Lupin Seed Supplementation as a Functional Feed Additive: In Vitro Ruminal Gas, Methane and Carbon Dioxide Production, Fermentation Kinetics, and Nutrient Degradability

Simple Summary Enteric greenhouse gas (GHG) emission remains a challenge in modern livestock production as it relates environmental perspectives to animal production. In view of this, constant attempts are made to use ingredients that reduce enteric GHG emission and improve energy efficiency. This study aimed to investigate the effect of lupin seeds as a functional feed additive on in vitro ruminal fermentation, and methane (CH4) and carbon dioxide (CO2) emissions. Lupin seed lowered the proportion of CH4 in the total biogas production. Also, lupin seed increased fiber digestibility, total short-chain fatty acids, acetic acid, propionic acid, and microbial protein. Lupin seed at 2.0% supplementation increased gas production, enhanced nutrient degradability, and reduced the proportion of methane in every volume of biogas produced. The results suggest that lupin seed can be an ecofriendly ingredient that can be added to ruminant diet. Abstract The inevitable enteric gas emission from ruminants is considered a modern-day problem from an environmental perspective. Addressing this problem requires nutritional approaches such as the use of phytogenic additives in ruminant diets. In this regard, lupin seed (LS) can be a useful additive due to its phytochemical constituents. Therefore, this study investigated the effects of lupin (Lupinus angustifolius) seed supplementation as a functional and sustainable feed additive in sheep diet (50:50 concentrate-to-forage ratio) on in vitro gas production (GP; mL/g DM), methane (CH4; mL/g DM) and carbon dioxide (CO2; mL/g DM) emissions, fermentation parameters, and nutrient degradability (g/kg DM incubated). Gas production and CH4 were measured per gram of incubated dry matter (DM), degradable DM (dDM), degradable neutral detergent fiber (dNDF), and degradable acid detergent fiber (dADF). Lupin seeds were included at 0 (control), 0.5, 1.0, 1.5, and 2% of the diet. The seeds contained 3.27% essential oils (DM basis), with eucalyptol as the main phytochemical. The highest GP per gram of DM and dDM was observed (p < 0.01) with 2.0% LS supplementation level. While 1.0% LS had the highest GP per gram of dNDF, 0.5% lupin diet had the highest GP per gram of dADF. Asymptotic GP and CH4 emissions linearly and quadratically increased (p < 0.01) with increasing LS level, while lag time decreased. Despite increased CH4 production, the proportion of CH4 in total biogas was lower (p = 0.008) for LS treatments than the control, with the 0.5% LS showing the lowest CH4 proportion. Production of CO2 increased with lupin seed treatments, with 0.5% LS producing the highest proportion (p = 0.027). Degradability of DM, NDF, and ADF was greater (p < 0.01) for the high LS supplementation level, while 0.5% supplementation level decreased ADF degradability. Total short-chain fatty acids, acetic acid, and propionic acid increased (p < 0.05) with LS supplementation level, leading to a reduced acetate:propionate ratio. Rumen pH decreased (p = 0.036) with LS supplementation, while ammonia-N decreased (p = 0.045) and estimated metabolizable energy increased (p < 0.001) linearly. Calculated microbial protein synthesis (p = 0.005) and gas yield (p = 0.047) increased with LS supplementation level. LS supplementation at 2.0% of diet (DM basis) increased GP and CH4 emission (mL/g DM) and enhanced nutrient degradability, suggesting its potential use as a functional feed additive for ruminants when supplemented at a 2.0% level into diet.


