Characterization of In Situ Ruminal Degradation of Crude Protein and Individual Amino Acids from Ryegrass

Abstract

In temperate pasture-based dairy systems, ryegrass (Lolium perenne L.) is a key forage due to its high crude protein (CP) content, yet its rapid ruminal degradation could limit the supply of rumen-undegraded protein and essential amino acids (EAAs) to dairy cows. This study aimed to investigate the in situ ruminal degradability of CP and individual amino acids (AAs) in fresh ryegrass at the vegetative stage. Three second-parity, rumen-cannulated Holstein Friesian cows (487 kg body weight, 16.5 kg milk/day) were used for the incubation of ryegrass samples collected in different seasons at the vegetative stage. The degradation kinetics were assessed using the Ørskov and McDonald model, with mathematical corrections for microbial contamination. Results showed that the effective degradability (ED) of AAs was generally higher than that of CP (p < 0.05), exceeding 2%, and that some EAAs, particularly lysine, exhibited an ED up to 5.5% greater than CP (p < 0.05). These differences underscore the need for caution when using CP as a proxy for AA degradation in dietary formulations. Given the high degradability of ryegrass AAs, it would be important to monitor and adjust their supply in diets with high ryegrass inclusion to prevent potential deficiencies that could impair milk production and reduce feed efficiency.

1. Introduction

In temperate pasture-based dairy systems, the efficient utilization of nitrogen (N) is a critical determinant of both economic viability and environmental sustainability [1]. Ryegrass (Lolium perenne L.) is commonly used as the main forage in these systems due to its low cost and high crude protein (CP) content, which ranges from 13 to 27% of dry matter (DM) [2]. However, the high ruminal degradability of ryegrass CP often limits the supply of rumen-undegraded protein (RUP), which has been reported to constitute only 10 to 25% of the CP, depending on specific growing conditions and management practices [2]. This high rate of degradation can lead to excessive ammonia production in the rumen, which is then converted to urea and excreted in urine, contributing to N losses to the environment [3]. These losses highlight a significant inefficiency in N utilization, as a substantial proportion of ingested N is lost rather than being used for microbial protein (MCP) synthesis, thereby reducing the potential outflow of ruminal amino acids (AAs) from both MCP and RUP to the intestine. Consequently, the limited RUP supply can exacerbate limitations in the supply of essential AAs like lysine (Lys) and methionine (Met), among others, which are important for protein synthesis and efficient milk production in lactating dairy cows [4].

Extensive research over the years has led to the development of mathematical models for diet formulation, transitioning from primarily empirical approaches to more complex, partially process-based systems [5,6,7,8]. These models aim to represent the biological processes of protein utilization, including ruminal degradation, microbial growth, intestinal digestion, and post-absorptive utilization [9,10]. However, a key limitation persists as these models assume that the amino acid profile of RUP mirrors that of the original feed, implying uniform degradation of both AAs and CP. Tamminga [11] discussed this assumption, highlighting evidence from in vitro studies [12] that indicate differences in ruminal degradation among individual AAs. Subsequent studies have confirmed that the amino acid profile of RUP changes during rumen exposure [12,13,14,15]. However, the extent of these changes varies significantly among different feeds, suggesting that current nutritional models may oversimplify these complex interactions. Given the limited data on the amino acid composition of the RUP fraction, which is restricted to only a few feedstuffs, further research is essential before these phenomena can be effectively integrated into nutrition models [16].

In pasture-based systems where ryegrass constitutes a significant portion of the diet, potential limitations in RUP and essential amino acid (EAA) supply can occur despite high CP levels. A meta-analysis by Morales et al. [17] found that diets predominantly composed of ryegrass contained 19.6% CP, with an average RUP content of 4.6 ± 0.97% of the diet. However, practical recommendations for lactating cows suggest 6 to 7.5% RUP on a DM basis [8]. Thus, a ryegrass pasture with, for example, 25% CP and 85% ruminal degradability of CP would yield an actual RUP value of only 3.75% on a DM basis, which is likely insufficient for high-producing dairy cows [18]. This discrepancy underscores the need to better characterize the nitrogen fractions of ryegrass and revisit assumptions about how they are utilized by dairy cows. Addressing these is crucial to avoid dietary limitations that can impact milk production, cow health, feed efficiency, and environmental sustainability [19]. Thus, this study hypothesizes that the in situ ruminal degradability of CP and AAs in ryegrass differs and that improving our understanding of these differences could lead to more precise diet formulations for dairy cows, thereby enhancing feed efficiency and promoting sustainable practices. The objective was to determine if there are differences in the in situ ruminal degradability of crude protein and amino acids, whether considered individually or as essential and non-essential groups, from fresh ryegrass in a vegetative stage.

2. Materials and Methods

2.1. Ethical Approval and Study Site

The study was conducted at the Agricultural Experimental Research Station of the Universidad Austral de Chile, located in Valdivia, Chile. The Institutional Animal Care and Use Committee of the Universidad Austral de Chile reviewed and approved all animal procedures under protocol number 407/2020, ensuring compliance with international standards for animal welfare.

