Impact of Neem Cake on In Vitro Ruminal Fermentation, Gas Production Kinetics, and Enteric Greenhouse Gas Emissions in Finishing Beef Cattle Diets

Abstract

Neem cake (Azadirachta indica) is a versatile plant with potential benefits for ruminant animals due to its effects on rumen modulation. This study aimed to evaluate the effects of increasing levels of neem cake and monensin on in vitro ruminal fermentation in cattle diets. Six treatments were tested: neem cake at 0, 240, 480, 720, and 960 mg/kg dry matter (DM) and monensin at 30 mg/kg DM. The basal diet consisted of a diet with a 15:85 roughage-to-concentrate ratio. Parameters evaluated included gas production kinetics, CH₄ and CO₂ emissions, pH, volatile fatty acids (VFAs), ammonia-N (NH₃-N), and organic matter digestibility. Neem cake increased ruminal pH compared to monensin (p < 0.01). The total VFAs increased linearly with neem cake inclusion (p < 0.01). The acetate proportion increased quadratically (p = 0.06). Propionate decreased linearly (p = 0.02), while branched-chain VFAs (BCVFAs) increased linearly (p = 0.09). The neem cake addition increased the NH₃-N concentration quadratically (p < 0.01). CH₄ and CO₂ concentrations were higher with neem cake compared to monensin (p < 0.05). Neem cake shows potential to reduce rumen acidosis and enhance fiber digestion, making it useful during the adaptation period for finishing diets in feedlots and for grazing animals. However, it was not effective in reducing greenhouse gas emissions in this in vitro system.

1. Introduction

Sodium monensin, an ionophore antibiotic, is widely used as a growth promoter in beef cattle feed due to its ability to influence the ruminal microbiota and enhance feed efficiency [1,2]. However, concerns about microbial resistance and residues in animal products have led some countries to ban antibiotics in animal nutrition, prompting the search for alternative additives [3]. In this context, natural compounds emerge as a promising alternative to monensin in beef cattle nutrition.

Azadirachta indica, popularly known as neem, is a plant of Asian origin belonging to the Meliaceae (mahogany) family. Neem has long been recognized for its herbal medicinal properties [4,5]. For example, its insecticidal effect can act against more than 430 species of pests that occur in several countries [6]. Although some trials have reported poor palatability and adverse performance effects of raw neem cake (which still contains the kernel) on farm animals, its industrial decortication—an effective strategy for maximum oil recovery and toxic compound removal—improves its utilization by ruminants [7,8]. In addition to providing bioactive compounds, the cake obtained after oil extraction and full kernel removal is a good source of nutrients, particularly protein (CP: 35–38%), with a relatively well-balanced amino acid profile [8,9].

The neem plant contains several bioactive compounds such as alkaloids, carotenoids, flavonoids, ketones, phenolic compounds, steroids, and triterpenes [10]. Additionally, azadirachtin, meliantrol, salanin, and vilasinin are specific bioactive secondary metabolites found in neem [11]. Azadirachtin, a complex tetranortriterpenoid limonoid, is particularly abundant in neem fruits and is responsible for its toxic effects on insects [12]. Neem seeds, bark, and leaves possess various beneficial properties, including anti-inflammatory, antimicrobial, anthelmintic, antioxidant, and immunostimulant effects [13], with its anti-inflammatory and antimicrobial effects attributed to its triterpenoid compounds [10].