Introduction
The roles of ruminant animals in food production and security cannot be overemphasized.However, ruminal fermentation has several disadvantages.It produces greenhouse gases, posing a significant environmental threat, and also causes loss of feed energy.The gas production and release are not a problem themselves but a consequence of raising ruminant animals.Therefore, the need to mitigate the harmful environmental impact of the produced and emitted greenhouse gases prompted the current study.The main produced gases are methane (CH 4 ) and carbon dioxide (CO 2 ).Methane generation is responsible for about 2-12% energy losses in ruminants [1], impacting productivity such as milk yield and growth.Additionally, enteric CH 4 emissions from ruminants account for approximately 17% of the global greenhouse gas emissions [2].
The Lupinus genus has been a part of animal feed in the Mediterranean region for centuries.The newly developed "sweet" lupin varieties are characterized by low alkaloid levels (previously responsible for bitterness) and high protein content.These new varieties with robust pods and reduced alkaloids have shifted lupin use as green manure and a soiling crop to a valuable source of nutritious seeds.This shift has reignited interest in lupin as a protein source in animal diets [3].Studies have shown that supplementing ruminants with lupins offers benefits that could improve growth and reproductive efficiency, comparable to cereal grain supplements [4].
Lupin seeds contain high levels of proteins, soluble fiber, and minerals, and a low content of starch, in addition to phytochemicals [5].Lupin species such as Lupinus albus, L. angustifolius, and L. luteus have an oil content ranging from 5-10%, which is relatively low.These species are free of anti-nutritional factors like trypsin inhibitors and saponins.Lupin seed oil is notably rich in unsaturated fatty acids, including oleic and linoleic acids, which can constitute up to 80% of the oil content [6].Furthermore, LS contains phenolic compounds, saponins, and flavonoids, whose concentrations have the ability to increase or decrease digestion [7] and mitigate CH 4 production [8,9].The degradability of LS protein in the rumen varies between 71 and 79%, depending on the variety.Additionally, lupin has a unique profile characterized by minimal starch, high levels of both soluble and insoluble non-starch polysaccharides (NSPs), and significant amounts of raffinose oligosaccharides.These properties can influence energy utilization and the digestion of other nutrients in a diet containing LS [4].Lupin contains NSPs, which are primarily composed of non-cellulosic polymers and pectic polysaccharides.It also contains tocopherol, lutein, α-carotene, β-carotene, and various polyphenols [10].In an in vitro experiment, Um et al. [10] showed that lupin flakes at 3, 6, and 9% of total diet enhanced rumen ammonia-N (NH 3 -N) concentrations and crude protein (CP) disappearance rate.Despite the benefits of lupin and its secondary metabolite contents, not much has been reported on its impact on greenhouse gas emissions and ruminal fermentation characteristics.However, lupin shares certain metabolites with eucalyptus plants.The main metabolite is eucalyptol, which can be used to measure the potential benefit of lupin on greenhouse gas emission.Eucalyptus leaf meal contains eucalyptol and p-cymene, which are active ingredients in lupins that decreased CH 4 production by 29% [11].Sallam et al. [12] and Kumar et al. [13] demonstrated that eucalyptus oils inhibited CH 4 production and decreased the number of ruminal protozoa.Additionally, supplementation of LS in dairy cow diets reduced ruminal protozoal population [14], which may indirectly lower methanogens by decreasing the population and activity of protozoal-associated methanogens [15].The active ingredients, such as monoterpenes (camphor and eucalyptol), in LS can halt the growth of some microbes, although their effect against certain rumen microbes may be limited [16].This demonstrates the ability of lupin and its secondary metabolites to manipulate rumen microbes.
The objective of this experiment was to assess the potential use of dried lupin (Lupinus angustifolius) seeds as a functional feed additive for ruminants, when supplemented at increasing levels to a total mixed ration (TMR), on in vitro gas production (GP), CH 4 and CO 2 emissions, in vitro ruminal fermentation (total and individual short-chain fatty acids (SCFAs), nutrient degradability, metabolizable energy (ME), ruminal NH 3 -N, and partitioning factor (PF 24 ) using rumen inoculum of sheep.It was hypothesized that at low levels of supplementation, the phytochemical constituents of the dried LS could increase ruminal microbial activities and growth (not measured), alter ruminal fermentation towards increasing ruminal propionate (C 3 ) and acetate (C 2 ), and increase nutrient degradability in sheep.

Ingredients and Treatments
A basal TMR containing [per kg of dry matter (DM)] 500 g of concentrate feed mixture, 400 g of berseem (Trifolium alexandrinum) hay, and 100 g of rice (Oryza sativa) straw was formulated and grounded.The incubated substrate or diet is the same as the control diet previously used by Kholif et al. [17][18][19].The basal TMR or substrate (control treatment) was supplemented with graded levels of dried LS.There were thus five treatments comprising the following: (1) 1 g substrate + 0 g LS; (2) 1 g substrate + 0.005 g LS; (3) 1 g substrate + 0.01 g LS; (4) 1 g substrate + 0.015 g LS; and (5) 1 g of substrate + 0.02 g LS.Following the treatments, the substrate and the respective doses of LS were carefully weighed into the filter bags using a Luna Analytical Balance (LAB 124e, Adam Scales & Balances, Thetford, UK).They were subsequently thoroughly mixed together and placed inside fermenter bottles prior to incubation.The nutrient compositions of the LS and the TMR are detailed in Table 1.  1 The concentrate feed mixture (CFM) used in the study consisted of the following components per kilogram of dry matter (DM): 170 g of soybean meal, 395 g of wheat bran, 395 g of maize, 20 g of limestone, 10 g of a vitamins and minerals mixture, and 10 g of salt. 2 The diets used in the experiment contained, per kilogram of DM, the following: 500 g of the concentrate mixture, 400 g of berseem hay (Trifolium alexandrinum), and 100 g of rice straw (Oryza sativa). 3The treatments were as follows: (1) 1 g substrate + 0 g LS; (2) 1 g substrate + 0.005 g LS; (3) 1 g substrate + 0.01 g LS; (4) 1 g substrate + 0.015 g LS; and (5) 1 g of substrate + 0.02 g LS.Following the treatments, the substrate and the respective doses of LS were carefully weighed into the filter bags using a Luna Analytical Balance (LAB 124e, Adam Scales & Balances, Thetford, UK).
Clean and dry LS were sourced from a local supplier in Egypt.Before use, the LS were ground and mixed thoroughly.To analyze the volatile compounds present in the LS, a GC-MS system (model 7890B from Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector was employed at the Central Laboratories Network, National Research Centre, Dokki, Giza, Egypt.The analysis was conducted according to Qin et al. [20] with some modifications.The separation process utilized a Zebron ZB-FAME column (dimensions: 60 m × 0.25 mm internal diameter × 0.25 µm film thickness).The analysis was conducted using hydrogen as the carrier gas at a flow rate of 1.8 mL/min in split-1:50 mode, with an injection volume of 1 µL.The temperature program was set initially at 100 • C for 3 min, and subsequently increased at a rate of 2.5 • C per minute to 240 • C, where it was maintained for 10 min.The temperatures for the injector and the flame ionization detector (FID) were set at 250 • C and 285 • C, respectively.The concentration of individual identified phytochemicals was expressed as mg/100 g DM seeds.

