A balance and sufficient supply of amino acids (AA) are driving factors to achieve highly efficient dairy cows′ productivity. In many practical situations, AA deficiency in high-producing dairy cows exists especially the supply of lysine (Lys) and methionine (Met) as the first and second limiting AA [1–3]. Thus, it is a common practice to supplement diets with synthetic Lys and Met. However, Lys and Met are easily degraded by rumen microbes and environment. Therefore, protecting them from rumen microbial and chemical degradation has been chosen as an efficacious strategy to effectively deliver the AA to meet the physiological requirements of lactating dairy cows [4,5]. In the rumen, non-protected AA and true protein are subjected to rumen microbial degradation, deaminated, and are used for rumen microbial protein (MP) synthesis together with carbon skeleton. This highly degradable AA in the rumen is unfavorable to deliver targeted amount of AA into small intestine for absorption and therefore protecting the AA from rumen degradation is critical. Commercially available protected AA products have been developed by industry achieving rumen stability and high bioavailable in the small intestine by coating or encapsulating with pH-sensitive copolymer that are resistant to rumen degradation [6]. The delivery of AA into small intestine as an absorptive site is aimed to increase metabolizable AA supply which is expected to increase milk protein synthesis while reducing N excretion to the environment [7]. Nitrogen (N) source from MP to delivers insufficient AA for optimal protein synthesis in the mammary gland because a certain amount of the AA-N is metabolized into urea-N in the liver. After being released from the liver, urea-N is either excreted via kidney clearance into urine or re-circulated to the gastrointestinal tract. The greater urea-N (plasma urea-N or PUN) in the circulation is an indication of inefficient N use because it has been positively correlated with urinary N excretion [8]. Excretory N, ammonia (NH₃) volatilization, and nitrous oxide formation are serious concerns for environmental sustainability.

Earlier, feeding post-ruminally available Met via rumen-protected Met (RPM) had been extensively carried out to improve production efficiency in dairy cows following the successful development of commercial RPM products. Progress in this topical study has been made, since first conducted in the 1980s [9]. Driven by the success of RPM development, the available product of rumen-protected Lys (RPL) followed shortly after. However, the production response to dietary RPL has been variable and inconsistent. For instance, improved milk yield due to RPL supplementation was observed in a few studies [7,10], but the latter was only observed during early lactation. Most RPL feeding studies reported a lack of statistically significant results on milk production [11–13]. Additionally, when fed a low crude protein diet (CP) or deficient Lys diets, no consistent results were found in response to RPL supplementation [14–16]. RPM, on the other hand, was demonstrated to linearly increase milk production in a recent meta-analysis [17], although they also provided evidence that RPM did not improve milk production in a discrete analysis. Furthermore, it was suggested that feeding RPLM is more promising to improve production traits and efficiency [18–20], but growing numbers of studies also pointed out little to non-substantial response to RPLM supplementation [21–23].

Lysine and methionine experience multifaceted ruminal microbial degradation and post-ruminal metabolism. Even though they are provided in protected form, interaction with various nutrients and conditions during metabolism makes the prediction of milk protein synthesis challenging. Despite this, it was postulated that there is a linear relationship between duodenal AA supply with plasma AA concentrations including Lys and Met [24], and differential rates of AA delivery into the duodenal sites greatly influence the effect on milk component biosynthesis. So far, knowledge of the accuracy of protected Lys and Met transfer into duodenum, plasma, and milk is often complicated with high variability among studies. The large variability in the substantial body of literature requires a comprehensive examination of possible factors that significantly contribute to the different outcomes. In general, RPL and RPLM supplementation are intended to either increase productivity or maintain productivity as complementary AA-deficient diets. In either case, it is important to assess the relationships of RPL alone or in combination with Met (RPLM) in various conditions, i.e., AA-deficient, or not AA-adequate diets, lactational stages, and supplementary levels on lactation performance. Supplying Lys and Met together as RPLM, theoretically, could provide more balance AA that might have higher effect to improve milk production efficiency. Accordingly, we hypnotized that RPLM supplementation would have greater effect to increase milk production and the efficiency of production and N utilization than RPL in dairy cows. Therefore, the present meta-analysis aimed to examine the relationships of supplemental RPL or RPLM on the plasma AA profile, nitrogen metabolism, and lactational performance of dairy cows.