2.2. Animals, Housing, and Diet

The animal-related portion of this study was carried out during December 2021. Three second-parity Holstein Friesian cows, each equipped with a rumen cannula (4-inch rumen cannula with stopper and U bolt, Bar Diamond Inc., Parma, ID, USA) and lactating during the period, were utilized. On 2 December 2021, at the start of the study, the cows had average values (mean ± SD) of 15.2 ± 1.39 kg of DM intake, 16.5 ± 1.11 kg of milk production per day, 487 ± 28.7 kg of body weight, 228 ± 13.6 days in milk, 30.0 ± 27.2 days of pregnancy, and a body condition score of 3.42 ± 0.14 on a 1 to 5 scale, as described by Ferguson et al. [20].

The cows were housed in the same tie-stall barn, equipped with individual feeders, automatic waterers, and stalls lined with rubber bedding to enhance comfort and reduce stress. They were milked twice daily at 07.00 and 16.00 h in a milking parlor located adjacent to the barn, involving a brief walk of approximately 50 m on flat terrain.

The cows had been consuming the same types of feed for two months, with only the quantities adjusted during a 15-day dietary acclimation period to ensure uniformity in the study outcomes [21]. Their selected diet, which included commonly used feedstuffs in pasture-based systems in Chile, consisted of a commercial pellet (Coagra S.A., Cayumapu, Chile) composed of corn grain, wheat, triticale, soybean meal, wheat milling by-products, and urea; imported soybean meal (Biosur SpA, Osorno, Chile); pelleted dry beet pulp (Iansagro S.A., Quepe, Chile); freshly harvested ryegrass (Lolium perenne L.); and pasture silage, previously harvested from the same research station (

Table 1. Intake and chemical composition of the experimental diet used.

	Commercial Pellet	Soybean Meal	Dry Beet Pulp	Fresh Ryegrass	Pasture Silage	Total Diet
Intake, kg DM/cow/d	2.5	1.0	5.0	6.5	2.0	17.0
Chemical composition 1
DM (%)	91.5	89.3	28.9	19.3	35.0	28.1
DE, Mcal/kg DM	3.24	3.88	2.93	2.86	2.56	2.64 2
CP (%)	21.7	48.0	12.8	21	14.1	19.5
NDF (%)	20.0	11.1	29.0	41.8	50.0	34.0
ADF (%)	7.10	7.19	19.1	21.8	30.1	19.0
Starch (%)	32.2	1.88	8.20	2.10	1.94	8.30
WSC (%)	16.9	13.0	2.98	14.6	7.29	10.6
Total fatty acids (%)	3.84	1.08	0.64	2.32	1.84	1.92

¹ Abbreviations: DM = dry matter; DE = digestible energy; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; and WSC = water-soluble carbohydrates. ² The energy content of the diet is presented as metabolizable energy as the balance was calculated using the NASEM software V8 R2024.05.05 [8].

). Additionally, a commercial mineral mix was included at 250 g as fed (Sal Mineral Alta Producción; Cooprinsem, Osorno, Chile). The overall diet was designed to ensure the applicability of the findings to local conditions. The cows were fed twice daily, after milking, at 0730 and 1630 h. Each feed was weighed separately and then manually mixed directly in the feeders just before being offered. Additionally, half a kilogram of commercial pellet was offered in each milking to facilitate the handling of the animals according to the routine of the farm.

2.3. Ryegrass Sampling, In Situ Incubation, and Laboratory Post-Incubation Residue Analysis

To ensure the ryegrass samples represented the vegetative stage consistently across different seasons, representative samples were collected from a perennial ryegrass pasture (Lolium perenne L. cv. Alto) on 15 January, 15 August, and 17 November 2021, corresponding to the summer (under irrigation), winter, and spring seasons, respectively. The pasture, approximately 3 hectares in size, had been established the previous year (fall 2020), resulting in a high percentage of ryegrass in the botanical composition (greater than 95%).

Samples were taken at an herbage mass of 2800 to 3000 kg DM/ha above ground level to align with the recommended pre-grazing herbage mass for dairy cows [22]. Herbage mass was estimated using a Filip’s Manual Folding Plate Meter (FPM; Jenquip, Feilding, New Zealand), with a total of 100 FPM measurements taken across the pasture. Herbage mass was then calculated using specific equations developed for each season in southern Chile, with R² values ranging from 0.71 to 0.77 depending on the season [23].

For sampling, grass was cut 4 cm above ground level with shearing scissors, using a sampling ring to randomly collect subsamples from 10 points across the pasture in each season. These subsamples were pooled to create a representative 1 kg sample for each season. After collection, the pooled samples were immediately frozen at −20 °C to preserve their integrity until further processing. Once all samples were collected, they were thawed under refrigeration at 4 °C and then oven-dried at 60 °C for 48 h. After drying, the samples were milled through a 2 mm sieve using a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA, USA). The choice of a 2 mm particle size was made to standardize the samples for in situ incubation as this size is commonly used in rumen degradation studies to simulate the physical breakdown of forage during chewing. Milling to this specific size ensures uniform exposure of the plant material to rumen microorganisms, thereby providing consistent results across all samples [24].

Samples of 5 g DM from the pooled ryegrass collected during each season were placed in individual Dacron bags (10 × 20 cm and pore size of 50 μm; ANKOM Tech. Corp., Macedon, NY, USA). Each bag was sealed three times with a heat sealer to ensure they remained closed during incubation. Six incubation times were selected (0, 4, 8, 12, 24, and 96 h) to capture key stages of protein degradation, focusing on early time points where degradation primarily occurs, while the 96-h point was included to assess the undegradable fraction. For each time point, duplicate samples from each season were placed in separate Dacron bags, resulting in a total of six bags per time point. The 0 h bags were not introduced into the rumen and were used to determine the soluble fraction by soaking them in warm water (40 °C) for 20 min. All bags for each incubation time per cow were placed inside a lingerie bag (30 cm by 40 cm) to facilitate handling.