In Vitro Fermentation and Biodegradation
The in vitro fermentation medium was prepared following the method outlined by Goering and Van Soest [21].A reducing solution containing 2 mL of sodium sulfide was added to the buffer shortly before the addition of rumen fluid.Each 250 mL bottle contained a mix of 20 mL ruminal inoculum and 80 mL buffer solution.
The ruminal inoculum was sourced from three sheep at a local slaughterhouse in Cairo, Egypt.Prior to slaughter, the sheep were fed a diet of concentrates, berseem hay, and rice straw in a 500:400:100 ratio (DM basis) ad libitum, with free access to water.Rumen contents were collected following the standardized procedure for sampling, storage, and use of ruminal contents as recommended by Fortina et al. [22].Sheep were fasted for 24 h before slaughtering.The time between the animal's death and rumen fluid collection was under 10 min.Approximately 150-250 g of rumen contents was hand-sampled and squeezed into a plastic beaker through a colander, and the process was repeated until about 1000 mL of rumen fluid was collected.The rumen fluid was then filtered through a two-layered cheesecloth to remove large feed particles, and particulate material was squeezed to collect microbes attached to the feed.The initial pH of the inoculum was between 6.8 and 6.9.All the treatments were tested in two incubation runs (statistical replicates), each with three bottles (analytical replicates).In each run, two bottles with inoculum but without substrate (blanks) were included to establish baseline fermentation GP (5 treatments × 3 replicates × 2 incubation runs + 2 blank bottles).
Approximately 1 g (±10 mg) of TMR was weighed into filter bags (ANKOM F57; Ankom Technology, Macedon, NY, USA) and placed into 250 mL ANKOM bottles (AnkomRF Gas Production System) equipped with an automatic wireless in vitro GP module (Ankom Technology, Macedon, NY, USA) with pressure sensors.Lupin seeds were included at 0 (control), 0.5, 1, 1.5, and 2% of the TMR before incubation.Pressure was recorded every 10 min for 48 h, and cumulative pressure was calculated from these values.Gas pressure was converted into volume (mL) at standard pressure and temperature, and the gas volume in the blank units was subtracted to obtain net GP.At 2, 4, 6, 8, 10, 12, 24, 36, and 48 h of incubation, 5 mL gas samples were taken from the sampling vent and analyzed using a Gas-Pro detector (Gas Analyzer CROWCON Model Tetra3, Abingdon, UK) to measure CH 4 and CO 2 concentrations.

Sampling and Analysis of Fermentation Variables
After 48 h of incubation, fermentation was halted by chilling the bottles on ice for 5 min, followed by immediate pH measurement using a pH meter (Thermo Scientific, Orion Star™ A121, Beverly, MA, USA).The ANKOM F57 filter bags were dried in a forced air oven at 55 • C for 48 h.The degradation of dry matter (dDM), neutral detergent fiber (dNDF), and acid detergent fiber (dADF) was determined by subtracting the weight of the dried residue from the initial weight of the dried substrate.Total gas and CH 4 production were normalized to dDM, dNDF, and dADF at the 48 h mark of incubation.
Samples of the fermented fluid supernatant (5 mL) from each bottle were collected in glass tubes for analyzing NH 3 -N concentration, as well as total and individual SCFAs.A 3 mL subsample, was preserved with 3 mL of 0.2 M hydrochloric acid solution to measure NH 3 -N concentration according to AOAC [23] guidelines.
A 0.8 mL aliquot was mixed with 0.2 mL of metaphosphoric acid solution (250 g/L) for SCFA analysis using steam distillation and titration.

Chemical Analysis
The LS and TMR samples were subjected to ash analysis by burning them in a muffle furnace at 550 • C for 12 h (method ID 942.05),CP analysis using the Kjeldahl method (method ID 954.01), and ether extract (EE) determination using diethyl ether in Soxhlet extractors (method ID 920.39) following AOAC methods [23].Neutral detergent fiber (NDF) content was determined without alpha amylase but with sodium sulfite, following the Van Soest et al. [24] procedure.Acid detergent fiber (ADF) content was analyzed according to AOAC [23] (method ID 973.18) and expressed without residual ash.Nonstructural carbohydrate, cellulose, hemicellulose, and organic matter (OM) concentrations were calculated.

