Quality of Silage with Different Mixtures of Melon Biomass with Urea as an Additive

Abstract

The objective of this study was to evaluate the silage produced with different mixtures of melon biomass with urea used as an additive. It adopted a completely randomized design in a 5 × 2 factorial scheme with five replications. The first factor was made up of five mixtures on an as-fed basis (AF) of the melon, including plant (branch and leaf) and fruit (culled melon, after harvest), where the amount of fruit varied as follows: 0% fruit, 5% fruit, 10% fruit, 20% fruit, and 100% fruit. The second factor was represented by the use of urea in the silage as follows: 0% and 1.5% urea AF. The highest silage dry matter (DM) content (200 g/kg DM) was observed in the silage with 20% fruit. Regarding the crude protein, the highest content (69.8 g/kg DM) was found in the silage with the addition of urea. The highest loss through the gas (GAS) was observed in the silages with 0% and 100% fruit (0.6 and 1.13%), with no addition of urea. The silages made with melon biomass and the addition of 20% and 100% fruit showed differences regarding the fermentative pattern, chemical composition, and aerobic stability, thus being the most indicated mixtures for silage making.

1. Introduction

Silage making is an efficient alternative to conserve feed for livestock, especially during periods of low availability of forage in pastures, guaranteeing roughage feed to the animals throughout the year [1]. This technique consists of the preservation of forage or wet grains, through anaerobic fermentation, where lactic acid bacteria metabolize soluble sugars into organic acids, which are mainly lactic acid, acidifying and preserving the mass [2].

Commercial processing of tropical fruits in northeastern Brazil produces a large volume of crop waste, and this material is often burned and dumped in landfills or has no appropriate destination, becoming harmful to the environment [3], or to the subsequent crop itself if left in the field [4]. According to [5], the biomass of fruits, such as banana, pineapple, papaya, and melon, is rich in nutrients such as vitamins, minerals, and fiber, and can be feed resources for ruminants, since besides reducing waste disposal, it could contribute to cost reduction, favoring the sustainability of the production system, [6] and also reducing the damage to the environment.

The melon plant (Cucumis melo L.) is one of the most important representatives of the Cucurbitaceae family, presenting high variability within different cultivars, in addition to excellent adaptation to the predominant soil and climate conditions in the northeast region [7]. The melon crop produces a large amount of biomass that has no use (melon plant: branch and leaves; and fruit: culled) after harvesting the commercial fruit.

A challenge regarding the production of silage with the remaining biomass of melon production is to offer a good quality feed to the animals, since the intrinsic factors of the plant, such as moisture content and proportion of soluble carbohydrates, cause high fermentability [8] increasing nutrient losses. This makes it necessary to use additives to improve these characteristics, thus enabling beneficial fermentation in the anaerobic phase (closed silo) and aerobic phase (opened silo) in the conservation and offer of silage.

Additives are added during ensiling and have as main objectives to improve beneficial fermentations and/or inhibit undesirable fermentations, and can also improve the nutritional quality of the ensiled material. Urea is among the most used additives in silages, presenting its beneficial effect associated with increased stability of the silage by the action on microorganisms and control of undesirable fermentations, as well as in controlling proteolysis or aerobic growth [9], which promotes lower fermentative losses. As reported by [9], in the presence of moisture and through the action of urease from plants and microorganisms, urea is hydrolyzed, producing two molecules of ammonia and one of carbon dioxide, changing the fermentative profile. The use of urea as an additive in silages is already well established, but its use in melon biomass has not yet been evaluated.

Thus, based on the fact that the recovery and valorization of agro-industrial residues are currently identified as key factors for the development of circular economy models [10] and looking for better utilization of the melon biomass after harvesting the commercial fruit, adding urea, it was hypothesized that this can improve the fermentative quality of this silage. Therefore, the objective of this study was to evaluate the silages produced with different mixtures of melon biomass and urea as an additive.