For the five ruminal incubation times, each lingerie bag was tied to a cotton cord, and all the cords were attached to a central cord that secured the bags to the stopper of the rumen cannula. This setup resulted in a total of thirty Dacron bags being ruminally incubated per cow. The samples were incubated in three fistulated cows using a “all in/staggered out” sequence, where all bags for different incubation times were placed in the rumen simultaneously. Bags were then gradually removed as each incubation time was completed, starting with the 4 h bags and continuing until the 96 h bags were removed last. This method ensures consistent rumen conditions across all time points and facilitates the collection of samples for each specific incubation period.

After removal from the rumen, the bags were washed under running cold water until the water was clear and rinsed in a domestic washing machine with cold tap water for 30 min at a “normal” wash setting, and then they were frozen at −20 °C. Incubation residues of the 108 samples were freeze-dried, weighed, and the duplicate samples were combined within cow and incubation time to increase the sample volume for each time point for subsequent analyses.

N in the incubation residues was determined at the Animal Nutrition Laboratory of the Universidad Austral de Chile (Valdivia, Chile) by combustion (Dumas method) and was used to calculate CP (990.03; AOAC, 2000) [25]. Amino acid analyses were carried out at Cumberland Valley Analytical Services (Chambersburg, PA, USA). Determination of sulfur AAs (Met and cysteine (Cys)) was assessed after performic acid oxidation for 16 h prior to acid hydrolysis (converting Met and Cys to the stable methionine sulfone and cysteic acid, respectively), then samples were hydrolyzed with 6 N HCl for 21 h and analyzed by High-Performance Liquid Chromatography (982.30 E(b); AOAC, 2006) [26]. Other AAs, with the exception of tryptophan (Trp; which was not determined), were not subjected to performic oxidation, but the same acid hydrolysis protocol was used (994.12; AOAC, 1997) [27]. Determination of CP content (also by Dumas) and amino acid profile of ryegrass pastures before incubation was carried out at Cumberland Valley Analytical Services.

2.4. Amino Acid Corrections

To better understand the differences between the measured amino acid content in ryegrass samples and values reported in the literature, specific correction factors were applied to the raw data [28]. These corrections were implemented to present both corrected and uncorrected values, providing additional context for comparison.

Hydration correction factors were used to account for the incorporation of water molecules during protein hydrolysis, which can lead to an overestimation of amino acid concentrations. The adjustment used the ratio of the anhydrous molecular weight of each amino acid to its hydrolyzed form. Additionally, a recovery correction was used to address potential losses or incomplete recovery of AAs during the hydrolysis process. For instance, the Lys content was adjusted using a global correction factor of 0.93. This factor results from multiplying 0.877, which accounts for the difference in molecular weight between the anhydrous and hydrolyzed forms by 1.06, which compensates for losses during the 24 h hydrolysis.

These corrections were applied only to the initial amino acid measurements to facilitate comparison with literature values. They were not applied in the estimation of ruminal degradation kinetics, as applying the same corrections to both pre- and post-incubation data would not influence the observed degradation patterns.

2.5. Calculation of Ruminal Degradation Kinetics

The in situ disappearance of CP and individual AAs was determined using the non-linear model described by Ørskov and McDonald [29]. The potential degradability (PD) was calculated according to the exponential model:

$$PD=A+B\left(1-e^{-kdt}\right)$$

where A is the soluble fraction (the fraction washed out at t = 0), B is the insoluble but potentially degradable fraction, kd is the fractional degradation rate (per hour), and t is the incubation time (h). The undegradable fraction (C, %) was estimated as 100 − A − B.

The effective degradability (ED) was calculated assuming a constant ruminal passage rate (kp) of 2% (ED2), 5% (ED5), and 8% (ED8) per hour according to the following equation:

$$ED=A+B\left(kd/\left(kd+kp\right)\right)$$

The kp value of 5%/h was selected based on the static value proposed for forages in the NASEM guidelines [8], while the extreme values of 2 and 8%/h were selected to evaluate their effects on ED.

2.6. Adjustment for Microbial Contamination in Incubation Residues

Bacterial N contamination of the in situ residual is a recognized challenge in ruminal degradation studies [30]. Direct methods for measuring microbial contamination, while highly accurate, are often labor-intensive and costly, which can limit their feasibility in routine studies [24]. Additionally, in this study, the high degradation of the pasture resulted in a limited amount of residual sample after incubation for some incubation times, necessitating the use of an alternative approach for correction.

To address this, we applied a correction model specifically developed for ryegrass, which was derived using ¹⁵N techniques [31]. This model estimates microbial contamination (M) of bag residues as a proportion of CP using the following equation:

$$M=m\left(1-e^{-ft}\right)$$

where m = 0.745 represents the microbial component of the undegradable fraction, and f = 0.076/h is the rate of microbial accumulation on feed particles. In our study, microbial contamination was estimated to be 19.5%, 33.9%, 44.6%, 62.5%, and 74.4% for incubation times of 4, 8, 12, 24, and 96 h, respectively. Given M, and considering that microbes contained 82.4% true protein according to the meta-analysis by Sok et al. [32], the microbial amino acid profile from the same study was used to calculate the contribution of each individual AAs from microbes and subtract that amount from the incubation residues. Concurrently, the total microbial protein contribution to the CP was calculated by applying the contamination percentages to the measured CP at each incubation time. This calculated microbial protein content was also subtracted from the total CP, aiming to adjust the CP and amino acid profiles to better reflect the actual content in incubation residues, thereby mitigating potential overestimations due to microbial contamination.