Calculations and Statistical Analyses
To analyze the kinetics of GP, CH 4 , and CO 2 , the data on total GP, CH 4 , and CO 2 (measured in mL/g of dry matter) were fitted using the NLIN procedure in SAS (version 9.4, SAS Inst., Inc., Cary, NC, USA), following the model proposed by France et al. [25]: , where y represents the volume of total GP, CH 4 , or CO 2 production at time t (in hours); A is the asymptotic GP, CH 4 , or CO 2 (measured in mL/g DM); c is the fractional rate of GP, CH 4 , or CO 2 (per hour), and Lag (/h) is the discrete lag time before any gas, CH 4 , or CO 2 release occurs.
The partitioning factor at 24 h of incubation (i.e., PF 24 ; in mg of dry matter: mL of gas production) was also determined [26].The gas volume produced (in mL per 200 mg DM) at 24 h of incubation (GY 24 ) was computed.Metabolizable energy (ME) was calculated following the method by Menke et al. [27], and microbial crude protein (MCP) production was estimated using the approach of Blümmel et al. [26].
Statistical analysis was performed using the GLM procedure in SAS, employing a completely randomized design with the model: Y ij = µ + L i + ε ij , where Y ij represents the observation, µ is the population mean, L i is the effect of LS supplementation level, and ε ij is the residual error.Data of each of the two runs of the same sample of the substrate were averaged prior to statistical analysis.Mean values of each individual run (two runs) were used as the experimental unit.Linear and quadratic contrasts were applied to assess the responses at different levels of LS supplementation.

Lupin Seeds
Lupin seed contained 3.27% essential oils (DM basis), with five volatile compounds identified: αand βpinene, eucalyptol, camphor, and trans-caryophyllene (Table 2).They were all C 10 except for trans-caryophyllene which was a C 15 compound.Eucalyptol was the most prominent (86.7%) bioactive compound in the LS while the least was trans-caryophyllene.

Biogas Production
Figures 1-3 show GP, CH 4 , and CO 2 production per gram DM, dDM, dNDF, and dADF.Lupin seed at 2% supplementation level of the incubated diet had the highest GP (mL/g DM; mL/g dDM) among the treatments.Whereas GP (mL/g dNDF) was greater in 1.0% LS treatment, 0.5% lupin diet produced the lowest gas.Conversely, 0.5% lupin produced the greatest gas for every gram of dADF.It is worth noting that the concentrations of CH 4 and CO 2 (Figures 1 and 2) did not appear to follow the same pattern as GP.This discrepancy arises because GP was measured every 10 min, whereas CH 4 and CO 2 were initially measured every 2 h until 12 h of incubation after which they were measured every 12 h (i.e., at 24, 36, and 48 h of incubation).
Supplementation with LS significantly impacted the asymptotic GP, CH 4 , and CO 2 production (Table 3).The asymptotic GP showed both linear (p < 0.001) and quadratic (p = 0.002) increases, whereas the hourly rate of GP exhibited a linear increase (p = 0.001) in response to increasing LS levels.Lag time was linearly (p = 0.021) reduced with increasing LS in the diet.Asymptotic CH 4 production linearly (p = 0.002) increased with increasing level of LS, and the lag time, i.e., the time taken for initial GP, was prolonged by the supplementation.Despite the increase in asymptotic CH 4 production of the treatments, the proportion of CH 4 in the total biogas was lower (p < 0.05) than the control, and 0.5% LS had the lowest proportion of CH 4 in total biogas production.The control treatment produced the highest gas for every gram of dNDF and dADF, while 0.5% produced the lowest (Figure 3). 1 Identification was conducted using authentic standards, spectra from the National Institute of Standards and Technology (NIST) library, and relevant literature sources. 2 RT, retention time (min). 3The concentration was determined based on the total areas of the identified peaks.