2. Materials and Methods

2.1. Statistical Design and Treatments

A completely randomized design was adopted in a factorial scheme (5 × 2) with five replications. The first factor consisted of five mixtures on as fed basis (AF) of melon biomass containing plant (branch and leaf) and fruit (culled melons after harvest) as follows: 0% fruit, 5% fruit, 10% fruit, 20% fruit, and 100% fruit. The second factor consisted of the use of urea, as follows: 0% and 1.5% of urea AF of the ensiled biomass. A total of 1.5% urea was used, based on the recommendations by Vilela et al. [11], where the authors observed higher quality in the silage produced with this amount of urea.

2.2. Biomass Collection from the Melon Plant

The melon biomass was collected after the harvest of the marketable fruit in a rural property located in Canto do Buriti, Piauí, Brazil, at latitude 08°06′41″ South, longitude 42°56′48″ West, and altitude of 258 m. The region has climate classification AW, with a drier season in winter according to Köppen’s classification of 1936, described by Alvares et al. [12], presenting a minimum temperature of 21 °C and maximum of 38 °C, with an average annual rainfall precipitation of 600 mm.

The collection of the material from the field occurred in September 2018 (the melon production season in the region occurs from July to October). The biomass harvest was performed 85 days after planting and after three harvests of the marketable melon. The melon biomass used in the study was composed of the plant (branch and leaf) and fruit (culled melon, after three harvests of the commercial fruit). The chemical composition of the material before ensiling is presented in

Table 1. Chemical composition of melon biomass before ensiling.

Analyses	Plant	Fruit	Seed
Dry matter (g/kg)	220.9	72.1	944.2
Ash (g/kg DM)	81.4	68.6	54.0
Ether extract (g/kg DM)	46.3	19.3	80.3
Crude protein (g/kg DM)	37.5	25.7	43.4
NDF(g/kg DM)	436.4	579.8	367.4
ADF(g/kg DM)	161.0	310.3	-
pH	5.5	6.7	-
N-NH(%)	0.55	0.98	-
WSC(g/kg)	206.0	257.8	-
BC	29.13	4.96	-

DM, dry matter; NDF, neutral detergent fiber; ADF, acid detergent fiber; WSC, water-soluble carbohydrate; BC, buffer capacity (e.mg NaOH/100 g/DM).

. The melon biomass used in the production of silage was collected at the farm of the company Itaueira Agropecuária S.A, melon variety was the ‘Gold Mine’, with a creeping growth habit with lateral branches, irrigated under dripping and cultivated in soil covered by mulching (in polyethylene of 25 microns and 1.20 m wide), fertilization and phytosanitary control were carried out according to the needs and recommendations for the crop, according to Silva et al. [13].

The evaluations were performed in the biomass collection where the green forage mass was measured by the PVC pipe square method, which consists of using PVC rectangles as mini silos: 0.5 m × 0.5 m (0.25 m²), and the weight obtained was converted to t/ha⁻¹; after the collection, the material was fractioned according to the treatments, and taken to an oven to determine the dry matter (DM). MS values were 137.8, 130.0, 122.0, 111.0, and 100.0 g/kg for treatments 0, 5, 10, 20, and 100% fruit, respectively. The calculation of the silage DM yield (DMY) considered the DM recovery (DMR). The silage yield used the following equation.

Silage yield = (DMY × DMR)/100

(1)

where: Silage yield (t/ha DM); DMY (T/ha); DMR (%).

2.3. Silage Making

After harvest, the melon biomass was ensiled in experimental silos (PVC tubes) with a capacity for 4 kg of silage, conditioned at a storage density of 500 kg/m³, with a Bunsen-type valve adapted to the lid, to allow the escape of fermentation gases. The urea was added homogeneously to the melon biomass in proportions corresponding to the treatments.

To characterize the quality of the silage, it was analyzed 90 days after ensiling through the following parameters: losses (gases and effluent) and DMR, BC and WSC content, pH and ammonia nitrogen (N-NH₃), volatile fatty acids, microbiological dynamics, chemical composition, and aerobic stability. The analyses were performed at the Animal Nutrition Laboratory and Microbiology Laboratory of the Professor Cinobelina Elvas campus (CPCE) of the Federal University of Piauí (UFPI), in Bom Jesus, Piauí, Brazil.