2.7. Statistical Analysis

The experimental unit in this study was defined as each individual Dacron bag after combining the residues from duplicate samples for each cow, each incubation time, and each season. Initially, 108 Dacron bags (3 cows × 6 incubation times × 2 replicates × 3 seasons) were used, but after combining the duplicates, 54 independent measurements were obtained. This combination was necessary to ensure sufficient material for subsequent analyses, as previously mentioned.

First, parameters of in situ degradation kinetics (A, B, and kd) for CP and each amino acid were derived using a non-linear model fitted through the “nlsLM” function in R (RStudio version 2024.04.2, RStudio Inc., Boston, MA, USA) with constraints ensuring non-negative values for all parameters. Subsequently, PD, and effective degradability at different passage rates (ED2, ED5, and ED8) were calculated based on these estimates, as described earlier. Following this, PD, ED2, ED5, ED8, and C were calculated based on these parameter estimates, as described earlier.

For the statistical analysis, a mixed-effects model was applied using the “lmer” function from the lme4 package in R. This model included nutrient type (CP and individual AAs) as a fixed effect, while cow and season (winter, spring, and summer) were treated as random effects to account for variability across animals and seasons. The response variables in this analysis were the kinetic parameters (A, B, C, and kd), along with PD, ED2, ED5, ED8. Post hoc multiple comparisons were performed using the Bonferroni correction with the “multcomp::cld” function from the multcomp package to assess significant differences between levels of fixed effects.

Additionally, customized contrasts were performed to compare CP vs. grouped AAs; CP vs. EAAs—arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), Lys, Met, phenylalanine (Phe), threonine (Thr), and valine (Val); CP vs. non-essential amino acids (NEAAs)—alanine (Ala), aspartate (Asp), Cys, glutamate (Glu), glycine (Gly), proline (Pro), serine (Ser), and tyrosine (Tyr); and EAAs vs. NEAAs. These contrasts were evaluated using the “contrast” function from the emmeans package, with a Bonferroni correction applied. A significance level of 95% (p < 0.05) was defined as the criterion for determining statistical significance.

3. Results

3.1. Crude Protein and Amino Acid Profile in Ryegrass

Table 2. Overview of crude protein and amino acid profiles in ryegrass at vegetative stage with non-corrected, hydration-corrected, and recovery-corrected values (mean ± SD) and literature reference values ¹.

CP and Individual AAs (% of CP)	Non-Corrected	Hydration-Corrected	Hydration + Recovery Corrected	Literature Values 4
				a	b	c
CP, % DM	25.8 ± 1.42			10.9	16.9	27.5
Essential Amino Acids
Arg	4.57 ± 0.12	4.09 ± 0.11	4.34 ± 0.11	4.74	8.30	4.10
His	1.89 ± 0.14	1.67 ± 0.12	1.79 ± 0.13	1.74	2.60	1.94
Ile	3.63 ± 0.21	3.13 ± 0.18	3.51 ± 0.21	3.71	2.20	3.96
Leu	7.42 ± 0.38	6.40 ± 0.32	6.82 ± 0.34	7.43	4.00	7.39
Lys	4.42 ± 0.43	3.88 ± 0.38	4.13 ± 0.40	4.42	5.10	4.85
Met	1.61 ± 0.10	1.41 ± 0.08	1.49 ± 0.09	1.74	1.00	1.64
Phe	5.20 ± 0.32	4.64 ± 0.29	4.92 ± 0.31	4.50	2.40	4.78
Thr	3.68 ± 0.10	3.12 ± 0.09	3.33 ± 0.10	4.11	2.10	4.10
Trp 2	— 5	—	—	—	1.80	2.09
Val	4.88 ± 0.17	4.13 ± 0.14	4.55 ± 0.16	5.14	3.10	5.22
Non-Essential Amino Acids
Ala	5.59 ± 0.06	4.47 ± 0.05	4.65 ± 0.05	6.16	4.60	—
Asp 3	7.64 ± 0.18	6.61 ± 0.16	6.74 ± 0.16	8.22	4.40	—
Cys	0.93 ± 0.03	0.79 ± 0.02	0.91 ± 0.03	1.11	0.50	—
Glu 3	10.9 ± 0.17	9.55 ± 0.19	10.1 ± 0.22	9.72	4.60	—
Gly	4.53 ± 0.08	3.45 ± 0.06	3.79 ± 0.07	4.90	4.20	—
Pro	4.21 ± 0.21	3.56 ± 0.18	3.69 ± 0.18	4.74	1.50	—
Ser	3.51 ± 0.13	2.91 ± 0.11	3.26 ± 0.12	3.95	2.30	—
Tyr	3.06 ± 0.27	2.76 ± 0.24	2.89 ± 0.26	2.69	1.80	—
Total AAs, % CP	77.6 ± 2.76	66.5 ± 2.40	70.9 ± 2.61	79.0	56.5	—