Biogas Production
Figures 1-3 show GP, CH4, and CO2 production per gram DM, dDM, dNDF, and dADF.Lupin seed at 2% supplementation level of the incubated diet had the highest GP (mL/g DM; mL/g dDM) among the treatments.Whereas GP (mL/g dNDF) was greater in 1.0% LS treatment, 0.5% lupin diet produced the lowest gas.Conversely, 0.5% lupin produced the greatest gas for every gram of dADF.It is worth noting that the concentrations of CH4 and CO2 (Figures 1 and 2) did not appear to follow the same pattern as GP.This discrepancy arises because GP was measured every 10 min, whereas CH4 and CO2 were initially measured every 2 h until 12 h of incubation after which they were measured every 12 h (i.e., at 24, 36, and 48 h of incubation).
Supplementation with LS significantly impacted the asymptotic GP, CH4, and CO2 production (Table 3).The asymptotic GP showed both linear (p < 0.001) and quadratic (p = 0.002) increases, whereas the hourly rate of GP exhibited a linear increase (p = 0.001) in response to increasing LS levels.Lag time was linearly (p = 0.021) reduced with increasing LS in the diet.Asymptotic CH4 production linearly (p = 0.002) increased with increasing level of LS, and the lag time, i.e., the time taken for initial GP, was prolonged by the supplementation.Despite the increase in asymptotic CH4 production of the treatments, the proportion of CH4 in the total biogas was lower (p < 0.05) than the control, and 0.5% LS had the lowest proportion of CH4 in total biogas production.The control treatment produced the highest gas for every gram of dNDF and dADF, while 0.5% produced the lowest (Figure 3).(A) Table 3.The kinetics of in vitro rumen gas production (GP), methane (CH4), and carbon dioxide (CO2) were influenced by increasing levels of lupin seeds.Means in the same column with different superscripts differ (p < 0.05).The p-value represents the observed significance level of the F-test for the treatment effect, while SEM stands for the standard error of the mean. 1 GP parameters: A is the asymptotic GP (mL/g DM), c is the rate of GP (/h), Lag is the initial delay before GP begin (h). 2 Methane (CH4) production parameters: A is the asymptotic CH4 production (ml/g DM), c is the rate of CH4 production (/h), Lag is the initial delay before CH4 production begins (h). 3 Carbon dioxide (CO2) production parameters: A is the asymptotic CO2 production (ml/g DM), c is the rate of CO2 production (/h), Lag is the initial delay before CO2 production begins (h). 4 The proportion at the end of incubation (48 h).

GP Parameters
Similarly, asymptotic CO2 and the rate of GP per hour linearly (p ≤ 0.001) increased with increasing levels of LS.The proportion of CO2 in the total GP was higher (p = 0.027) Figure 3.In vitro ruminal carbon dioxide (CO 2 ) production: mL/g incubated DM (A), mL/g degradable DM (B), mL/g degradable NDF (C), mL/g degradable ADF (D) of a total mixed ration supplemented with different levels of lupin seeds.Table 3.The kinetics of in vitro rumen gas production (GP), methane (CH 4 ), and carbon dioxide (CO 2 ) were influenced by increasing levels of lupin seeds.Means in the same column with different superscripts differ (p < 0.05).The p-value represents the observed significance level of the F-test for the treatment effect, while SEM stands for the standard error of the mean. 1 GP parameters: A is the asymptotic GP (mL/g DM), c is the rate of GP (/h), Lag is the initial delay before GP begin (h). 2 Methane (CH 4 ) production parameters: A is the asymptotic CH 4 production (ml/g DM), c is the rate of CH 4 production (/h), Lag is the initial delay before CH 4 production begins (h). 3 Carbon dioxide (CO 2 ) production parameters: A is the asymptotic CO 2 production (ml/g DM), c is the rate of CO 2 production (/h), Lag is the initial delay before CO 2 production begins (h). 4 The proportion at the end of incubation (48 h).

GP
Similarly, asymptotic CO 2 and the rate of GP per hour linearly (p ≤ 0.001) increased with increasing levels of LS.The proportion of CO 2 in the total GP was higher (p = 0.027) in the treatments than in the control, with 0.5% lupin having the highest proportion of CO 2 in total biogas produced.In Figure 3, 1.0% lupin produced the highest volume of CO 2 for every gram of dNDF, 0.5% produced the highest volume of CO 2 for every gram of dADF, and the diet without lupin produced the lowest in both cases.

Degradability and Fermentation
Table 4 presents the rumen fermentation profile of diets with increasing levels of LS supplementation.dDM increased (p < 0.001) with increasing levels of LS in the diet.Furthermore, dNDF and dADF increased (p < 0.01) with increasing levels of LS, with the diet containing 2.0% LS being the most degraded.However, dADF was lowest (p < 0.001) for 0.5% LS among the treatments.Total SCFAs, C 2 , and C 3 linearly (p < 0.01) increased with the level of LS in the diet.However, C 2 :C 3 ratio linearly (p = 0.015) reduced as the LS level in the diet increased.Although butyrate (C 4 ) was not (p = 0.155) influenced by LS addition to the diet, it linearly (p = 0.025) increased with increasing LS levels.
Rumen pH decreased (p = 0.036) with increasing levels of LS.The decrease was linear (p = 0.015) except for the diet containing 0.5% of lupin, which had a higher pH than the control.While ruminal NH 3 -N linearly (p = 0.008) decreased, ME linearly and quadratically (p < 0.001) increased with increasing levels of LS.The partitioning factor was not (p = 0.093) affected by LS, but there was a linear (p = 0.009) decrease in the grams of feed degraded per volume of GP.Microbial crude protein (MCP) was linearly (p = 0.001) and quadratically (p = 0.018) affected by treatment, with diet containing 1.0% LS having the highest MCP.However, LS-supplemented diets had higher (p = 0.005) MCP compared to the control.Gas yield at 24 h (GY 24 ) linearly (p = 0.006) increased with the doses of LS.