As mentioned, A. indica is a multipurpose plant that has also been used in ruminant animals due to its effects on rumen microbial fermentation, animal health and performance, and enteric CH₄ emissions [14,15,16,17]. El-zaiat et al. [16] reported no detrimental effect on the ruminal fermentation profile, nutrient intake, or digestibility when feeding neem leaf powder (40 g/kg of dry matter (DM)) to growing lambs. However, Jack et al. [18] found that adding 5% water-washed neem fruit to the diet of West African dwarf rams increased average daily gain and final body weight due to greater nutrient digestion efficiency. Yang et al. [19] investigated the effects of neem cake addition (20 and 40 g/kg DM basis) in finishing diets using a continuous culture system. They reported greater bacterial N efficiency and bacterial N synthesis in neem-supplemented diets compared to the control, although they observed a linear reduction in the digestibility of organic matter (OM) and crude protein (CP). Akanmu et al. [15] evaluated the effects of natural compounds, such as A. indica crude extract, on in vitro digestibility and CH₄ production, finding that 50 mg/kg of A. indica extract reduced CH₄ production and improved digestibility of a forage-based diet without significantly reducing in vitro OM digestibility. Additionally, Patra et al. [20] reported that water extracts from neem seeds reduced total VFA synthesis in the rumen and affected protozoa activity in an in vitro study.

Altogether, these results demonstrate that neem cake may modulate ruminal fermentation, improving the feed efficiency of ruminant diets. Nonetheless, although several studies have reported the effects of A. indica on ruminal fermentation, animal performance, and enteric CH₄ emissions, few studies have demonstrated the effects of increasing levels of neem cake supplementation on in vitro ruminal fermentation, gas production kinetics, and enteric greenhouse gas emissions. Thus, in this study, we hypothesized that the components present in neem cake by-products may modulate rumen fermentation similarly to monensin, enhancing fermentation efficiency and reducing greenhouse gas emissions. Therefore, the objective of this study was to assess the effects of increasing levels of neem cake and a fixed level of monensin (positive control) on in vitro ruminal fermentation, gas production kinetics, and enteric greenhouse gas emissions in feedlot cattle finishing diets.

2. Materials and Methods

2.1. Ethics, Experimental Designs, and Chemical Analysis

The experiment was conducted at the Instituto de Zootecnia, Beef Cattle Research Center, located at Sertãozinho, São Paulo, Brazil. The experiment was approved by the Animal Use Ethics Committee of the Instituto de Zootecnia (no. 363-2022).

The experiment was conducted aiming the evaluation of increasing levels of neem cake (A. indica;

Table 1. The chemical composition of the neem cake.

Item	Neem Cake—Chemical Composition
Dry matter, g/kg as fed	857
Ash, g/kg of DM	200
Organic matter, g/kg of DM	800
Crude protein, g/kg of DM	198
Crude fat, g/kg of DM	370
Phosphorus, g/kg of DM	5.87
Calcium, g/kg of DM	5.51
Magnesium, g/kg of DM	2.80
Sulfur, g/kg of DM	4.47
Zinc, mg/kg of DM	78.7
Copper, mg/kg of DM	12.6
Manganese, mg/kg of DM	30.8
Iron, mg/kg of DM	1846

) and compared them with monensin supplementation (positive control). Thus, five different levels of neem cake were tested independently, resulting in six treatments: neem cake at levels of 0 (negative control), 240, 480, 720, and 960 mg/kg DM) and monensin as positive control at 30 mg/kg DM. Monensin was chosen as the positive control because it is the primary additive used to modulate rumen fermentation. We selected the monensin level of 30 mg/kg DM because it is a common dosage used in commercial beef cattle diets.

The neem cake was obtained from the company Base Fertil (Cravinhos, SP, Brazil). The product was produced through a micronization process designed to release active compounds from the seed. Briefly, the process involves micronization in a ball mill or concrete mixer. In this method, a steel cylinder, filled halfway with heavy balls, rotates in a circular motion, grinding seeds, cake, or neem almonds combined with oil and an organic solvent through cold friction. The dosages were calculated proportionally considering a rumen of 80 L according to manufacturing recommendations. The basal diet consisted of a typical finishing beef cattle’s diet (15:85 roughage/concentrate ratio) and was formulated to meet the nutrient requirements of a non-castrated steer with an average daily gain of 1.8 kg/d (

Table 2. The ingredients and chemical composition of the basal diet.

