Fermentation and Nutritive Value of Pineapple Stubble Silage Supplemented with Leucaena Hay

Abstract

The increase in pineapple production has generated a large amount of waste, which, when incorporated into the soil, can lead to contamination and environmental degradation. In this context, this study highlights the innovative combination of pineapple waste and Leucaena hay, offering benefits for animal nutrition while reducing input costs. The objective of this study was to determine the nutritional value of silage from ‘Pérola’ pineapple waste (PS) with different levels of Leucaena hay. A completely randomized design was adopted with six treatments (pre-wilted PS and PS with 0%, 10%, 20%, 30%, and 40% inclusion of Leucaena hay (LH)). The dry matter content of the silage, crude protein, lignin, and pH increased linearly (p < 0.05) as a function of the inclusion of Leucaena hay, while buffering capacity, soluble carbohydrate content, and effluent losses showed an inverse relationship. As LH levels increased, ruminal DM disappearance decreased. Wilting the pineapple plant did not alter the in vitro gas production, in vitro digestibility of organic matter, partition factor (PF), and methane production when compared to the silages added with Leucaena hay. It is recommended to add 20% Leucaena hay in PS silage.

1. Introduction

Over the last decade, the world production of pineapple has grown by 12.52%, mainly due to the performance of major Asian-producing countries and the growth of Nigeria and Puerto Rico [1]. Brazil is one of the largest producers of pineapples in the world. In 2022, the country accounted for approximately 9% of the global pineapple production, ranking third globally, behind only Costa Rica and the Philippines. The country significantly increased the destination of natural pineapple—prepared and processed in addition to the fruit juice—including potential consumer markets, such as America and Europe [1]. As a consequence of this increase, a large amount of waste is produced, both from fruit and plant, since when starting a new production cycle, the residual mass—that is, the plants—must be removed. Usually, these plants are incorporated into the soil, which can favor the dissemination of pests and diseases, and/or are burned for the implementation of the new crop, which causes great damage to the environment. There are several studies about the use of by-products from the pineapple fruit in the formulation of animal diets [2,3]. However, no reports can be found regarding the use of crop stubble. Thus, it becomes imperative to search for a destination for such waste causing less environmental impact.

On the other hand, most of the agricultural farming and animal production in South America is located in the AW (tropical Savannah) and CFa (humid subtropical) climates of the Koppen classification, with “rainy season” and “dry season” [4], which leads to an oscillation in forage production, which makes it necessary to conserve forage for the dry season, and the silage of this by-product can be an interesting alternative to circumvent the problem of seasonality in producing regions and reduce the effects of environmental pollution. Kiggundu et al. [2] showed the potential of this plant in terms of nutritional value, highlighting the TDN content (62.8%) and soluble carbohydrate contents (36 to 13.16%), which are above the required levels for good-quality silage. However, it presents a low dry matter content (15%), which is below the minimum required to ensure a good fermentation process (28–35%) [5]. Consequently, when subjected to ensiling, there may be clostridial fermentation and reduced development of lactic acid bacteria, leading to nutrient losses through effluents [6,7]. This not only reduces the quality of the ensiled mass but can also result in soil contamination.

Thus, it becomes essential to use absorbent additives or wilt the forage to raise the dry matter content. Leucaena is a legume that is widely used as a protein bank or legume plant because of its high contents of protein, minerals, and beta carotene, which is the precursor of vitamin A, which is highly important in the dry season when the pasture is usually dry and the Leucaena is green. Leucaena hay is known for its high crude protein content, which ranges between 20% and 30%. This high protein content is crucial for growth, milk production, and overall health of ruminants. Additionally, Leucaena has high digestibility, making nutrients more readily accessible to the animals. This results in better feed conversion and greater efficiency in nutrient utilization. It can also be used in the form of hay and fed to various animals or be used as a supplement in mixed silages, aiming to increase the crude protein content of the silage [8,9].