Increasing Lys and Met availability in a balanced manner for dairy cows is expected to increase efficiency of milk protein synthesis or maintain productive performance. It is because Lys and Met are the first two limiting AA in dairy cows [77] in which deficient supply or imbalance between those two would unfavorably change AA metabolism and energy partitioning which eventually disrupts milk synthesis. Supplying Lys and Met in the form of RPL or RPLM is therefore essential to meet the AA requirement for optimum milk protein synthesis in the mammary gland and improves milk production [62]. Nevertheless, evidence from the first meta-analysis examining supplementation of post-ruminally available Lys or Met showed that the response to both increasing Met and Lys intakes was a declining efficiency of milk protein synthesis [1]. As studies investigating the production response to RPL and RPLM evolved, comprehensive examination of their effects on production traits and milk component synthesis is required. Our findings revealed that the status of AA or MP deficiency and lactation phase greatly influenced the prediction models. Under deficiency of Lys or Met, or when dietary CP or NE_L were reduced, RPL supplementation was most likely to maintain production performance while fewer studies reported decreasing in milk production and milk component [21,38].

In the present meta-analysis, higher supply of intestinally available RPL in early lactating cows resulted in linear increase in milk production (kg/d), FCM, ECM, and overall dairy efficiency as measured by milk yield/DMI and ECM/DMI. Linear increases were also observed for the yield of milk protein, milk fat, and milk lactose production (g/d). Estimation using the result of regression equation indicated that an increase of 1.30 kg/d milk yield and 53.5 g/d milk protein was obtained by supplementing 100 g/d of intestinally available RPL when Lys supply from basal diet held constant or considered as adequate, assuming the absorption rate or bioavailability to be at least as guaranteed by producers (~50%). Experiments that directly measure the bioavailability of Lys are scarce and considerable variability was reported, such as [77] who reported the bioavailability of Lys ranged between 11% to 67%, in which one of three products had lower bioavailability than the company recommendation. Discrepancies of Lys bioavailability were also reported by different studies [78,79] even for products with similar protection methods, which might, in part, explain the effect of RPL on milk production and component synthesis. The variability of RPL and RPLM bioavailability might also be attributed to the protection methods. There are several methods that have been developed by manufacturers to protect Lys and Met from rumen microbes and environment such as coating the Lys and Met using ethyl-cellulose, pH-sensitive co-polymer, calcium soaps, hydrogenated fatty acids, and triglycerides [78]. The RPL and RPM escapes degradation in the rumen because the coating method especially using pH-sensitive co-polymer is effective preventing rumen bacterial attack and it is highly resistant to the rumen pH and rumen environment [78] and therefore increases their availability to meet the animal requirement. Different co-polymers could possibly affect the bioavailability.

The increased milk production was in line with the finding of Fehlberg et al [62] who reported a higher milk increase than that of the estimated value (+3 kg/d) by delivering 99.3 g/d of absorbable RPL for early lactating dairy cows. Similarly, Robinson et al [52] also reported +2 kg/d increase in milk production of dairy cows during early lactation. The lower estimated result from our meta-analysis was expected due to the variability within studies. Such variability could also be due to the different length of RPL supplementation. Several studies, for instance, demonstrated that 90 d and 280 d RPL supplementation increased lactational performance [32,33] while shorter supplementation period produced inconsistent findings [11,23,55]. Our findings revealed that lactation stage influenced the outcome variables. Supplementation of RPL in early but not in mid or late lactating dairy cows is likely to increase milk production plausibly because the cows in the early postpartum are most likely deficient in nutrient supply (negative energy balance) including MP and AA. Therefore, providing intestinally available Lys at this period would help meeting the needs of Lys for milk production as evident from several studies [10,62]. In mid-lactating cows, on the other hand, protected Lys and other AAs supplementation resulted in none to small effect on production traits as the cows have sufficient AA supply from the diet [21,23,80]. In this situation, it is not surprising if supplementary Lys or Met increased plasma Lys [23] but not milk protein yield because the plasma Lys is re-routed into body protein flux. Studies have observed a decrease of Lys and Met efficiencies for milk protein synthesis especially when the supplemental doses exceed the requirement, as previously suggested [1,5]. This was supported by the decrease of the utilization of Lys (from 39% to 25%) and Met (44% to 12%) for milk protein synthesis in response to increasing Lys and Met supply [1] and an overall lower N utilization efficiency when dietary CP increases [81]. There are several factors that could explain the efficiency of Lys and Met supply to lactating dairy cows: energy and MP levels or status, AA balance, DMI, protecting efficiency, and supplementation methods. After absorption, metabolizable AA undergoes complete oxidation for energy production and the rest would be utilized for milk production [5]. In addition, incomplete endogenous AA reabsorption and oxidation of AA in the epithelial cells also contribute to the AA losses that lead to low efficiency of AA utilization [1,82]. Under energy deficiency status, cows would utilize the AA for energy production via gluconeogenesis and AA breakdown would increase in the condition of excessive AA supply resulting in higher urinary N and lower N use efficiency [83]. Imbalance of AA supply could also prevent the absorption of other AA, as suggested by Baumrecker [84].