Item	Basal Diet, g/kg of DM
Ingredients
Corn silage	100
Braquiaria hay	50.0
Corn finely ground	690
Soybean meal	150
Urea	10.0
Chemical composition
Dry matter, g/kg as fed	556
Organic matter	964
Crude protein	134
Starch	445
Neutral detergent fiber	248

) [21].

A 33-bottle automated in vitro GP system (Ankom Technology, Macedon, NY, USA) equipped with wireless pressure sensors connected to a computer was used to evaluate the ruminal fermentation pattern of tested diets. Thus, treatments were evaluated in four 48 h fermentation incubations to assess the in vitro GP profiles, and ruminal fermentation parameters. In each incubation, treatments were incubated individually in 250 mL bottles, which were randomly arranged in the incubator. Therefore, each fermentation run (3 runs total) had 5 replicates of each treatment plus 3 blanks (only rumen/mineral/buffer solution), totaling 99 observations.

All ingredients were ground through a 1 mm screen using a Wiley mill (TE 650; Tecnal^® Piracicaba, SP, Brazil) for performing all incubations and analyses. Samples were analyzed for dry matter (DM; method 930.15 [22]), ash (method 942.05 [22]), crude protein (Dumatherm^®; GerhardtGmbH & Co, Königswinter, Germany; method 990.13 [23]), and ether extract (method 2003.05 [22]). The organic matter (OM) was calculated as the difference between DM and ash contents. For neutral detergent fiber, samples were treated with alpha thermo-stable amylase without sodium sulfite [24] and adapted for a Fiber Analyzer (TE 149; Tecnal^®, Piracicaba, SP, Brazil).

2.2. Ruminal Fluid Collection and Buffer Solution Preparation

The rumen fluid was collected from two Nellore steers cannulated in the rumen (average body weight of 640 kg). Steers were maintained on a total mix diet of 60% of corn silage and 40% of concentrate (ground corn grain, citrus pulp pellet, soybean meal, and mineral mixture). Two hours before feeding, 2000 mL of rumen fluid were collected from each animal, immediately filtered through four layers of cheesecloth, placed into pre-warmed (39 °C) thermal bottles, and immediately transported to the laboratory, according to Yáñez-Ruiz et al. [25].

The buffer mineral solutions of all experiments were prepared according to Menke and Steingass [26]. The buffer solution was kept in a water bath at 39 °C and purged continuously with N for 30 min. Resazurin was used as a color indicator to control the buffer pH and N saturation (oxidation–reduction potential). The rumen fluid was mixed with the buffer solution (1:2 v/v) in a water bath at 39 °C under anaerobic conditions by flushing N.

2.3. In Vitro Gas Production

The Ankom^RF system bottle valves were set to be vented. Each bottle (250 mL) was filled with 1.0 g of each diet. Samples were hydrated with deionized water to avoid particle dispersion. Bottles were inoculated with 150 mL of rumen/buffer solution keeping the headspace of bottle continuously flushed with N. After inoculation, bottles were closed and placed in the air-ventilated shaker incubator (EI-450T, ENGCO, Piracicaba, SP, Brazil) under controlled temperature (39 °C) and agitation (95 rpm). The data acquisition software (Gas Pressure Monitor, Ankom Technology, Software GEN 3, Macedon, NY, USA) was set to monitor the cumulative pressure every 5 min, and data were recorded every 60 min for 48 h for both experiments. Valves were set to automatically release the gas when the pressures reached 3.4 kPa [27].

The cumulated gas pressures—total gas production—at 24 and 48 h were converted into mL according to Tagliapietra et al. [28] as follows:

GP, mL = (Pc/Po) × Vo,

where Pc is the cumulated pressure change (kPa) in the bottle headspace, Vo is the bottle headspace volume (95 mL), and Po is the atmospheric pressure read by the equipment at the beginning of the measurement.