In addition, when added in the form of hay, it is an excellent moisture sequestrant, favoring DM increase [8]. However, it should be noted that the use of legumes in the form of silage, as the main source, presents some inherent factors such as the high buffering power and low content of soluble carbohydrates [10]. Therefore, the hypothesis is that the inclusion of Leucaena in the ensiling of pineapple crop stubble could be an effective strategy to reduce the environmental impact of pineapple waste while producing high-quality and nutritious animal feed. The objective of this study was to evaluate the chemical composition, fermentation parameters, and in situ degradability of ‘Pérola’ pineapple stubble silage with the addition of increasing levels of Leucaena hay and the wilted plant.

2. Materials and Methods

2.1. Location and Ethics Committee Protocol Number

The experiment was carried out in the facilities of the Forage Crops sector of the Center for Agricultural and Environmental Sciences at the IV campus of the Federal University of Maranhão, located in Chapadinha, MA, Brazil (03°44′33″ S, 43°21′21″ W). Experimental procedures were approved by the university’s Ethics Committee for Animal Experimentation under number 23115.011059/2015-26.

2.2. Experimental Design and Treatments

It adopted a completely randomized design with six treatments, with treatment one being pineapple plant, and the other five treatments consisting of pineapple plant silage with the addition of Leucaena hay (LH) at levels of (0%, 10%, 20%, 30%, and 40% in DM). The selection of inclusion levels of Leucaena hay (LH) between 0% and 40% in dry matter (DM) was based on balancing the amount needed to enhance the nutritional properties of the silage while avoiding levels that could negatively affect the silage’s physical and fermentative characteristics, such as density, aerobic stability, and palatability. Inclusion levels above 40% could potentially compromise these qualities and lead to an excess of undesirable components, such as tannins, which can affect digestibility and animal intake.

2.3. Handling and Silage Preparation

The legume used was Leucaena (Leucaena leucocephala), which was harvested from the protein bank of the Forage Crops sector of the Federal University of Maranhão, 60 days after the last harvest. Branches up to 0.80 cm in diameter were collected and then chopped in a mechanized chopper into 2 to 3 cm particles and sun-dehydrated until reaching the hay point. The pineapple plant was collected in the town of São Domingos do Maranhão—MA, fifteen days after fruit harvest (

Table 1. Chemical–bromatological composition of ‘Pérola’ pineapple stubble and Leucaena hay [Leucaena leucocephala (Lam.)] used for silage making.

Chemical Composition (% DM)	Pineapple Stubble	Leucaena Hay
Dry matter (% as fed basis)	19.11	91.17
Crude protein	6.26	17.81
Soluble carbohydrates	11.68	6.54
Neutral detergent fiber	51.40	43.60
Acid detergent fiber	26.90	23.90
Hemicellulose	24.50	19.70
Lignin	4.87	9.74
Ash	15.57	13.82
Buffering capacity *	51.67	22.48

* = e.mg NaOH 100 g·DM⁻¹.

). The material was chopped in a mechanized chopper into 3-mm particles, and part of it was separated for the pre-wilted treatment (exposed to the sun for eight hours), and the rest was immediately homogenized with the proper proportions of Leucaena hay and ensiled (Figure 1).

The material was ensiled in experimental silos made from PVC tubes of 0.10 m diameter and 0.35 m length, using wooden sticks for compaction at a density of 600 kg m⁻³ (as fed). After filling, the silos were sealed with PVC lids (taps) covered with plastic adhesive tape. Silage and sand were separated by two nylon screens, avoiding direct contact and allowing only the drainage of effluent. Before ensiling the pineapple plant, Leucaena hay and the material mixed with hay were sampled for further chemical analysis.

2.4. Silo Opening and Sample Preparation for Analyses

Sixty days after being sealed, the silos were opened and 10 cm from the top and bottom removed, and a silage sample of 500 g was collected for analysis. The samples were packed in paper bags, weighed, and dried in a forced ventilation oven at 65 °C, then they were ground in a knife-type mill with the sieve of 1 mm porosity chemical analysis, while for the degradability trial, a sieve of 2 mm porosity was used.

