Post-ruminal effects of rumen-protected methionine supplementation with low protein diet using long-term simulation and in vitro digestibility technique

Proteins, specifically amino acids profiles, are the most limiting nutrient for milk production in ruminants with high genetic merit. To solve this problem, dietary supplementation with rumen-protected amino acids (RPAA) is a suitable approach (Elek et al. 2008; Osorio et al. 2013). The main objective of any nutritional program is to formulate diets that match the nutritional requirements of the animal to their age and stage. Moreover, it is important to promote the production of short-chain fatty acids (SCFAs), which are known as key bacterial metabolites, for synthesis by dietary carbohydrate fermentation in the gastrointestinal tract of animals (Tan et al. 2014). In ruminants, 70–80% of energy for the rumen epithelia and 50–70% for the overall energy requirements are provided by SCFAs and a variety of physiological functions are maintained by the rumen epithelium (Bergman 1990; Tufarelli et al. 2009). Furthermore, digestibility of nutrients is expressed as the amount that is absorbed by the body of an animal and the utilization of those nutrients for growth, reproduction, and other functions. Data suggest that dry matter intake (DMI) and digestibility of nutrients are major factors influencing nutrient utilization in dairy cattle, yet in lactating dairy cows, DMI is driven by the production of milk but sometimes limited by physical fill and metabolic effects (Allen 2000). Feed is a rich source of proteins; however, it is hard to digest and animals cannot maintain the amino acids (AA) balance between the high production of milk and supplementation of free AA and the balance of essential AA, which limits the key methyl donor, methionine, in dairy ruminants. In this situation, RPAA supplementation is a common method to counteract AA deficiency in dairy cow diets (Abbasi et al. 2018). The AA products are produced from ruminal fermentation and aim to enable maximum flow of AA concentrations in the small intestine. Supplementation of RPAA can increase the flow of AA in the duodenum and absorption also significantly promotes production performance in dairy cattle (Lara et al. 2006). Methionine is a sulfur AA that plays a major role in many pathways, e.g., synthesis of phospholipids, carnitine, creatine, and polyamines, and a source of the methyl donor S-adenosyl methionine (Abbasi et al. 2018). Moreover, methionine is also utilized for protein synthesis and can donate methyl groups for a different reaction and to deliver sulfur groups from the synthesis of cysteine. Therefore, it is important to balance and maintain all these methionine-dependent key functions in dairy ruminants via the supply of additional methionine into a protected form (i.e., rumen-protected methionine, RPM) for post-ruminal maximum absorption, particularly in the small intestine. In many studies, methionine has been reported as being one of the most important AA required for growth of small and large ruminants (Wiese et al. 2003). It is involved in many vital metabolic processes including serving as a glycogenic AA (Brosnan 2003), as a precursor to cysteine, and as a constituent of various vital proteins, e.g., glutathione peroxidase and apolipoprotein B-100 (Brosnan 2003). Additionally, methionine also plays a direct role in low density lipoprotein synthesis in bovines and decreases plasma ketones during early lactation (Girard and Matte 2005; Abbasi et al. 2017). Elevating blood plasma methionine levels might promote better liver function, increase antioxidant levels, decrease inflammation, and improve oxidative stress capacity (Osorio et al. 2013). If maximum degradation of methionine in the rumen or free supply occurs, then this causes a limitation of methionine in dairy ruminants. Therefore, a negative methyl donor balance is also a significant challenge for peripheral dairy cows. Several techniques have been developed to ensure the proper supply of methionine including encapsulation or matrix protection of methionine (Patton 2010) into the diets of ruminants to balance energy by increasing glucose production, promote hepatic oxidation of AA, or directly endorse protein synthesis.

Our hypothesis was that rumen-protected DL-methionine was degraded more slowly by rumen microorganisms, moderately in the abomasum, and fully in the small intestine of the ruminant, thereby providing a steadier supply of methionine to the small intestine. Thus, the main purpose of the present study was to examine the effects of feeding different levels of rumen-protected DL-methionine supplementation with low crude protein (CP) on post-ruminal fermentation, disappearance, production of volatile fatty acids, and ammonia nitrogen (NH₃-N) production.

Mammals are depending on macro and micronutrients for the regulation of physiological functions, health, production, and the prevention of diseases (Abbasi et al. 2014; Stover et al. 2017). Amino acids are natural compounds in many of the aforementioned biological courses (He et al. 2011). Methionine is a sulphur-containing AA that plays a key position in many pathways, i.e. synthesis of phospholipids, carnitine, creatine, polyamines, phosphatidylcholine, precursor of succinyl-CoA, homocysteine, cellular methylation (Soares et al. 2017), deliver sulphur groups for the synthesis of cysteine and reduce dietary cysteine requirement (Mackay et al. 2012). The present study was designed to measure which concentration of RPM with low dietary CP effects maximum post-ruminal, to help for further application in vivo study. For maximum VFA production, disappearance and essential AA at ileum to maintain dairy performance and solve environmental problems linked to high protein feeding in ruminant’s industry.