¹ Amino acid content is presented as non-corrected values, and values corrected for hydration or hydration and recovery according to Lapierre et al. [27]. ² Tryptophan was not determined in this study. ³ Asp corresponds to the sum of Asp + asparagine (Asn), while Glu it is the sum of Glu + glutamine (Gln). ⁴ Literature values correspond to (a) Edmunds et al. [33]; (b) Dineen et al. [34]; and (c) NASEM [8]. ⁵ “—” indicates values that were not available.

presents the CP and AAs profiles from ryegrass at vegetative stage, including non-corrected, hydration-corrected, and recovery-corrected values, alongside literature references. The average CP content across the samples was 25.8% DM. Non-corrected values showed higher percentages for EAAs and NEAAs compared to values corrected for hydration and recovery. Discrepancies were noted both in amino acid concentrations and their recovery percentages (the percentage of AAs relative to the total CP) when compared with some values previously reported in the literature. The coefficient of variation (CV), calculated as the percentage of the standard deviation (SD) divided by the mean (CV = (SD/Mean) × 100), indicated that all analyzed AAs had a CV below 10%, suggesting moderate to low variability in the amino acid content of the ryegrass samples collected in a vegetative stage.

3.2. Kinetic Parameters of In Situ Degradation of Crude Protein and Amino Acids in Ryegrass

Table 3. Kinetic parameters of in situ crude protein and individual amino acids ruminal degradation in ryegrass at vegetative stage.

Nutrient	A Fraction, % 1	B Fraction, % 2	C Fraction, % 3	kd, %/h 4
CP	48.3 gh	51.4 a	0.44 a	11.8 a
Arg	35.5 a	65.2 g	0.13 a	14.8 bcd
Met	36.3 ab	64.7 fg	0.08 a	14.2 abc
Ile	37.4 abc	63.5 efg	0.09 a	17.2 de
Leu	38.5 abcd	62.0 defg	0.16 a	15.1 bcd
Gly	38.7 abcd	61.8 defg	0.15 a	14.5 bc
Cys	39.2 abcd	60.4 cdef	0.43 a	15.8 bcd
Val	40.3 bcde	60.2 cdef	0.14 a	15.3 bcd
Ser	41.3 cde	58.9 cd	0.13 a	15.3 bcd
Thr	41.6 cde	58.9 cd	0.08 a	16.8 cde
Tyr	41.9 cde	59.1 cde	0.03 a	18.7 e
Asp	42.4 def	58.3 cd	0.05 a	16.9 cde
Pro	42.5 def	57.8 bcd	0.21 a	14.0 ab
His	42.8 def	57.8 bcd	0.20 a	16.0 bcd
Ala	44.2 efg	56.2 bc	0.11 a	15.6 bcd
Lys	44.4 efg	56.4 bc	0.03 a	19.2 e
Phe	47.0 fgh	53.6 ab	0.09 a	15.4 bcd
Glu	49.6 h	50.8 a	0.08 a	16.7 bcde

¹ A (%) represents the soluble fraction. ² B (%) represents the insoluble but potentially degradable fraction. ³ C (%) represents the undegradable fraction. ⁴ kd (%/h) is the fractional degradation rate, per hour. Nutrients were arranged starting with crude protein (CP), followed by individual amino acids, each listed in ascending order based on the percentage of the A fraction. This ordering is intended to facilitate comparisons across different kinetic parameters for each nutrient. Within columns, parameters sharing a common letter are not significantly different (p < 0.05).

presents the kinetic parameters of in situ degradation of CP and individual AAs in ryegrass at vegetative stage. The A fraction of CP showed a value of 48.3%, which was generally higher than that of most AAs, except for Lys, Phe, Glu, and Ala. Arg had the lowest A fraction (35.5%), being significantly lower than most other AAs, except for Met, Ile, Leu, Gly, and Cys. The C fraction was minimal across all nutrients, with no values exceeding 0.5%, and no significant differences were observed among the AAs. Given that the C fraction was negligible, the order and magnitude of the B fractions for CP and AAs were largely the inverse of the A fractions. In terms of kd, CP had the lowest value, which was only comparable to Met and Pro, while Lys showed the highest kd value at 19.2%/h. There was considerable variability in the kd values across different AAs, indicating differences in their degradation rates.

3.3. Extent of Degradation of Crude Protein and Amino Acids in Ryegrass

Table 4. Extent of degradation of crude protein and individual amino acids of ryegrass at vegetative stage predicted from in situ incubations.

	PD, % 1		ED2, % 2		ED5, % 2		ED8, % 2
CP	99.6 a	CP	91.4 a	CP	83.2 a	CP	77.7 ab
Cys	99.6 a	Arg	92.6 b	Met	83.7 ab	Met	77.2 a
Pro	99.8 a	Gly	92.6 b	Arg	83.8 abc	Arg	77.3 a
His	99.8 a	Cys	92.7 b	Gly	84.1 abcd	Gly	78.0 abc
Leu	99.8 a	Met	92.7 b	Pro	84.6 abcde	Leu	78.5 abcd
Gly	99.9 a	Pro	92.7 b	Leu	84.6 abcde	Pro	78.7 abcd
Val	99.9 a	Leu	92.9 bc	Cys	84.8 bcde	Cys	79.0 abcde
Ser	99.9 a	Ser	93.2 bcd	Val	85.2 cdef	Val	79.3 bcdef
Arg	99.9 a	Val	93.2 bcd	Ser	85.4 def	Ser	79.7 cdefg
Ala	99.9 a	Ala	93.7 cde	Ile	86.1 efg	Ile	80.2 defgh
Ile	99.9 a	His	93.9 de	Ala	86.4 fg	His	80.9 efgh
Phe	99.9 a	Ile	94.0 de	His	86.4 fg	Ala	80.9 efgh
Glu	99.9 a	Thr	94.0 def	Thr	86.6 fg	Thr	81.1 fghi
Met	99.9 a	Phe	94.2 efg	Asp	87.0 gh	Asp	81.6 ghi
Thr	99.9 a	Asp	94.3 efg	Phe	87.1 gh	Phe	81.9 hij
Asp	99.9 a	Glu	94.8 fgh	Tyr	88.2 hi	Tyr	82.9 ij
Tyr	99.9 a	Tyr	95.1 gh	Glu	88.4 hi	Glu	83.7 j
Lys	99.9 a	Lys	95.2 h	Lys	88.7 i	Lys	83.7 j