2.4. Silage Chemical Composition

In order to determine the chemical composition of the biomass of the melon plant and silage, the contents of DM (method No. 934.01), Ash (method No. 942.05), crude protein (CP, method No. 920.39), and an ether extract (EE, method No. 920.29) were analyzed according to AOAC [14]. Analyses to determine neutral detergent fiber (NDF) and acid detergent fiber (ADF) were performed according to Van Soest et al. [15].

2.5. Amassment of Losses and Recovery of the Silage Dry Matter

The experimental silos were weighed at closing and opening, to determine DM losses in the form of gases and effluents, and DMR according to the equations described by Zanine et al. [16].

The loss through gases was obtained by the difference in weight of the dry forage mass.

G = (FWc − FWo) ÷ (FMc × DMc) × 10,000

(2)

where: G: losses through gas (g/kg DM); FWc: full silo weight at closure (kg); FWo: full silo weight at opening (kg); FMc: forage mass at closure (kg); DMc: forage dry matter content at closure (g/kg).

The losses through effluent were calculated by the equation below, based on the weight difference of the sand related to the fresh forage mass at closure.

E = [(EWo − St) − (EWc − St)] ÷ FMc × 100

(3)

where: E: effluent yield (kg/ton of silage); EWo: empty silo weight + sand weight at opening (kg); EWc: empty silo weight + sand weight at closure (kg); St: silo tare; FMc: forage mass at closure (kg).

The following equation was used to estimate the DM recovery (DMR):

DMR (%) = [(FMo × DMo) ÷ (FMc × DMc)] × 100

(4)

where: DMR (%): DM recovery (DMR in g/kg); FMo: Silage forage mass at opening (kg); DMo: Silage DM content at opening (g/kg); FMc: Fresh forage mass at closure (kg); DMc: Forage DM content at closure (g/kg).

2.6. Buffer Capacity and Soluble Carbohydrates

The buffer capacity (BC) was determined according to the methodology of Mizubuti et al. [17], by using 10 to 20 g of macerated silage with 250 mL of distilled water. The macerate was titrated to pH 3.0 with 0.1 N HCl to release bicarbonates, such as carbon dioxide. It was then titrated to pH 6.0 with 0.1 N NaOH, and the volume of NaOH spent to change the pH from 4.0 to 6.0 was recorded. The BC was calculated by the equation:

$$\mathrm{BC}=\frac{0.1\times (\mathrm{Va}-\mathrm{Vi})}{\mathrm{SW}}\times 100$$

(5)

where: BC = buffer capacity in e.mg NaOH/100 g DM; 0.1 = Normality of NaOH; Va = volume of NaOH spent to change the sample pH from 4.0 to 6.0; Vi = volume of NaOH spent to change the blank pH from 4.0 to 6.0; SW = dry sample weight = [ (sample weight × DM) ÷100].

The concentration of water-soluble carbohydrates (WSC)), was obtained by the concentrated sulfuric acid method, described by Dubois et al. [18], with adaptations by Corsato et al. [19]. The organic compounds were extracted in the ethanol solution. The proportion of WSC, in g/100 mL, was calculated based on the solution and subsequently adjusted based on the DM of each sample used.

2.7. Silage Fermentation Indicators

The pH values and ammonia nitrogen concentration (N-NH₃) of the silages were determined at the opening of the silo after 90 days. The pH determination in distilled water was performed in duplicate by collecting 25 g of sample of the ensiled material of each treatment and adding 100 mL of water. After 1 h, the reading was performed according to the methodology described by Bolsen et al. [20], with a pH meter.

For the determination of N-NH₃ of the samples, the methodology described by [20] was used, where in 25 g of fresh sample was added 200 mL of H2SO4 solution at 0.2 N. After 48 h of refrigerated resting, the mixture was filtered in filter paper, for estimation considering the DM content of the silage, according to Detman et al. [21].

2.8. Silage Microbial Analyses

The microbiological evaluation was performed according to the methodology described by González et al. [22]. The population of lactic acid bacteria (LAB), enterobacteria, molds, and yeasts were evaluated. A 25 g of fresh silage sample was collected, to which 90 mL of distilled water was added to and disintegrated in a blender for approximately 1 min. Then, 1 mL of the mixture was taken and pipetted with the appropriate dilution (10⁻¹ to 10⁻⁹). The plating was performed in duplicate for each culture medium. Populations were determined by the selective technique of cultures in an anaerobic medium, and the following were used:

• Rogosa Agar medium for counting LAB, after incubation for 48 h in an oven at 37 °C;
• The PDA (Potato Dextrose Agar) medium acidified with 1% tartaric acid to count molds and yeasts after 48 h of incubation at room temperature;
• Brilliant Green Bile Agar medium for enterobacteria count, after incubation for 24 h at 35 °C.