Another part of the sample was taken to determine pH by a pH meter, following the methodology described by [11] For the determination of buffer capacity, approximately 15 g of macerated sample was used together with 250 mL of distilled water. Using a potentiometer, the material was first titrated to pH 3.0 with 0.1 N HCl to release bicarbonates as carbon dioxide. Then, it was titrated to pH 6.0 with 0.1 N NaOH, recording the volume of NaOH used to change the pH from 4.0 to 6.0 as described by [12]. The dry matter losses in the silages as effluents and gases were quantified by the weight difference using the equations described by Zanine et al. [13]

GL = (WFp − WFo)/(FOMc × DMp) × 1000

where GL = gas losses (% of dry matter), WFp = weight of the filled silo at closing (kg), WFo = weight of the filled silo at opening (kg), FOMc = forage mass at silo closing (kg), and DMc = dry matter content at silo closing (%).

EL = [(WEf − Tb) − (WEp − Tb)]/FOMc × 100

where EL = effluent losses (kg ton⁻¹ fresh matter), WEp = weight of the empty silo + sand at closing (kg), WEf = weight of the empty silo + sand at opening (kg), Tb = weight of the empty silo (kg), and FOMc = forage mass at silo closing (kg).

Dry matter recovery was estimated based on the difference in the dry matter mass before and after ensiling using the equation:

DMR = (FOMo × DMo)/(FOMc × DMc) × 100

where DMR = dry matter recovery rate (%), FOMo = forage mass at silo opening (kg), DMo = forage dry matter content at silo opening (%), FOMc = forage mass at silo closing (kg), and DMc = dry matter content at silo closing (%).

Dry matter—DM (AOAC, 2005, method number 930.15 [14]), ash (AOAC, 2005, method number 942.05), and crude protein—CP (AOAC, 2005, method number 984.13) were determined. Neutral detergent fiber (NDF; INCT-CA method F-002/1) use of alpha-amylase and sodium sulfite, acid detergent fiber (ADF; INCT-CA method F-004/1), cellulose (CEL), hemicellulose (HCEL), and lignin (LIG; INCT-CA method F-005/1 [15])

2.5. In Situ Degradability

In situ dry matter degradability was obtained through incubation in three gyr (Bos indicus) race males rumen-fistulated bovine, with an average body weight of 700 kg at 24 months of age, feeding on a diet based on chopped elephant grass (Pennisetum purpureum) and concentrate consisting of corn, soybean meal and mineral salt, formulated according to NRC for beef cattle [16] (

Table 2. Percentage of ingredients in the diet of fistulated animals.

Ingredients	Proportions (% DM)
Elephant grass	70
Corn	21
Soybean meal	8.4
Mineral mix	0.6

DM = Dry matter.

and

Table 3. Chemical composition of ingredients used in the diet.

Item	Ingredients (% DM)
	Elephant Grass	Corn	Soybean Meal	Mineral Mix
Dry matter	22	88	90	98
Crude protein	8	9.0	45	-
Neutral detergent fiber	65	8.0	10	-
Acid detergente fiber	40	5.0	12	-
Ether extract	3	4	2	-
Total digestible nutrients	55	86	80	-
Calcium	0.3	0.02	0.02	0.30
Phosphorus	0.2	0.30	0.7	10

DM = Dry matter.

). The silage samples were packed in 12 × 8 cm nylon bags of 50 µm porosity [17].

To determine the material disappearance at time zero (soluble fraction) the bags were placed in a water bath for one (1.0) hour at a temperature of 39 °C [18]. After that, the bags were washed together with the samples incubated in the rumen, until the water was clear, then they were placed in a forced air circulation oven for 72 h at a temperature of 55 °C and after pre-drying, they were weighed for dry matter content determination.

In situ degradation parameters of DM (a, b, and c) were estimated through the model proposed by Ørskov and McDonald [19]: PD = A−B.e^−c.t, where PD = actual percentage of nutrient degraded after t hours of incubation in the rumen; A = maximum potential degradation of the material within the nylon bag; B = potentially degradable fraction of material kept in the nylon bag after time zero; c = degradation rate of the remaining fraction in the nylon bag after time zero; and t = incubation time.

DM effective degradability (ED) was estimated considering three rumen passage rates 2, 5, and 8% h⁻¹, through the equation described by [19], ED = a + (b × c/c + k), where: a = soluble fraction; b = potentially degradable fraction, c = degradation rate of fraction b, k = passage rate.