A higher increase of ECM during early lactation as a result of dietary RPL is validated in our meta-analysis, indicated by the higher slope of ECM than that of milk yield from the regression equation. This was mainly explained by the pertinent increase in milk fat yield in response to dietary RPL. It was suggested that milk fat synthesis increased when Lys or Met are oversupplied [34] via diverting the excessive AA into de novo milk fat synthesis. Increasing milk fatty acids in the study conducted by Robinson et al [52] and by Fehlberg et al [62], likely resulted from greater de novo milk FA production as suggested by Woolpert et al [85] exacerbate this hypothesis. This was similar to our result where elevating RPL levels linearly increased milk fat yield at the rate of 0.928 g/d as per 1 g/d Lys increase. However, it should be noted that an enhancement in milk production and synthesis of protein and fat would be observed only when MP supply met physiological requirements of dairy cows, as evidenced by the contrasting results found by Lee et al [11] and Fehlberg et al [6] in which they had different MP supply from their formulated diet (800 vs 1,190 g/d). Additionally, the increase in milk lactose yield could be explained by gluconeogenesis of AA into lactose similar to the fat when Lys or Met supply excess of the minimum requirement.

Regarding RPLM, we found similar results with RPL where increasing RPLM level produced a linear increased on milk yield and milk protein synthesis (Table 5). In many studies, lack of effect on milk production was reported with RPM alone [17,59], probably because Met was not the first limiting AA. Therefore, the production response was more sensitive to RPL supplementation as Lys is known to be the first limiting AA. Many other plausible reasons might be related to the basal MP or Met level, diet composition, and adequacy status [2,43,52]. This might also explain why the improvement effect was little to none in RPLM compared to RPL. However, it might be taken into consideration that it does not necessarily compare their effects due to the scarcity of studies that directly compared RPL and RPLM. In most studies included in our meta-analysis, RPL and RPLM were obtained from independent studies except in a few in very few experiments. Additionally, RPLM levels failed to increase milk fat and milk lactose but did increase milk protein yield. Earlier studies reported higher milk protein production from cows in two different locations supplemented with RPLM [10,86] with the condition of adequate CP in the basal diets for microbial growth. A greater nutrient supply especially MP and AA exceeding the requirement for milk protein synthesis would be circulated for body protein at a different priority. This is especially true in the case of postpartum and early lactating cows where increasing protein or AA supply would reduce alveolar cell apoptosis and is important to maintain mammary cell functions for milk synthesis [87]. It is probably also relatable to question whether the efficiency of RPL or RPLM depends on the protection methods. However, our meta-analysis did not compare the protection methods as they were not very heterogeneous; more than 90% of the studies described similar protection methods regardless of the differences among producers, despite discrepancies existing for this. Fagundes et al [66] demonstrated that three different forms of commercially protected Lys at recommended doses did not affect milk performance, component, and efficiency of mid-to-late lactating dairy cows with a potential increase in N use efficiency [66]. They also found a lack of increasing AA delivery to plasma EAA concentrations, although the supplementary levels of MP were higher with relatively balanced Lys for treatment group.