Lupin Seeds
Bioactive components of a plant can serve as either antinutritional or nutraceutical agents, influencing nutrient absorption and availability.Currently, there are limited studies on the bioactive components of LS.In the present study, five compounds were identified, with four containing C 10 carbon and one containing C 15 carbon.Compounds such as αand β-pinene and camphor had 16 hydrogen atoms (H 16 ), while others like eucalyptol and trans-caryophyllene had 18 (H 18 ) and 24 (H 24 ) hydrogen atoms, respectively.Phytochemical constituents are known to possess antimicrobial, antioxidant, and anti-inflammatory properties [8].Lupin seeds in this study contained a higher proportion of eucalyptol compared to the other four compounds combined.Therefore, eucalyptol, with the concentration of 2835 mg/100 g DM, was the major bioactive compound, accounting for 86.7% of total phytochemicals in the LS.
Previous phytochemical studies on lupin focused on alkaloids [28,29], without considering other compounds evaluated in the current study.The concentration of eucalyptol, which has the ability to positively or negatively influence ruminal microbes [30], in LS is similar to that found in peppermint leaves.The presence of phytochemicals in LS highlights their potential as a functional feed additive.These phytochemicals can positively influence ruminal microflora activity and growth, leading to improved nutrient digestion and animal performance.Different responses with the evaluated levels of LS in different experiments may be mainly due to different concentrations of phytochemicals.

Gas Production
Generally, GP is used to measure the rate of fermentation in anaerobic conditions, and a greater rate of fermentation can lead to gases accumulation.Though gas accumulation is of paramount importance, the proportion of the gases produced in total GP is also of interest.This is because sometimes the proportion of environmentally unhealthy gases may comprise a larger portion of the total gases.In the present experiment, the rise in asymptotic GP of the lupin supplemented diets could stem from its contents of phytochemicals which perhaps fostered microbial activities.It appears that certain phytochemical constituents of the LS enhanced microbial proliferation and activity, resulting in an increased fermentation rate and microbial protein synthesis, ME content, and fiber digestion [31].The prevalence of β-galactan in lupins might positively impact rumen microbial populations [14].The accelerated rate of GP per hour and reduced lag time for GP suggest faster adaptability of rumen microbes to lupin-supplemented diets.Although lupin seeds are known to contain quinolizidine alkaloids, which may be unfavorable to animals, the improved fermentation (increased GP, nutrient degradability, and SCFAs) may be because the variety of lupin used in the present experiment had reduced alkaloid contents [32,33].
Methane production accounts for substantial dietary energy losses, which can impact ruminant productivity significantly.Additionally, enteric CH 4 emissions from ruminants contribute to approximately 17% of global greenhouse gas emissions [2].Although CH 4 from ruminants is biogenic in nature, the short-term environmental impact calls for the need to reduce its emission.Though the volumes of CH 4 produced were higher for the LS supplemented diets than for the control diet except for the diet containing a 0.5% level of LS, a closer look at the proportion of CH 4 in the total biogas shows a tradeoff in that the proportion of methane in the biogas decreased.It can be seen that all the treatments had lower proportions of CH 4 for every milliliter of total biogas.This result can be plausibly attributed to the antimicrobial property of the phytoconstituents of LS, which perhaps reduced ruminal microbes, such as protozoa and methanogens, responsible for methanogenesis.Bryszak et al. [34] showed that dietary inclusion of lupin, particularly Lupinus angustifolius seed meal at 80 and 100 g/kg in a complete diet containing a concentrate-to-forage ratio of 448:552, reduced total bacteria, archaea, and total protozoa in an in vitro experiment.Methanogenesis arises from the interactions between hydrogen-producing microorganisms (bacteria, protozoa, and fungi) and methaneconsuming microorganisms (methanogens) in the rumen, maintaining environmental homeostasis [34].Thus, the reduced CH 4 output could be due to lower bacterial and methanogen activities or a shift in methanogen populations towards less efficient methaneproducing species [35].Notably, the lupin fatty acid profile includes C12:0 and C14:0 acids, which exhibit antimicrobial effects against protozoa [36,37].Bryszak et al. [34] demonstrated that feeding lupin seeds at 2 kg/day to cows fed a total mixed diet containing concentrate and forage at 462:538 reduced the total count of methanogens, including Methanobacteriales and Methanomicrobiales classes, leading to decreased methanogenesis.Methanogens possess the capability to convert CO 2 to CH 4 by utilizing various substrates during fermentation [38].Unfortunately, we did not measure H 2 concentration to elucidate the proper picture of how lupin affected CH 4 emissions.Nevertheless, it was conjectured that they probably achieved this by positioning themselves on the outer layers of the biofilm, allowing them to access H 2 diffusing from the carbohydrate fermentation site [39].Subsequently, they possibly combined H 2 with CO 2 to generate CH 4 .A further look at the CO 2 showed that there was a reduction in the combination of CO 2 and H 2 to form CH 4 .It is pertinent to say that the 0.5% LS supplementation level, with the lowest proportion of CH 4 , had the highest proportion of CO 2 .This suggests that as the metabolic pathways for the formation of CH 4 were hindered, the materials for CH 4 formation, such as CO 2 and H 2 , became the alternatives which were eructated.