2.6. In Vitro Gas Production Measurements

The semi-automatic in vitro gas production technique [20,21] was used, with a pressure transducer and data logger (Pressure Press Data 800, 147 LANA, CENA-USP, Piracicaba, Brazil). Rumen content was collected via the rumen fistula before the animals were fed. The liquid fraction was obtained through a probe and stored in thermoses previously heated to 39 °C. The solid fraction, on the other hand, was obtained with the aid of tweezers, and stored in plastic bags in Styrofoam boxes. The rumen inoculum was obtained after homogenizing the solid and liquid fractions for 10 s and filtering in four layers of cotton fabric [22]. The inoculum was kept at 39 °C, and saturated with CO₂ to maintain anaerobiosis until use [20].

The substrate was weighed into samples of 0.5 g in pre-weighed bags and inserted in fermentation bottles with a capacity of 160 mL. Then, 75 mL of the inoculum was added to the bottles, which were sealed with rubber stoppers, and kept in an oven at 39 °C for 24 h. The gas pressure was measured 24 h after incubation, and then the total gas produced in the bottles with the samples was subtracted by the gas contained in the bottles with no sample (control). The amount of gas was calculated according to [6]:

V = 7.365 × p, where V is the volume of gas produced (mL) and p is the measured pressure (psi).

After the incubation period (24 h), the bottles were placed in trays with water and ice to cease microbial activity. The samples were removed from the bottles, washed in running water, treated with the neutral detergent solution for one hour, and then washed in hot distilled water and acetone. The bags were placed in the hood for acetone evaporation, then in an oven at 105 °C for 16 h and incinerated in a muffle furnace at 550 °C for four hours. To estimate the methane (CH₄) production, 2.5 mL of gas was collected from each bottle after 24 h of incubation and stored in test tubes of 10 mL capacity.

The collection was performed by a 5 mL syringe. After collection, the internal pressure of the bottles was released with a needle, then shaken and placed back in the oven. Methane was determined using a gas chromatograph (Shimadzu GC2014, Tokyo, Japan), equipped with a Shincarbon ST 100/120 packed micro column (1.5875 mm OD, 1.0 mm ID, 1 m length; Ref 19.809; Restek, Bellefonte, PA, USA). The temperatures of the column, injector, and flame ionization detector were 60, 200, and 240 °C, respectively. The carrier gas was Helium (10 mL/min) and the CH₄ concentration was determined by external calibration obtained with pure CH₄ (White Martins PRAXAIR Gases Industrial Inc. Osasco—SP, Brazil; 99.5% purity). The methane produced during incubation was calculated according to:

CH₄ mL = (total gases, mL + headspace, 85 mL) × CH4%.

The concentrations of propionic and butyric acids were obtained by gas chromatography, following the methodology of Nocek [23]; Palmquist, and Conrad [24], with changes in gas chromatograph (Shimadzu 2014, Tokyo, Japan) equipped with a GP 10% SP-1200/1 H3PO4 80/100 Chromosorb WAW column (Cat. No. 11965; 6′ × 1/8” stainless steel, Supelco, Bellefonte, PA, USA).

2.7. Statistical Analysis

The data were subjected to analysis of influential values and outliers through Residual Studentized analysis, where data presenting residual values >±2.5 were excluded. Then, the data were subjected to normality tests to verify the basic prerogatives of analysis of variance.

Data were explored in two ways, firstly mean comparisons were established by the Dunnett’s test (T1-T2; T1-T3; T1-T4; T1-T5 and T1-T6) through the MIXED procedure of SAS^® statistical program (University Edition, SAS Institute Inc., Cary, NC, USA, CODY 2015) using the following statistical model:

Yik = μ + Si + εik,

where Yik is the dependent variable of the experiment measured on experimental unit “k” of silage “i”; μ is the general constant; Si is the effect of silages “i”; and εik is the effect of random error. Means were obtained through the LSMEANS command at the level of p < 0.05.