The bottles’ final GP volumes were corrected for inoculum contribution by subtracting the final GP of the blank bottles. For total GP over time, the cumulative pressure values were adjusted to assess biological values using the following dual-pool model [29]:

Vt = [V1/(1 + e² + 4 × [K1 × (L − Time)])] + [V2/(1 + e² + 4 × [K2 × (L − Time)])],

where Vt = gas volume produced up to the specific time, mL; V1 and V2 = maximum gas volume achieved from complete digestion of each pool, mL; K1 and K2 = specific rate of digestion of each pool, h⁻¹; and L = lag time, h.

Since the treatments were applied to the same basal diet with a common chemical composition, the in vitro OM digestibility (IVOMD) was calculated according to Menke and Steingass [26], as follows:

IVOMD (g/kg DM) = 31.55 + 0.8343 × GP₂₀₀,

where GP is the net gas production (mL/200 mg DM) at 24 and 48 h.

For ammonia-N (NH₃-N) and volatile fatty acids (VFA) analysis, subsamples of 15 mL from the rumen/buffer solution before incubation and from each bottle at 48 h were filtered through four layers of cheesecloth. Then, 0.2 mL of a 500 mL/L H₂SO₄ solution was added for the determination of NH₃-N and VFA. The VFA concentrations were determined using gas chromatography (Nexis GC-2030, Shimadzu, Kyoto, Japan) equipped with a glass capillary (Supelco Nukol^TM, Bellefonte, PA, USA, 30,000 cm × 0.53 mm i.d.) and coupled with a flame detector ionization, and N was used as a carrier gas. The NH₃-N concentration was determined by colorimetry as described by Chaney and Marbach [30]. The total VFA and NH₃-N concentrations were calculated by subtracting the values measured on the initial content of the components in the rumen/buffer solution from the final concentrations of each bottle [31].

2.4. Enteric Carbon Dioxide and Methane

Here, the Ankom^RF system bottle valves were set to be closed. All other procedures and designs were the same as the total GP essay. After inoculation, bottles were sealed and then placed into an air-ventilated shaker incubator (39 °C). At the end of each fermentation batch (48 h), CO₂ and CH₄ production were measured from the headspace using a gas chromatograph (Nexis GC-2030) equipped with a Gas Solid-Carbonplot capillary (Agilent Technologies, Inc., Santa Clara, CA, USA 30,000 cm × 0.32 mm i.d.) coupled with a discharge ionization detector, and helium (999.9 mL/L) was used as the carrier gas. The gas samples were collected from the headspace of the bottles using an 8 cm, 20-gauge needle attached to a section of stainless-steel tubing with a valve. This assembly was connected directly to the gas chromatograph. The CH₄ and CO₂ content were measured as concentrations and then converted to millimoles per gram of digestible organic matter (OMD). The bottles’ enteric CH₄ and CO₂ productions were corrected for inoculum contribution by subtracting the final GP of the blank bottles. The solution pH was measured (Accumet™ AP61, Fisher Scientific, Atlanta, GA, USA) at the beginning and at the end of each incubation (48 h).

2.5. Statistical Analysis

All statistical analyses were performed in SAS v9.4 (SAS Institute Inc., Cary, NC, USA). All results were assessed for residual normality and variance homogeneity. For all experiments, data were collected and analyzed in a completely randomized design by fitting the data using generalized linear mixed model (GLIMMIX procedure) and considering neem cake inclusion as fixed factors and fermentation batch as a random factor. The fermentation batches were considered experimental units. Non-linear model (NLIN procedure) was used to estimate fermentation rate and gas pool size. The parameters of the nonlinear functions as well as all other variables were fitted through generalized mixed models (GLIMMIX procedure) and compared using Dunnet test (neem levels vs. monensin) and polynomial regressions (neem levels). Additive levels were analyzed for linear and quadratic responses using the following model:

Yij = B0 + B1Xi + B2Xi2 + Pj + eij,

where Yijk is the observed measurement of the ith level of additive in the diet of the jth incubation; i = 1, 2, 3, 4, and 5 (levels of inclusion of additive); B0, B1, B2 = regression parameters of the model; Xi = effect of ith level of fixed quantitative factor (inclusion of neem cake); Pj = random effect of fermentation batch assuming Pj ~ N (0, Pj2); and eij = residual error, assuming eij ~ N (0, σe2). For all the analyses, differences detected at p ≤ 0.05 were considered significant, and differences at 0.05 < p < 0.10 were considered a tendency toward statistical significance.