¹ PD (%) represents the potential degradability. ² ED (%) represents the effective degradability, which was calculated using a constant ruminal passage rate (kp) of 2%/h (ED2), 5%/h (ED5), or 8%/h (ED8). Nutrients were arranged starting with crude protein (CP), followed by individual amino acids, each arranged separately for each parameter—PD, ED2, ED5, and ED8—. Each column is ordered in ascending percentage values for that specific degradation parameter. This ordering is intended to facilitate the identification of shifts in the ranking of nutrients across different degradation parameters. Within columns, parameters sharing a common letter are not significantly different (p < 0.05).

presents the extent of degradation for CP and individual AAs in ryegrass at the vegetative stage, estimated under different ruminal passage rates. Potential degradability for CP and all AAs exceeded 99%, with minimal variation across nutrients. For effective degradability, CP exhibited one of the lowest values across the different passage rates. At ED2, CP degradation was lower than all other AAs, reaching 91.4%, while Lys had the highest value at 95.2%. At ED5, CP degradation was 83.2%, placing it among the lowest, alongside Met, Arg, Gly, Pro, and Leu. At ED5, EAAs such as Val, Ile, His, Thr, Phe, and Lys exhibited significantly higher degradation rates compared to CP. Lys notably showed the highest degradation rate across all scenarios, with a 5.5% difference above CP at ED5. Additionally, at ED8, CP degradation was 77.7%, while Lys reached 83.7%.

3.4. Comparison of Crude Protein and Amino Acid Degradation Kinetics in Ryegrass

Table 5. Contrast comparisons (least square means differences ± SE) among crude protein, essential amino acids, and non-essential amino acids in ruminal degradation kinetics of ryegrass at vegetative stage.

	CP vs. Aas 1	p Value	CP vs. EAAs 2	p Value	CP vs. NEAAs 3	p Value	EAAs vs. NEAAs	p Value
A fraction, % 4	6.92 ± 0.92	<0.01	7.89 ± 0.94	<0.01	5.83 ± 0.95	<0.01	−2.06 ± 0.43	<0.01
B fraction, % 5	−7.77 ± 0.89	<0.01	−8.88 ± 0.92	<0.01	−6.52 ± 0.92	<0.01	2.37 ± 0.42	<0.01
C fraction, % 6	0.31 ± 0.10	<0.01	0.33 ± 0.10	<0.01	0.29 ± 0.10	<0.01	−0.04 ± 0.05	0.38
kd, %/h 7	−4.21 ± 0.53	<0.01	−4.24 ± 0.54	<0.01	−4.18 ± 0.54	<0.01	0.01 ± 0.25	0.80
PD, % 8	−0.32 ± 0.10	<0.01	−0.34 ± 0.10	<0.01	−0.30 ± 0.10	<0.01	0.04 ± 0.05	0.38
ED2, % 9	−2.27 ± 0.17	<0.01	−2.27 ± 0.17	<0.01	−2.27 ± 0.17	<0.01	−0.01 ± 0.08	0.99
ED5, % 9	−2.77 ± 0.30	<0.01	−2.62 ± 0.30	<0.01	−2.94 ± 0.31	<0.01	−0.33 ± 0.14	0.02
ED8, % 9	−2.56 ± 0.38	<0.01	−2.30 ± 0.39	<0.01	−2.85 ± 0.39	<0.01	−0.55 ± 0.18	<0.01

¹ CP: crude protein and AAs: amino acids. ² EAAs: essential amino acids, corresponding to the sum of Arg, His, Ile, Leu, Lys, Met, Phe, Thr, and Val. Trp was not determined. ³ NEAAs: non-essential amino acids, corresponding to the sum of Ala, Asp, Cys, Glu, Gly, Pro, Ser, and Tyr. ⁴ A (%) represents the soluble fraction. ⁵ B (%) represents the insoluble but potentially degradable fraction. ⁶ C (%) represents the undegradable fraction. ⁷ kd (%/h) is the fractional degradation rate, per hour. ⁸ PD (%) represents the potential degradability. ⁹ ED (%) represents the effective degradability, which was calculated using a constant ruminal passage rate (kp) of 2%/h (ED2), 5%/h (ED5), or 8%/h (ED8). The contrasts were made in the order the nutrients are presented. A positive value indicates that the former nutrient has a greater value for the parameter in question compared to the latter nutrient. Conversely, a negative value indicates that the latter nutrient has a greater value than the former. p values are provided to indicate the statistical significance of the differences (p < 0.05).

presents the contrast comparisons between CP, EAAs, and NEAAs across ruminal degradation kinetics parameters and measures of the extent of degradation in ryegrass at the vegetative stage. CP exhibited significantly higher A fraction values than AAs, with a difference of 6.92%, and this difference was slightly larger when comparing CP to EAAs (7.89%). EAAs showed lower A fraction values compared to NEAAs (−2.06%).