The inclusion levels of Leucaena hay were explored by orthogonal contrasts through the REG procedure. Parameters a, b, and c and the in situ degradation curves were determined according to the Gauss–Newton method through PROC NLIN of SAS^® (University Edition, SAS Institute Inc., Cary, NC, USA, CODY 2015).

3. Results

3.1. Fermentation Characteristics and Chemical Composition of Silages

The pH was affected (p < 0.05) by the inclusion levels of Leucaena hay and by the pineapple plant wilting (

Table 4. Dry matter, pH, buffering power, soluble carbohydrates, gas production, effluent production, dry matter recovery, and propionic and butyric acids of ‘Pérola’ pineapple stubble silage with different inclusion levels of Leucaena hay.

Variable	Wilted Pineapple	Levels of Inclusion (%)					SEM	R2
		0	10	20	30	40
pH 1	3.81	3.73 *	3.82	3.90 *	3.97 *	4.06 *	0.02	0.94 ***
Buffering capacity (e.mg NaOH 100 g·DM−1) 2	98.35	102.42	79.59 *	56.16 *	52.23 *	40.44 *	4.56	0.87 ***
Water-soluble carbohydrates (%) 3	3.62	3.91	3.05	2.41	1.81 *	1.12 *	0.23	0.56 ***
Gas losses (%) 4	3.73	3.28	3.67	1.54 *	1.22 *	0.82 *	0.27	0.83 ***
Effluent losses (%) 5	2.97	11.11 *	3.98	2.29	1.67	0.63 *	0.70	0.71 ***
Dry matter recovery. (%) 6	90.73	82.04 *	91.06	95.30 *	96.80 *	97.64 *	1.05	0.93 ***
Propionic acid (ppm) 7	2.45	2.44	2.42 *	2.44	2.43	2.45	0.004	NS
Butyric acid (ppm) 8	2.09	2.06	1.95	1.98	1.86 *	1.95	0.04	NS

Means followed by an asterisk (*) differ from treatment PWPS. (***) indicates high significance at a 0.1% level (p < 0.001), according to the Dunnett test (p < 0.05). ¹: y = 0.008x + 3.736; ²: y = −1.513x + 96.434; ³: y = −0.061x + 3.492; ⁴: y = 0.088x + 3.463; ⁵: y = −0.232x + 8.592; ⁶: y = 0.915x + 82.455; ⁷: y = 2.11; ⁸: y = 1.76. NS = Not significant.

). The levels of 20, 30, and 40% of hay resulted in higher pH values when compared to the wilted material. Adding 10% of hay reduced the buffering power in comparison to the wilted material, which kept reducing as the inclusion levels increased.

Adding 30 and 40% Leucaena hay favored the reduction in soluble carbohydrates (CHOs) content when compared to the wilted material, while the use of 10 and 20% conserved the same amount of CHOs as the silage from wilted material. Adding 20, 30, and 40% Leucaena hay provided lower gas and effluent losses and higher dry matter recovery when compared to the silage from wilted material.

As the inclusion levels of Leucaena hay increased in the pineapple silages, pH values also increased, in a way that for each 1% inclusion of hay, pH increased by 0.008. Increasing the levels of Leucaena hay reduced the silage buffering power (BP) (p < 0.05), which ranged from 102.42 to 40.44 e.mg NaOH 100 g/DM at levels 0 and 40%. There was a 1.5% decrease in BP for each 1% Leucaena hay added to the silage (Table 4).

The content of water-soluble carbohydrates (WSC) decreased as the levels of Leucaena hay increased (p < 0.05), ranging from 3.91% to 1.12% for silages with 0 and 40% Leucaena hay, respectively. A reduction of 0.061% was observed for each 1% of Leucaena hay added. Gas losses (GL) and effluent losses (EL) were reduced by 0.088 and 0.232% for each 1% Leucaena hay was added to the pineapple silage. On the other hand, dry matter recovery increased by 0.915% for each 1% inclusion of Leucaena hay (Table 4).

Levels 20, 30, and 40% inclusion of hay provided greater dry matter contents when compared to the wilted material, and a similar response was observed for the CP content (

Table 5. Chemical composition of ‘Perola’ pineapple stubble silage with different inclusion levels of Leucaena hay.