3. Results

3.1. Effects of Neem Cake Inclusion on Rumen In Vitro Fermentation Kinetics and Characteristics

The effect of neem cake inclusion on in vitro gas production kinetics and characteristics are presented in

Table 3. The effect of neem cake inclusion and monensin on in vitro kinetic variables in a gas production system.

Item a	Neem Cake Inclusion, mg/kg DM					Monensin	Pooled SEM	p-Values
	0	240	480	720	960			Treatment b	Linear c	Quadratic c
V1	98.9	106	86.4	79.3	88.2	81.9	11.5	0.55	0.19	0.72
V2	95.0	75.7	92.0	101	94.7	76.3	9.77	0.31	0.43	0.62
K1	0.11 *	0.11 *	0.11 *	0.14	0.11 *	0.15	0.001	<0.01	0.20	0.31
K2	0.03	0.03	0.04	0.05	0.03	0.04	0.007	0.36	0.57	0.38
L	1.19	0.91	0.73	0.81	1.68	1.03	0.377	0.49	0.49	0.09

^a V1 and V2 = Maximum gas volume of each pool, mL; K1 and K2 = specific rate of digestion of each pool, h−1; L = lag time, h. ^b All doses were compared against monensin using the Dunnett test at * p < 0.05. ^c The negative control treatment was used as having 0 mg of neem cake addition in polynomial regressions.

, Figure 1, and

Table 4. The effect of neem cake inclusion and monensin on ruminal fermentation variables in a gas production system.

Item	Neem Cake Inclusion, mg/kg DM					Monensin	SEM	p-Value
	0	240	480	720	960			Treatment a	Linear b	Quadratic b
Gas production 24 h, mL/g DM	184	164	163	175	179	155	10.1	0.28	0.97	0.13
Gas production 48 h, mL/g DM	194	176	173	181	186	158	12.5	0.40	0.78	0.26
IVOMD, g/kg	666	634	629	644	652	599	22.4	0.33	0.79	0.25
pH	6.36 *	6.36 *	6.36 *	6.35 *	6.34 *	6.19	0.02	<0.01	0.47	0.66
Total VFA, mM	79.0 *	80.2 *	80.9 *	84.4	83.5 **	87.9	1.26	<0.01	<0.01	0.78
VFA profile, mol/100 mol
Acetate	46.0	45.9	48.7 **	48.9 *	46.7	45.3	0.98	0.04	0.14	0.06
Propionate	28.2 *	28.6 *	27.3 *	25.7 *	27.8 *	34.3	0.49	<0.01	0.02	0.11
Butyrate	19.3 *	18.9 *	18.1 *	19.0 *	18.9 *	15.8	0.55	<0.01	0.69	0.27
Iso-valerate	2.04 *	2.00 *	1.91	2.27 *	2.31 *	1.56	0.11	<0.01	0.03	0.17
Iso-butyrate	2.10 *	2.09 *	1.82 *	1.87 *	1.98 *	1.30	0.08	<0.01	0.06	0.06
Valerate	2.41 *	2.42 *	2.15	2.19 *	2.31 *	1.83	0.10	<0.01	0.19	0.15
BCVFA, mM	3.28 *	3.28 *	3.02 **	3.49 *	3.57 *	2.51	0.15	<0.01	0.09	0.12
Acetate:Propionate	1.64 *	1.61 *	1.79 *	1.91 *	1.69 *	1.33	0.05	<0.01	0.02	0.03
N-NH3, mg/dL	13.3	16.7 *	20.6 *	14.5	6.28	8.73	1.99	<0.01	0.02	<0.01

^a All levels were compared against monensin using the Dunnett test at * p < 0.05 and ** p < 0.10. ^b The negative control treatment was used as having 0 mg of neem cake addition in polynomial regressions.