The relationships observed in the B fraction were similar to those in the A fraction but with inverse differences, indicating that while CP had a higher A fraction (representing greater solubility), it had a smaller B fraction available for further degradation compared to the AAs. For the C fraction, CP showed slightly higher values compared to AAs, with no differences between EAAs and NEAAs.

In terms of kd, CP had lower values compared to AAs, EAAs, and NEAAs, with no observed differences between EAAs and NEAAs. For ED2, ED5, and ED8, CP consistently exhibited significantly lower degradation rates than AAs, with differences exceeding 2%. Additionally, at ED5 and ED8, EAAs degraded at significantly lower rates compared to NEAAs.

4. Discussion

4.1. Factors Affecting Crude Protein and Amino Acid Profiles in Ryegrass

The elevated CP content of ryegrass, as observed in the study, aligns with previously reported values for pastures in a vegetative stage [2]. In systems that predominantly utilize pasture, substantial CP levels in ryegrass can complicate dietary formulation, often leading to CP levels that exceed nutritional requirements of cattle and adversely affect N use efficiency [18]. The amino acid concentrations (%CP) observed in ryegrass were consistent, indicating low variability in amino acid profiles when the forage is in a similar phenological stage. Although information comparing amino acid profiles in similar phenological stages of ryegrass is limited, available data suggest minimal variation in amino acid composition within these stages, which aligns with our findings and supports the stability of amino acid profiles in ryegrass [35,36]. Once characterized, this uniformity can serve as a useful reference for diet formulation in similar phenological stages.

Phenological stage of the plant could also potentially affect the amino acid profile. Although no studies have detailed changes in the amino acid profile of ryegrass as the plant matures, research comparing fresh grass with grass silage suggests that certain AAs, such as Lys, may decrease as the plant matures [33]. The study by Dineen et al. [34], conducted during summer, reported lower CP and amino acid content for most AAs except Arg, His, and Lys. They attributed this lower CP content to drought conditions, which could suggest that their ryegrass was at a more advanced stage of maturity compared to the ryegrass in our study. This advanced maturity, combined with the dynamic nature of the non-protein nitrogen (NPN) fraction in fresh forages, might contribute to the observed differences. In contrast, our study found a higher proportion of amino acid nitrogen (AAN) recalculated as a percentage of CP (77.6%) compared to Dineen et al. [34] (56.5%), which aligns more closely with values reported by Edmunds et al. [33]. To better understand these variations, future research should investigate the amino acid profiles of ryegrass across different stages of maturity to assess their impact on nutritional content. Additionally, differences in analytical methodologies and correction factors may contribute to variations in amino acid profiles, making it crucial to apply appropriate correction factors for accurate estimation. For instance, when AAs are reported as grams of AAN per 100 g of total N, the values should be divided by 0.93 to account for the loss of one atom of N from Asn to Asp and from Gln to Glu during the hydrolysis process [32]. This adjustment would increase the recovery values reported by Dineen et al. [34] by approximately 7.5%.

Moreover, the methodological procedures used during amino acid hydrolysis also influence the results. Lapierre et al. [28] developed correction factors for hydration and recovery during a 24 h hydrolysis, which adjust for the addition of a water molecule across each peptide bond and account for AAs that are not fully released or are partially degraded during hydrolysis, respectively. While these factors are essential for accurate amino acid estimation, recovery correction factors are not available for all feeds. According to Dineen et al. [34], recovery corrections may not be necessary for ryegrass, except for specific AAs like Ile and Val. Interestingly, the essential amino acid profile obtained in this study was quite similar to that used in the NASEM library [8], likely because these values are derived from averages across multiple laboratory analyses.

4.2. Variability in Ruminal Degradation Kinetic Parameters of Crude Protein and Amino Acids in Ryegrass

Previous studies evaluating the ruminal degradation of dietary AAs have often employed in vitro procedures [12] or utilized fixed incubation times with techniques like the mobile nylon bag method [33,37]. Consequently, most research has assessed amino acid degradation at a single time point (e.g., 12 or 16 h), limiting the understanding of degradation kinetics over time [37]. Studies incorporating multiple incubation time points are scarce [38,39], likely due to the increased labor and cost involved. Moreover, we are not aware of previous studies that have included sufficient incubation times and replicates to allow for the determination of the actual rate of disappearance of individual AAs in the rumen, as our study has.

Studies of ruminal degradation kinetics have primarily focused on CP, recognizing distinct nitrogenous fractions, such as NPN and true protein, each with specific chemical properties and solubilities that influence their degradation [8]. While analyzing individual AAs may not traditionally follow this fractional approach, applying it can offer valuable insights into the behavior of AAs during ruminal degradation. Our results revealed that the A fraction of CP was significantly higher than that of most individual AAs, suggesting variability in the solubility of specific AAs, which could be attributed to their chemical and physical properties, as well as their distribution within the protein matrix of ryegrass. Although AAs themselves do not possess distinct subfractions like CP, they may behave as if they do, with certain portions being more or less soluble depending on how they are released from the protein matrix during ruminal digestion [9,40]. Tamminga [11] noted that the solubility of feed protein is influenced in part by the relative proportions of albumins and globulins, which are highly soluble, and prolamins and glutelins, which are less soluble. Although this area has not been extensively studied, there is evidence suggesting that the content of albumins and globulins can vary across different ryegrass cultivars and in response to fertilization levels [41]. Future research focused on characterizing the specific types of proteins present in ryegrass, along with their associated amino acid profiles, could improve predictions of both the nutritional composition and ruminal degradation behavior.