Variable	Wilted Pineapple	Levels of Inclusion (%)					SEM	R2
		0	10	20	30	40
Dry matter (%) 1	27.00	21.9 *	27.65	31.80 *	36.25 *	43.50 *	1.34	0.97 ***
crude protein (%) 2	6.49	6.24	9.33 *	10.90 *	12.79 *	12.92 *	0.54	0.90 ***
Neutral detergent fiber (%) 3	52.01	54.54	53.30	52.89	52.99	51.29	0.41	0.24 NS
Acid detergent fiber (%) 4	29.70	31.08	30.73	30.12	28.77	28.81	0.33	0.24 NS
Hemicellulose (%) 5	22.31	22.30	22.57	22.77	23.22	22.48	0.17	0.02 NS
Lignin (%) 6	4.63	5.01	7.12 *	8.09 *	9.13 *	9.96 *	0.37	0.93 ***
Ash (%) 7	14.28	14.82	14.78	14.90	14.42	14.32	0.14	0.07 NS

Means followed by an asterisk (*) differ from treatment PWPS, (***) indicates high significance at a 0.1% level (p < 0.001), according to the Dunnett test (p < 0.05). ¹: y = 0.519x + 21.86; ²: y = 0.182x + 6.927; ³: y = 54.36; ⁴: y = 31.20; ⁵: y = 22.48; ⁶: y = 0.119x + 5.478; ⁷: y = 14.92. NS = Not significant.

). There was no difference (p > 0.05) in the contents of NDF, ADF, hemicellulose, and ash between silage of wilted pineapple plant or added with Leucaena hay (Table 5).

Among the inclusion levels of Leucaena hay, a positive linear response (p < 0.05) was observed for dry matter and CP contents, which increased by 0.52 and 0.182%, respectively, for each 1% Leucaena hay added in the pineapple plant silage (Table 3). There was no effect (p > 0.05) of the inclusion levels on the contents of NDF (Y = 54.36), ADF (31.20), hemicellulose (Y = 22.48), and ash (14.92). A positive linear effect (p < 0.05) was found for lignin content, which showed an increase of 0.12% for each 1% of Leucaena hay inclusion (

Table 6. Rumen degradability parameters (a, b, and c), potential degradability (A), and effective degradability (ED) of dry matter at passage rates 2, 5, and 8%/hour of pineapple stubble silage with different levels of Leucaena hay.

Treatments	a (%)	b (%)	c (%/h)	A	R2	Effective Degradability (%)
						2%/h	5%/h	8%/h
Wilted pineapple	35.72	43.75	2.46	79.47	92.49	59.85	50.15	46.01
0	30.46	42.97	2.44	73.43	89.73	54.07	44.55	40.50
10	31.65	53.59	1.43	85.24	91.84	53.99	43.57	39.78
20	29.89	36.13	2.87	66.02	92.01	51.18	43.07	39.43
30	29.22	35.42	2.46	64.64	75.10	48.76	40.90	37.55
40	29.51	34.09	0.03	63.60	92.68	48.45	40.87	37.63

a = water soluble fraction; b = water insoluble fraction, but potentially degradable; c = degradability rate of fraction b; R2 = coefficient of determination, A = potential degradability.

).

3.2. In Situ Degradability

The values of the fraction representing the portion readily available to the rumen microorganisms (fraction a) were like each other, except for the silage of the wilted pineapple plant, which showed a higher fraction (Table 6). The lowest degradation rate (c) was observed for the silage with the addition of 40% hay. Potential degradation (A) decreased as the inclusion of Leucaena hay increased (Table 6). A similar response was observed for the effective degradability (ED), since as the passage rate increased, degradation also decreased (Table 6).

3.3. In Vitro Gas Production

Wilting the pineapple plant did not alter the in vitro gas production (mL g⁻¹ of DM; OM), in vitro digestibility of organic matter, partition factor (PF), and methane production (CH₄;

Table 7. In vitro gas production parameters of pineapple stubble silage with different levels of Leucaena hay.