. The highest digestion rate for the first pool was observed at a dose of 720 mg/kg of neem cake (p < 0.05), comparable to the effect of monensin. A tendency of reduced quadratic effect was detected for lag time response as neem cake increased (p = 0.09), in which no inclusion and maximum level of neem cake supplementation (960 mg) had higher lag time values but did not differ from monensin.

No differences were detected in neem cake inclusion on total gas production (24 and 48 h after fermentation) and IVOMD. However, the negative control and neem cake levels were able to increase the rumen fluid pH after 48 h of fermentation compared to monensin (p < 0.0001). Also, the total VFAs increased linearly according to neem cake inclusion (p = 0.001); however, the total VFA concentration for all neem cake levels was lower than the monensin treatment (p < 0.0001). Acetate proportion increased quadratically as neem cake increased (trend; 0.06) and neem cake at levels 480 and 720 had greater concentrations, compared to monensin (p < 0.0001). Propionate decreased linearly as neem cake increased (p = 0.02), and compared to monensin, neem cake had lower propionate proportions (p < 0.0001). Otherwise, butyrate proportion was higher in negative control and neem cake treatments compared to monensin (p = 0.0002). Iso-valerate and iso-butyrate increased linearly and quadratically as neem cake increased (p = 0.03 and trend p = 0.06, respectively) reflecting a linear increase (tendency p = 0.09) in the BCVFA concentration. In addition, iso-valerate and iso-butyrate proportions and the BCVFA concentration of neem cake treatments were greater than monensin (p < 0.0001, p < 0.0001, and p < 0.0001, respectively), except iso-valerate at 480 mg/kg DM, which did not differ from the monensin result (p > 0.10). Valerate had a higher proportion in neem cake treatments than monensin (p = 0.0006), except at level 480 mg, which was not different from monensin (p > 0.10). The acetate-to-propionate ratio was higher for the negative control and neem cake treatments compared to monensin (p < 0.0001), and a quadratic increase was detected as neem cake increased (p = 0.03). NH₃-N increased quadratically as neem cake increased (p = 0.0002), but the negative control treatment and neem cake inclusion at levels 720 and 960 mg/kg DM did not differ from the monensin result (p > 0.10).

3.2. Effects of Neem Cake Inclusion on Enteric Methane and Carbon Dioxide Production

The effects of neem cake inclusion on enteric CH₄ and CO₂ production are presented in

Table 5. The effect of neem cake inclusion and monensin on enteric greenhouse gases in a gas production system.

Item	Neem Cake Inclusion, mg/kg DM					Monensin	SEM	p-Value
	0	240	480	720	960			Treatment a	Linear b	Quadratic b
CH4, mmol/g OMD	50.0 *	55.9 *	59.9 *	53.4 *	54.8 *	29.7	1.54	<0.01	0.14	<0.01
CO2, mmol/g OMD	220 *	231 *	235 *	226 *	221	230	1.59	<0.01	0.70	<0.01

^a All levels were compared against monensin using Dunnett test at * p < 0.05. ^b The negative control treatment was used as having 0 mg of neem cake addition in polynomial regressions.

. The intensity of CH₄ concentration expressed as mmol/g OMD was higher for neem cake levels compared to monensin (p < 0.05). In addition, neem cake quadratically increased CH₄ emission by OMD (p = 0.001), having the highest CH₄ emission per OMD at levels 240 and 480 mg. Furthermore, the intensity of CO₂ emission expressed as mmol/g OMD for neem cake levels was higher compared to monensin treatment (p < 0.05).