The considerable variability in kd values among different AAs suggests differences in their degradation rates within the rumen. This variability highlights the importance of considering individual amino acid kinetics rather than relying solely on CP degradation rates when formulating diets to meet the amino acid requirements of ruminants [16]. Notably, greater variability among AAs was observed in the A fraction compared to the kd values, indicating that solubility may play a more significant role than the degradation rate in differentiating the ruminal degradation of individual AAs. Feeds with higher soluble fractions may lead to more pronounced differences in the amino acid profile of the RUP compared to the original feedstuff. This could partially explain the contradictory results obtained in the past regarding the existence of differences between the amino acid profile of the original feedstuff and the corresponding amino acid profile of the RUP. Feeds with a low A fraction are likely to show little or no difference in the RUP amino acid profile, while the kd will likely only impact some AAs [37]. Given the high values observed for the A fraction and kd of the AAs in ryegrass, it is likely that most amino acid degradation occurs within the first few hours of ruminal incubation [39]. As a result, studies that rely on fixed incubation times—which are predominant in the available literature—may be underestimating the true differences in the degradation of individual AAs.

4.3. Effective Degradability of Crude Protein and Amino Acids in Ryegrass

The ED of CP and AAs in ryegrass revealed significant differences in their ruminal degradation. With a kp of 5%/h, the ED of CP was 83.2%, similar to previous reports [2] and placing it among the lower values compared to most AAs, suggesting that ryegrass in the vegetative stage may provide a limited supply of RUP. Based on the results of this study, ryegrass containing 25.8% CP with 83.2% rumen-degradable protein (RDP) will provide only 4.33% RUP on a DM basis, which is likely insufficient for dairy cows despite the elevated CP content. This falls below the practical recommendations for lactating cows, which suggest ~6% to 7.5% RUP on a DM basis, depending on milk production [8,18].

It is important to note that all ED values for CP and AAs exceeded 80% at ED5 and 90% at ED2, indicating that overall degradability is very high in ryegrass during the vegetative stage. This could be partially compensated in predominant-pasture diets due to the elevated CP content, which results in a greater supply of AAs. However, this brings several associated challenges, such as decreased fertility, predisposition to metabolic diseases, and negative environmental impacts, with the excess of N in diets currently being a significant concern [18,42]. Lys exhibited the highest ED, being 5.5% greater than that of CP at ED5, which is particularly important as Lys is frequently identified as one of the most limiting AAs in dairy cow diets [43]. Additionally, EAAs such as Val, Ile, His, Thr, and Phe also showed significantly higher ED compared to CP. Given that many of these AAs are often referred to as limiting for dairy cows [43,44], their inclusion should be especially prioritized when formulating diets based on high ryegrass inclusion.

4.4. Comparison of Ruminal Degradation Kinetics Between Crude Protein and Amino Acids

The results from this study indicate that significant differences exist in the ruminal degradation kinetics between CP and individual AAs in ryegrass. These differences highlight the importance of analyzing AAs independently rather than relying solely on CP kinetics when evaluating protein sources for dairy cows [45]. Although AAs are part of CP, the results show that the ED of grouped AAs is faster than that of CP as a whole, primarily because some AAs have higher kd values than CP, meaning they degrade more quickly than other CP components. This variability is likely associated with the type of feed, chemical properties, and distribution within the feed matrix, as previously discussed [9,40]. While these factors explain part of the observed differences, additional mechanisms may also influence AAs ruminal degradation, such as the amino acid profile of the feedstuff [46], differences in microbial amino acid transport [11], ruminal amino acid interactions (Chalupa, 1976), ruminal AAs concentrations [47], and rumen microbiome [48], which could further contribute to the observed differences.

The lower ED of EAAs compared to NEAAs observed in this study aligns with previous findings [15]. However, the differences were small, suggesting that it is not a group-wide distinction but rather specific to certain AAs [46]. Overall, the information available on ruminal degradation of CP and AAs is inconsistent across studies but tends to be feed-specific, suggesting that differences in degradation are influenced primarily by the type of feed [13,14,15,46]. Therefore, a proper characterization of feeds used in pasture-based systems is necessary to enhance nutritional models by incorporating these processes, ultimately improving diet formulations and nutrient efficiency. Future studies are necessary to confirm these findings in a production setting. Building on this work, conducting feeding trials would help validate the practical implications of the observed AA degradation patterns.

5. Conclusions

This study demonstrates that there are differences in the ruminal degradation kinetics between CP and AAs in ryegrass at the vegetative stage, highlighting that relying solely on CP degradation can lead to errors in estimating AA degradation. In particular, the effective degradability of AAs was higher than that of CP, generally exceeding 2%. However, this difference was more pronounced for several EAAs, reaching up to 5.5% in the case of Lys. Given the high degradability of these AAs in ryegrass, special attention should be paid to their supply when formulating diets with high ryegrass inclusion.