Degradability and Fermentation
Dry matter degradability (i.e., dDM) of a feed or forage is a key indicator of its nutritional quality and potential to provide energy to animals [40,41].The dDM, along with dADF and dNDF, reflects the extent to which different components of a feed are broken down or degraded by rumen microorganisms.The increased dDM, dADF, and dNDF with increasing levels of lupin indicates the importance of defining the optimal level (dose) of LS supplementation.This may be attributed to the inherent characteristics of lupin, especially phytochemicals which possibly enhanced microbial proliferation (growth and activity), leading to the improved colonization and degradation of the diet [42].The inherent phytochemical components of the LS make it an appealing functional supplement in ruminant diets.It is pertinent to say that the increases in DM, NDF, and ADF degradability were not due to the nutritive attribute of the LS but rather the functional properties or phytogenic compounds, which were likely responsible for enhanced microbial activities.Thus, the phytochemicals of the LS likely aided digestibility, which increased with higher doses or levels [8,9].The increased degradability of dDM, dADF, and dNDF is beneficial as it connotes the potential of LS to enhance nutrients and energy availability to animals.
Lupin seed supplementation increased C 2 and C 3 concentrations.Each of C 2 , C 3 , and C 4 are SCFAs produced during rumen fermentation in ruminants.The increased C 3 is important because it serves as a major precursor for glucose synthesis in the liver through gluconeogenesis, helps to regulate blood glucose levels, and contributes to overall energy metabolism, which is important for lactating animals to meet their energy requirements for milk production [43].Moreover, the increased C 2 production is also an important result, especially in dairy animals, because it serves as a precursor for fatty acid synthesis, particularly in adipose tissue and milk [44,45].Gas production is directly linked to SCFA levels, with higher GP indicating higher SCFA levels [46,47].Consequently, increased SCFA and ME values are associated with higher GP and digestibility.The levels of SCFAs reflect energy availability and can contribute up to 80% of the animal's daily energy requirement [48], showing a direct correlation with ME and degradable OM [27].Additionally, SCFA levels can be used to establish connections between feed composition, production parameters, and the net energy value of feeds [49].The higher SCFA concentration of the treatments is obviously the result of increased fermentability and nutrient availability for ruminal microbial growth and activity for enhanced degradability [50].The increased SCFAs, C 2 , and C 3 of the LS treatments align with the finding of Um et al. [10], who reported that diets containing lupin cake at 3, 6, and 9% showed higher production of propionate, butyrate, and total volatile fatty acids in an in vitro experiment.The results, however, contradict the work of Bryszak et al. [34], where increased LS meal decreased total and individual SCFAs.This variation in results may be attributed to the lupin varieties and doses used in different studies, since different varieties have different phytochemicals (type and concentrations) [32,33].The lower C 2 :C 3 ratio with increasing levels of lupin is nutritionally beneficial as it indicates that LSs favor an increase in the production of gluconeogenic acids.The lower C 2 :C 3 ratio may also contribute to reduced CH 4 production [43] and a decrease in the activity of cellulolytic bacteria such as R. albus and F. succinogenes [51].The linear increase in the C 4 acid with increasing levels of lupin suggests that lupin seeds, a functional feed additive, can support both dairy and beef production systems and can be used in ruminant nutrition for altering ruminal fermentation and affecting the production of individual SCFAs.
Rumen pH is a crucial measure that reflects the balance between acids and bases in a solution, playing a central role in understanding nutritional diseases and nutrient digestion in ruminants [52].Recent studies suggest that diet type may not be the sole reason for fluctuations or depressions in rumen pH.Rather, the increase in dissolved CO 2 (dCO 2 ) concentration in rumen liquor has been identified as a key factor [53].While rumen dCO 2 concentrations are typically low and stable, modern feeding practices can lead to CO 2 holdup, defined as a decrease in CO 2 fugacity due to changes in rumen liquor's physicochemical properties.This gas holdup can contribute to elevated rumen dCO 2 concentrations, leading to a subsequent decline in pH [53].In the present experiment, ruminal pH decreased with increasing levels of LS, but obtained values were within the normal pH reported by Kamra [54] and Ososanya et al. [55] for optimal rumen function.The decreased ruminal pH with increasing LS levels indicates rapid fermentation of the readily fermentable carbohydrate content of the diets to volatile fatty acids [56,57], indicating the importance of defining the optimal level of LS supplementation.Ruminal pH is related to SCFA production, which is associated with OM degradability, primarily carbohydrates (both readily degradable and structural carbohydrates) degradability in the rumen environment.Therefore, a lower pH, as a consequence of greater DM degradability, is expected.As previously mentioned, the increases in DM digestibility were not due to the nutrients in the LS but rather the changes in microbial activity as a consequence of the phytogenic compounds.A positive correlation between readily fermentable carbohydrate and volatile fatty acids, and a negative correlative between pH and volatile fatty acids [58] have been reported.Ammonia-N plays a crucial role in the digestion and metabolism of feed materials by rumen microorganisms.Ammonia-N is a byproduct of the breakdown of proteinaceous compounds such as plant proteins and microbial proteins.It is a key step in protein degradation within the rumen and serves as a crucial nitrogen source for rumen microbes.Microbes assimilate NH 3 -N to synthesize microbial protein, which is then utilized by the animal upon microbial cell death or passage from the rumen to provide a significant portion of the dietary protein requirements for ruminants [59].The decrease in NH 3 -N with increasing levels of lupin implies efficient N utilization.Ammonia serves as a N source used by rumen bacteria to meet their nitrogen requirement for body protein synthesis.However, ciliate protozoa do not utilize ammonia.The recycling of bacterial N in the rumen increases in the presence of ciliate protozoa, and the number of ruminal bacteria capable of utilizing NH 3 -N decreases with increased ruminal breakdown of dietary protein.Hence, the decrease in NH 3 -N concentration could be partially attributed to lupin's ability to reduce protozoa numbers [34] and decrease hyper-ammonia producing bacteria due to its phytochemical constituents [8].
Inclusion of up to 6% lupin flakes in the diet of cows improved the efficiency of N utilization and decreased ruminal NH 3 -N concentration [60].The improved efficiency of N utilization reduces NH 3 -N concentration, which is used by rumen microbes for their proliferation, resulting in increased MCP.This perhaps explains the reason for the increased MCP and reduced rumen NH 3 -N concentration of the LS treatments compared to the control.Microbial crude protein supplies amino acids to the small intestine for absorption, underscoring its usefulness to ruminant animals.The synthesis of MCP depends on various factors such as the sources of carbohydrates and proteins, the synchronization of rumen functions, rumen microbial recycling, and the presence of anti-nutrients in consumed feeds [61].Moderate quantities of carbohydrates that are readily fermentable in a diet can sometimes increase microbial protein synthesis [62].This increase occurs due to the enhanced availability of substrates and the growth rate of bacteria associated with the liquid phase of digestion [61,62].Likewise, there is an increment in the enhancement of degradable OM.Therefore, it seems that LSs show potential in eliminating some microor-ganisms that are not beneficial in the rumen of ruminant livestock.Simultaneously, they create a conducive environment for the growth and proliferation of beneficial microbes, thereby increasing feed digestibility.Phytochemical-rich LS enhanced the ME, PF 24 , and MCP levels, indicating an ideal combination of energy and protein, leading to increased microbial protein synthesis and PF 24 [18].The phytochemicals in LS may interfere with the biosynthesis of aromatic amino acids, as synthesis pathways are linked through phytochemicals from the seeds [19].Results from increased MCP indicate that most of the NH 3 -N and SCFAs were used for MCP synthesis [17][18][19].
Gas yield for every gram of DM digested after 24 h (GY 24 ) shows more gases were produced per gram of DM digested as LS increased.The higher GY 24 of the LS supplemented diets thus indicates an enhanced incubation environment and fermentability [19].