4. Discussion

Plants and their extracts have the potential to function as effective “green” solutions for reducing environmental pollution and enhancing the digestibility of feed in ruminant animals [32,33]. In this context, neem cake, a by-product of neem seed, may provide beneficial effects on rumen fermentability due to its high levels of limonoids (tetranortriterpenoid azadirachtin, salannin, nimbin, and gedunin [34]) as well as alkaloids, flavonoids, saponins, tannins, and phenols. These compounds may act as a rumen modulator through their anti-microbial properties. Additionally, neem cake has high levels of proteins, aminoacids, phosphorous, calcium, trypsin inhibitor, and lysine, which can serve as nutrients [35,36,37,38,39].

Considering the high-grain diet used in this study, the effects of neem on in vitro rumen fermentation variables were minimal compared to those of the monensin treatment. However, the neem cake exhibited comparable or even improved effects relative to the 0 mg/kg DM treatment. Compared to monensin, neem cake inclusion, except to the 720 mg/kg DM level, reduced the rate of digestion of the first (rapid) pool of digestion. This effect may have influenced the media pH values, which were higher for neem cake supplementation compared to monensin treatment. This effect may be beneficial, considering the inclusion of neem cake as an additive to prevent rumen acidosis in high-grain diets. However, in this study, we did not evaluate lactate production to confirm such information. At the 480 mg/kg DM level, neem cake was able to reduce the lag time value, which can improve diet fermentability. Before the initiation of digestion, bacteria need to adhere to cell walls derived from the substrate diet; however, this attachment process may face limitations, as both feed particles and bacteria exhibit a negative charge, thereby reducing the degradation rate [40]. Nonetheless, although a reduced lag time has been detected for neem cake inclusion at the 480 mg/kg DM level, no effects on gas production and IVOMD were observed. However, it is important to emphasize that IVOMD was calculated using a linear equation that incorporates gas production as a component. Therefore, it is expected that both results will follow the same pattern. Nevertheless, these findings could indicate that neem cake may modulate microbial population of the rumen; however, our results did not demonstrate improved ruminal fermentability with increased energy expenditure from a high-grain diet substrate.

The linear increase of VFA concentration with increasing neem cake supplementation suggests enhanced microbial activity and nutrient fermentation [41]. The quadratic effect revealed a predominant acetate metabolic pathway in the rumen with increasing levels of neem cake supplementation, up to 720 mg/kg, despite the low forage inclusion in the diet. This could be due to neem cake stimulating the production of enzymes involved in fiber degradation. Indeed, water extracts from neem seed kernel cake have been reported to modulate in vitro enzyme activity in rumen bacteria [42]. Specifically, enzymes associated with fiber degradation in the rumen (carboxymethylcellulase and xylanase) were stimulated by cold and hot water extracts of neem (A. indica) in a level-dependent manner [42]. Conversely, the same study found that enzymes associated with starch, protein, and urea degradation (α-amylase, protease, and urease, respectively) were inhibited. The trend of increased proportions of BCVFA observed in this study aligns with the increases in acetate and butyrate proportions, as BCVFA components may enhance fiber degradation [43]. This occurs because these VFA are produced from the deamination of branched-chain amino acids, and both BCVFA and NH₃-N are essential nutrients for the growth of cellulolytic bacteria [44]. The significant increase in NH₃-N concentrations supports the trend of increasing BCVFA. Overall, neem cake may serve as an additive during the adaptation period of finishing diets in feedlot systems and for grazing animals, as BCVFA is associated with improved fiber digestion. However, further studies are needed to confirm this hypothesis. Considering the increased proportions of BCVFA and the degradation kinetics observed in this study, we suggest that neem cake could be used as an additive during the adaptation period of finishing diets in feedlot systems to prevent acidosis. Additionally, it may benefit grazing animals by improving fiber digestion due to its association with BCVFA.