Parameters	Wilted Pineapple	Levels of Inclusion (%)					SEM	Equation	p Value
		0	10	20	30	40
Gas production
mL g−1 DM	133.9	134.4	130.8	133.7	129.1	132.5	1.566	y = 133.15 − 0.05x	0.92
mL g−1 OMD	62.7	61.9	60.2	62.7	62.0	61.8	1.048	y = 61.39 − 0.01x	0.98
Nutrient degradability (g kg−1)
OMD	480.3	472.8	460	484.4	479.4	476.4	2.743	y = 469.3 − 0.26x	0.14
PF	1.5	1.5	1.4	1.6	1.5	1.5	0.028	y = 1.47	0.47
Methane production (CH4)
mL g−1 DMD	4.3	4.7	5.0	6.3	5.7	6.6	0.238	y = 4.794 + 0.04x *	0.12
mL g−1 OMD	2.0	2.2	2.3	3.1	2.7	3.1	0.120	y = 2.268 + 0.02x *	0.10

DM = Dry matter, OMD = Organic matter degradability; PF = Partition factor; SEM = standard error of the mean; * = p < 0.05.

) when compared to the silages added with Leucaena hay. Among the inclusion levels, a reduction in vitro gas production based on dry matter and organic matter of 0.05 and 0.01 was observed for each 1% inclusion of Leucaena hay, respectively (Table 7). Organic matter digestibility decreased by 0.26 g kg⁻¹ for each 1 kg of Leucaena hay inclusion. The partition factor was not affected by the hay levels, with values ranging from 1.4 to 1.6 (Table 5). CH₄ production increased by 0.04 for each 1% inclusion of Leucaena hay (Table 7).

4. Discussion

Silage from pineapple crop stubble is an excellent forage option, being an additional alternative for the farmer, besides reducing the negative impacts of environmental pollution, despite the high buffering power of pineapple stubble.

As for the Leucaena hay (Table 1), it did not prevent the pH drop of the silages, whose values ranged from 3.73 to 4.02, staying within the optimal range for silages which is 3.8 to 4.2 which suggests that the fermentation process was adequate [6]. As the levels of Leucaena hay increased, the buffering power decreased, which is linked to a dilution effect, since the buffering power of the legume is lower than that of the pineapple plant. It is worth noting that the higher buffering power of the pineapple plant is due to the greater amount of K, Ca, and Mg, which are on average 26 g Kg⁻¹, 3.5 g Kg⁻¹, and 4.5 g Kg⁻¹, respectively [25] (Table 1). The same response was seen for the contents of soluble carbohydrates, which reduced as the inclusion levels of LH increased. Therefore, it was expected that the soluble carbohydrate content would increase. Legumes have low soluble carbohydrate content. The studies [2,26] evaluated pineapple by-product silage with a legume (Canavalia ensiformis) as an additive and observed a similar response to that recorded in the present study for the soluble carbohydrates content, and the reduction was attributed to a dilution effect.

Gas and effluent production decreased as the inclusion of LH increased, while the contrary was observed for dry matter recovery. These results were expected because the DM content of these silages increased linearly with the increasing inclusion of LH, so it can be inferred that the LH possibly reduced secondary fermentations that positively affected DM loss, and reduced gas production and consequently effluent loss, corroborating our hypothesis that the factors that hinder the ensiling process of the pineapple plant alone, are minimized with the addition of LH. Although there was no significant effect for butyric acid contents, the values obtained were high, being above 1%. According to Borreani et al. [21], butyric acid should not be found in properly fermented silages, as it indicates the activity of clostridia, which in addition to increasing dry matter losses, can cause proteolysis. Propionic acid contents were also not affected by the inclusion of LH; however, the values found were considered high, since according to Kung Jr. et al. [27] those values should be lower than 0.3%, which may indicate undesired fermentation due to the possible action of harmful microorganisms, commonly observed in silages with a predominance of clostridial fermentation.

