [38
]. Further quantitative evaluation of amino acid degradation in TMR silages is essential to add more information on accurate animal feeding and production when RY is introduced. Overall, increasing a substituted level of RY in TMR silages decreased NPN, peptide-N, and FAA-N content, thereby effectively protecting true protein from hydrolysis. Yet, it did not prevent the deamination of FAA because of the increasing NH3
The ensiling process did not affect the proportion of the B3 fraction, but increased the proportions of A and C fractions, while decreasing the proportions of true protein as well as B1 and B2 fractions, suggesting that the ensiling process not only negatively facilitated proteolysis, but also decreased the protein quality of TMR silages by converting true protein into soluble NPN. The higher fraction C in all TMR silages than fresh materials might mainly result from the heat accumulation owing to the Maillard reaction, as previously reported by Guo et al. [38
]. It should be noted that the shifts within the fraction B1 from 0 to 56 days showed differentiation among different treatments. There was a declining variation in fraction B1 before and after ensiling with an increasing proportion of RY in TMR silages. This indicated that the addition of RY to TMR silages minimized the degree of proteolysis and prevented the degradation of soluble true protein into NPN. Increasing the proportion of RY increased the true protein content in TMR silages, which was mainly associated with the increase in fraction B2 content. According to the CNCPS, fraction B2 is fermented only at a degradation rate of 50–150 g/kg h, and a large amount escaped to the lower gut [17
]. It can be predicted that the amount of true protein flowing to the lower gut increases with an increased proportion of RY based on fraction B2 data. Fraction C contains lignin, tannin-protein complexes, and Maillard reaction products that are indigestible in the rumen and intestine [17
]. The addition of RY did not affect the proportions of C fractions, but increased the B2 fraction and true protein content, while decreasing the non-protein nitrogen content of TMR silages, implying a potential beneficial effect from RY and leading to higher protein quality than that of the control silages.
A nutritional value of feeds is primarily determined by digestibility for the ruminant and an in vitro culture has been developed as a common technique owing to its high correlation with in vivo digestibility and convenient operation [21
]. Digestibility mainly depends on the chemical composition of forages, and high CP and low plant cell wall contents are beneficial for improving DM digestibility. However, increasing the proportion of RY in TMR silages resulted in an increase in CP content and a decrease in NDF content and DM digestibility in the TMR silages in the present study, which might be partly attributed to the high tannin content of RY. Cummins [39
] observed negative effects of tannin content on in vitro DM digestibility of a high-tannin hybrid of sorghum compared to a low-tannin hybrid. The decreased CP digestibility in TMR silages with an increased level of RY was a result of the composition of CP of RY being less digestible than that of alfalfa, which was reflected in the lower soluble CP and greater tannin contents in TMR silages containing higher level of PM, resulting in a reduction of total degradable CP fractions. Xu et al. [9
] used Suffolk Wethers to compare the digestibility of TMR silages containing different levels of green tea grounds rich in tannin and found that the DM and CP digestibility of silages with 150 g/kg concentrations of green tea grounds were lower than those with 0 and 50 g/kg green tea grounds.
The study of Si et al. [3
] indicated that substituting 10% and 15% of RY silage for a portion of whole corn silage, alfalfa hay, and oat hay improved milk quality, and immunity and antioxidant activity of dairy cows. Tao et al. [4
] reported that diets supplemented with 15% RY silage improved the meat quality and growth performance of beef cattle. This study indicates that the addition of 18% RY was the most optimal for formulating TMR silage because of the balance of fermentation quality, feed-nutritional value, protein degradation, and in vitro digestibility. Future research is needed to investigate intake and production, particularly nitrogen efficiency and urinary nitrogen excretion, when mixed TMR silage is fed to dairy cows.