Although an increased NH₃-N concentration has been detected at 480 mg of neem cake inclusion, there was a reduction in NH₃-N concentration when high neem cake levels were added. These results may indicate a decrease in CP digestibility. This reduction has been linked to the adverse effects of phenolic compounds, which form indigestible complexes with proteins [45]. Additionally, an incubation study revealed that the presence of tannin-rich leaves (a compound of neem cake) enclosed in nylon-gauze bags within the rumen resulted in an increased glutamate NH₃-N ligase activity [46], which can reduce NH₃-N availability in the rumen. The increased NH₃-N concentration detected for neem cake inclusion at the 480 mg/kg DM level demonstrated a high proteolytic activity in the rumen, corroborating the lag time result.

Neem cake has been recommended as a beneficial supplement for ruminants. Faniyi et al. [47] investigated the effect of standard maize substrate treated with selected herb and spice extracts on ruminal environmental biogas production and pressure during fermentation in an in vitro gas production system. They detected a level-dependent increase in gas volume and pressure, along with a reduction in biogas emissions, when increasing levels of neem leaf extract were fed. These findings indicate that increasing levels of neem leaf extract have the potential to influence the efficiency of ruminal fermentation, offering a means to mitigate greenhouse gas production. Moreover, this environmentally conscious approach underscores the inherent antimethanogenic properties of these plant extract, contributing to the eco-friendly goals of ruminant farming while concurrently enhancing productivity. Therefore, in this present study, we hypothesized that all these components present in neem cake by-products may modulate rumen fermentation due to their likely effects on ruminal microbial populations, improving fermentation efficiency and reducing greenhouse gas emission.

Contrary to our results, Akanmu et al. [15] detected reduced CH₄ emission when incubating A. indica extract with different subtracts (ranging from low to high quality). Additionally, Patra et al. [20] observed reduced CH₄ emission with increasing levels of A. indica incubated with a 1:1 mixture of wheat straw and concentrate. The quadratic increase in CH₄ and CO₂ per OMD, which peaked at the 480 mg/kg DM level, can be associated with lag time and NH₃-N levels, which reached the lowest and highest concentrations, respectively, at this level. Another factor that could explain the higher CH₄ production for this treatment is the shift from propionate to acetate production, which increased the acetate/propionate ratio. Pathways to acetate release substrates (CO₂ and H) for CH₄ production, while propionate pathways act as an H sink and do not release CO₂ [48]. One possible reason for the contrasting results mentioned above may be because in the present study, we closed the vent system of the bottle to obtain a homogeneous sample from the headspace for CH₄ and CO₂ analyses. However, according to Cattani et al. [49], in vitro systems that are not vented may alter gas composition and underestimate total GP. Nonetheless, we have no biological explanation for these results; however, to our understanding, neem cake at 480 mg/kg DM may have modulated the rumen microbiota, leading to these outcomes. Finally, the observed increase in CH₄ and CO₂ emissions could be closely related to the increased VFA production, as these gases are byproducts of VFA production pathways. However, greater VFA production may indicate better diet utilization and, consequently, more efficient nutrients use.

5. Conclusions

Neem cake initially decreased the rate of digestion of the rapid pool. At 720 mg/kg DM, it exhibited a similar rate of digestion and total VFA concentration as monensin. The BCVFA levels increased linearly with neem cake, surpassing those with monensin. Neem cake also increased ruminal fluid pH more than monensin and increased NH₃-N quadratically without affecting greenhouse gas emissions. Our findings indicate that neem cake could serve as an effective feed additive for animals on high-concentrate diets, as it helps maintain a higher pH level compared to monensin. For high-forage-fed cattle, neem cake could improve fiber digestion and increase acetate production. Additionally, it may enhance protein digestion (as indicated by BCVFA) and increase VFA production. Although in vivo studies are needed to confirm its effectiveness on different diets, neem cake (at 720 mg/kg) could be an alternative to reduce rumen acidosis and improve fiber digestion. Therefore, it could be a viable alternative during the adaptation period of finishing diets in feedlots and for grazing animals.