Regarding the chemical composition, although wilting increased the content of DM, this value (27%) is considered below the ideal range to achieve good-quality silage; however, the inclusion of more than 20% hay provided a DM content greater than 30%, this being within the ideal range. This result is proven by the indicators of fermentation losses that were reduced since the hay provided greater moisture absorption capacity for wet pineapple plant silage [21]. The increase in dry matter content with the addition of hay supports the hypothesis that Leucaena hay is a good alternative as a moisture absorbent [9].

CP values of wilted silage with no added LH are below 8% and may limit the development of rumen microorganisms and consequently reduce fiber degradation [15,27]. The wilted and wet ensiled pineapple plant did not show divergences in CP content, thus the hypothesis of intense proteolytic activity can be ruled out. In addition, when the silos were opened, there was no characteristic odor of excess ammonia. It is worth mentioning that this crop presents low crude protein content, which is very limiting for animal performance, besides being a nutrient of high cost.

It should be highlighted that the inclusion of LH was efficient in increasing the silage CP content, starting with the inclusion of 10% from which a CP content higher than 9% was recorded, and the greatest value was obtained with the inclusion of 40% hay (12.92% CP). This result is because legumes have a high protein content. The increase in CP content is another point that corroborates the hypothesis that proteolytic activity was reduced [28]. Gonçalves et al. [29] observed similar behavior for pineapple by-product silages with Canavalia ensiformis leaves as an additive.

When adopting the inclusion of a particular feedstuff as an additive, it must not lead to nutritional losses. The inclusion of increasing levels of LH did not alter the contents of NDF, ADF, and hemicellulose in the silages. On the other hand, it is known that legumes have high phenolic compound content in their cell wall, 50% higher than that found in the pineapple plant, which resulted in increased lignin in the silages. Costa et al. [8] observed significant increases in the lignin content of sorghum silages as the inclusion levels of LH increased. This response resulted in lower dry matter degradation because large amounts of lignin can be a limiting factor in the digestion of polysaccharides [30].

Another factor that may have corroborated the reduction in the soluble fraction is the low content of soluble carbohydrates in the legume, which by increasing its inclusion also reduced the soluble carbohydrates in the silages. This response was reflected in all parameters of in situ dry matter degradation. The reduction in effective degradation as passage times increased is due to the shorter contact time of microorganisms with the substrate.

Following the same behavior, gas production was reduced due to the reduction in soluble carbohydrate concentration, considering that this variable is an indirect measure of carbohydrate degradation, and similar results were observed [9]. Gas production tends to follow the same behavior of residual soluble carbohydrates in silages because of the lower contents that are found in Leucaena. In addition, there may be a negative correlation of CP with gas production because it is associated with altered fatty acid molar ratios and high in vitro ammonia accumulation [31]. This may also be due to the increase in the lignin content of silages, to prevent the action of microorganisms to degrade the feed, minimizing gas production resulting from the higher proportion of indigestible fiber [2].

The higher amount of fiber, mainly of low quality, causes higher acetate production, resulting in increased methane production [32]. This response may explain the result observed in the present study. It is worth noting that methane gas, besides causing environmental damage, is responsible for increasing energy expenditure, which is around 2 to 15% of all gross energy ingested by the animal [9]. Therefore, studies with information on alternative feeds must be complemented with methane evaluations.

The use of pineapple crop stubble with the addition of LH in the form of silage in pineapple-producing regions is a way to reduce the negative environmental impacts, but more studies are needed with the use of complementary additives. The increase in DM recovery from ensiled plants and reduction in effluent losses already bring great environmental benefits, and consequently, there is an increase in feed production for animals with the use of cleaner technologies.

5. Conclusions

Our findings indicate that the inclusion of 20% Leucaena hay in pineapple plant silage is the most effective based on the evaluations conducted. The process of wilting the pineapple plant before ensiling is recommended, particularly when it is not feasible to use a moisture-absorbing additive. This approach not only provides a sustainable solution for the disposal of pineapple crop residues by converting them into silage and, subsequently, into animal protein, but it also helps to reduce environmental impacts.

To further substantiate our results, it would be highly beneficial to explore microbial composition analysis in future studies, which could offer a more robust scientific foundation. Additionally, investigating different Leucaena cultivars and assessing animal performance in response to this silage would be crucial steps to validate and expand the practical applications of this research.