Conclusions
Lupin seeds contained certain bioactive compounds such as αand β-pinene, eucalyptol, camphor, and trans-caryophyllene, which can significantly modulate rumen fermentation and GP.Lupin seed supplementation of ruminant diets led to dose-dependent increases in GP, with higher levels of lupin seeds resulting in increased asymptotic gas, CH 4 , and CO 2 production.Interestingly, despite the increase in CH 4 production, the proportion of CH 4 in total biogas was reduced with lupin supplementation compared to the control treatment, suggesting a potential mitigation effect on CH 4 emissions.Furthermore, lupin seed supplementation enhanced dDM, dADF, and dNDF, indicating improved nutrient utilization.The increased SCFAs and ME with lupin seed treatments suggest higher energy availability.Overall, the findings of this study underscore the potential of lupin seeds supplementation of a total mixed ration at 2.0% (DM basis) for ruminants to improve ruminal fermentation efficiency and nutrient utilization, and mitigate CH 4 emissions.Further research should be conducted in vivo to validate the findings of the current study.

Table 1 .
Chemical composition of lupin seeds and incubated diet (g/kg DM).

Table 2 .
Volatile compounds in lupin seeds identified by GC-MS analysis.

Table 4 .
In vitro rumen fermentation profile of diet with increasing levels of lupin seeds.
24ans in the same column with different superscripts differ (p < 0.05).The p-value represents the observed significance level of the F-test for the treatment effect, while SEM stands for the standard error of the mean.1dDMstandsfordrymatterdegradability, dNDF represents neutral detergent fiber degradability, and dADF indicates acid detergent fiber degradability (measured in g/kg of incubated material).2SCFAstandsforshort-chain fatty acid, where C 2 represents acetate, C 3 denotes propionate, and C 4 indicates butyrate. 3 3 -N refers to ammonia-N (measured in mg/g of dry matter), GY 24 represents gas yield at 24 h (measured in mL of gas per gram of dry matter), ME stands for metabolizable energy (measured in MJ/kg of dry matter), PF24indicates the partitioning factor at 24 h of incubation (measured in mg of degradable dry matter: mL of gas), and MCP denotes microbial crude protein production (measured in mg/g of dry matter).