Furthermore, the linear increase in plasma Lys concentrations which concurrently increased milk protein yield in linear manner support previous hypothesis that level of deliverable Lys in the intestinal absorptive sites would be metabolized in the plasma and mammary gland for milk protein and milk lactose synthesis [10]. A greater relationship between plasma Lys and milk protein suggests that the effect was almost entirely postabsorptive, similar when glucose was supplemented in the duodenal site. Nevertheless, the overestimation dietary absorbable Lys or Met might result in an underestimation of milk production and component synthesis. For instance, Robinson et al [10] found 49% to 63% lower intestinal deliverable Lys from the calculated target. They reported different responses of early vs mid-lactating cows whereas the RPL increased the production of milk, fat, and true protein in the early lactation but similar production traits during mid-lactation. Also, a meta-analysis estimated that constant increase in either Lys or Met is likely to decrease the efficiency of AA conversion into milk protein [1]. Furthermore, Lys is categorized as Group II AA where the conversion of Lys into per unit of milk protein requires more the requirement which is plausible to explain the underestimation of milk protein as a function of plasma Lys concentrations although the relationship was considered as strong (R² = 0.708). For the conversion of Lys as measured by U:O, however, discrepancies exist in the literature from 1.27 [82] to 1.38 and 1.42 [88] depending on CP levels of the diets and the physiological state of the cows.

As a major net user of EAA supply, the mammary gland plays a greater role in EAA partitioning than other organs even though overall routes of EAA from intestine, bloodstream, liver, and mammary gland itself are not straightforward [82]. Lys and Met are converted into milk protein components at different rates according to the individual AA uptake-to-output ratio in the milk protein. After intestinal absorption, Lys is metabolized rapidly into the mammary gland for protein synthesis especially under Lys-deficient diet. Therefore, it is not surprising that in many studies plasma Lys concentration did not change with RPL supplementation [50,53,57]. Protein synthesis in the mammary gland is also dependent on dietary non-starch carbohydrate or energy and the availability of glucose for absorption. It is well known that energy sources determine the yield of MP in the rumen which contributes to approximately 50% of intestinal protein flux [89]. In addition, the availability of absorbable glucose in the intestine was suggested as a driving factor of ribosomal protein S6 (rpS6) activation in the mammary gland and influence metabolic process of milk protein synthesis [90]. Therefore, in a study designed to increase AA delivery into the intestine, it is critical to consider the proportion of AA to starch or energy supply to predict the mechanistic effect more accurately.

Despite this study suggesting a linear relationship for most of the parameters in response to increasing dietary RPL, it should be understood that the applicability of the equation is restricted to the maximum RPLM or RPL used in the dataset. In continuous incremental supply, however, the logistic model and or segmented regression would explain better predictability when the maximum AA supply beyond and under the breakpoint would result in constant efficiency [91]. In our modeling, linear models had better performance than quadratic, logistic, and segmented regression models.

In the present meta-analysis, the effects of supplementary levels of RPL and RPLM on N metabolism were not found by using full dataset. The insignificant effect was mainly due to the high cross-studies variance. In addition, it was also driven by lower N intake especially when RPL or RPLM substituted to MP-deficient diet, as reported in several studies [53,58,66]. Although it was suggested that supplementation of RPL or RPLM on non-deficient diets could improve N use efficiency, it is a limitation of the present meta-analysis having relatively small sample size for N metabolism data. Future modelling studies to assess the relationship between rumen protected AA supplementation and N use efficiency would help directing how to improve the efficiency and lower N excretion.

In conclusion, our meta-analysis demonstrates that increasing either intestinally available RPL or RPLM during early lactation of dairy cows increased lactational performance as shown by the linear increase of milk yield, feed efficiency (ECM/DMI), and milk protein synthesis in which RPLM had a better prediction model than RPL. The linear increase observed on the lactation performance by RPL and RPLM was not different between adequate and AA-deficient diets. In addition, higher efficiency of post-absorption effects of RPL and RPLM on milk protein synthesis as shown by a strong linear relationship between plasma Lys and milk protein synthesis. Therefore, it is recommended to supplement RPL or RPLM to help improve lactational performance of dairy cows when Lys or Met is deficient in the diet, but it is not recommended in adequate AA diets.