In the current study, RPM supplementation significantly promotes disappearance and fermentation of diets, as the concentration increases from 0.11 and 0.81 g/kg (DM basis). The post-ruminal disappearance of CP and aNDF were significantly higher in LPLM and LPHM, but no effects were found on DM, ADF, and GE with RPM supplementation. Like our current findings, previously reported that dry matter intake averaged 25.4 kg/day was not affected by RPM treatment (Zang et al. 2006). Consistent with these results, in other trials it was observed no DMI improvement through supplementing RPM (Lee et al. 2015). A DMI response to RPAA supplementation was recorded negligible in studies with early-lactation cows (Lara et al. 2006). Conversely, Lodman et al. (1990) recorded a numerical improvement in the extent and rate of DM and NDF digestion, because of urea and methionine supplementation. The post-ruminal production of total VFA was improved significantly, particularly, SCFAs, (acetate, propionate, and butyrate) were significantly improved by supplementation of RPM. Our findings support earlier observations that, the SCFAs, primarily acetate, propionate, and butyrate, are organic acids produced in the intestinal lumen by bacterial fermentation of mainly undigested dietary carbohydrates, specifically resistant starch and dietary fiber and, to a lesser extent, dietary and endogenous proteins (Fan et al. 2015). However, it was noted that propionate originating from fermentation is the major precursor of glucose in cows which contributes up to 60% of glucose flux rate (Larsen and Kristensen 2013). For ruminants, glucose is the important source of milk lactose synthesis. It is mostly provided by the liver (up to 90%) through gluconeogenesis process (Nafikov and Beitz 2007). Furthermore, Mulligan et al. (2002) stated that both total VFA and synthesis of acetate associated with a NDF degradability. However, in current study, at ileum the production of valerate was significantly increased with high supplementation of RPM, the results was similar to previous study of Mackie et al. (1991), who reported that, with supplementation of DL-Met the significant increase in iso-valerate and valerate observed which contributed to a greater total VFA, because of branched-chain VFA (Leu, Ile, Val) originates from the bacterial deamination moreover, greater branched-chain VFA indicates that methionine analog supplements increased the hydrolysis of protein. The pH plays a significant role to maintain normal homeostasis of the gastrointestinal tract. Maximum and balance pH is important for normal digestibility of experimental diets in simulation apparatus. The NH₃-N concentration in the Rusitec effluent depends on the degradation and N uptake efficiency by post ruminal microorganism, and production of NH₃-N play the main role in MCP synthesis (Bach et al. 2005). In the present study, the post-ruminal concentration of NH₃-N was unaffected significantly by RPM supplementation compared to the control groups. However, previous studies indicating that ammonia absorption (0.9 g/day with the silage diet) occurred between the rumen and duodenum and probably from the omasum (Engelhardt and Hauffe 1975). Ammonia that entered the ileum was mostly absorbed before the digesta reached the ileum (Siddons et al. 1985). Results indicated that RPM supplementation did not significantly alter the Rusitec fermenter pH before changing experimental feed or after changing from 0 to 24 h compared to the control group. The result agreed with Papas et al. (1984) who reported that, rumen-protected methionine was 94% stable in acetate buffer pH 5.4 in the rumen, however, polymeric coatings are usually pH-sensitive and intended to maintain their structural integrity at the pH normally encountered in the rumen, furthermore, in citrate buffer pH 2.9, which simulates the abomasal environment, and 90% of methionine was released. The lower pH of the post ruminal (abomasum and ileum) activates the breakdown of the coating and consenting release of amino acids for absorption. Furthermore, the current finding supported by Silva et al. (2016) who reported that dietary CP levels not altered ruminal pH in finishing beef cattle. The discrepancy between the in vivo and in vitro three-step procedure estimates may partly be due to the protein digestion, RPM supplementation and post-ruminal effects (Calsamiglia and Stern 1995). Moreover, another cause of the discrepancy between the in vivo and in vitro three-step procedure might be the incubation time in the Rusitec.

In conclusion, different levels of ethyl-cellulose RPM supplemented with low CP and their post-ruminal effects showed that the LPHM group promoted disappearance, improved fermentation, increased post-ruminal fiber degradation, and promoted the total tract digestibility of nutrients. Future research should proceed in vivo to address the mechanism involved in producing such effects by RPM supplementation and the way that oxidative status might affect subsequent reproductive and productive performance of dairy ruminants.