Petri dishes that presented between 30 and 300 colony-forming units (CFU) were considered susceptible to counting; the averages of the plates of the selected dilution were considered. The differentiation between yeasts and molds was made by the physical structure of the colonies, which was visually perceptible because yeasts are unicellular and molds are multicellular.

2.9. Organic Acids Determination

In order to determine the concentrations of organic acids (lactic, acetic, propionic, and butyric), 10 g of each silage was weighed in triplicate, then 90 mL of distilled water was added, homogenized in a blender for 1 min, and then, filtered in a syringe filter with pore 0.22 µm. Subsequently, a 10-mL sample was taken from the filtrate, which was placed in tubes to be centrifuged and 1.0 mL of metaphosphoric acid and two drops of the 50% sulfuric acid were added and the solution formed was centrifuged for 15 min at 13,000× g. After this process, the supernatant was collected in Eppendorf tubes, and frozen for the determination of organic acid concentrations using the high-performance liquid chromatography technique (HPLC; SHIMADZU, SPD-10A VP) [23]. The HPLC apparatus was equipped with an UltraViolet Detector using an Aminex HPX-87H column (BIO-RAD, Santa Clara, CA, USA) with the mobile phase containing 0.005 M sulfuric acid, a flow rate of 0.6 mL/min, and wavelength of 210 nm. The analyses were performed in the Animal Nutrition and Soil Laboratory of UFPI.

2.10. Silage Aerobic Stability

At the opening of the silo, the silage mass was homogenized and 2.5kg of the material was returned to the original silos for evaluation of stability in aerobiosis. The silage samples were exposed to air at a controlled room temperature (25 °C), similar to the evaluations performed by Johnson et al. [24]. The internal temperature of the silage was measured by an INCOTERM^® skewer type digital thermometer, inserting the stainless steel tip in the center of the material, while the surface temperature was measured by a BENETECH^® infrared laser sighted digital thermometer (−50 °C to 420 °C).

The temperature of the material was measured every 4 hours, during 96 h of exposure to air. The parameter for evaluating aerobic stability was the 2 °C increase in temperature of the silage in relation to the environment, after opening the silos [25]. To evaluate the pH in the aerobic stability of silage, 100 g of silage was collected from each silo at 1, 12, 24, 48, and 96 h of exposure to air.

2.11. Statistical Analysis

The data were subjected to analysis of variance with a significance of p < 0.05. The means were analyzed through Tukey’s test and compared with a significance of p < 0.05. The data were analyzed in the software SISVAR version 5.0.

The statistical model adopted was:

Yijk = μ + τi + γj + (τγ)ij + εijk

(6)

where: Yijk = observation referring to the different mixtures of melon biomass i with additives j; μ = general constant; τi = effect of the different mixtures of melon biomass with additives I, where i = 1, 2, 3, 4, and 5 (0% fruit, 5% fruit, 10% fruit, 20% fruit, and 100% fruit); γj = is the effect of additives 1 and 2 (0% and 1.5% urea); (τγ) ij = effect of interaction between the different mixtures of melon biomass i with additives j; εijk = random error associated to each mixture of melon biomass with additives.

3. Results

3.1. Silage Yield

It was found that there was an effect (p < 0.05) of the different biomass mixtures (increased inclusion of melon in the silages, 0, 5, 10, 20, and 100% AF) on silage yield and addition of urea (0% and 1.5% urea AF) (

Table 2. Yield of silages with different mixtures of melon biomass and addition of urea.

UR	Percentage of Fruit (PF)					Mean	SEM	p-Value
	0%	5%	10%	20%	100%			UR	PF	UR × PF
	Silage yield (t/ha DM)
0%	3.95	6.71	5.04	5.12	1.78	4.58	0.4	0.52	<0.01	0.61
1.5%	3.76	5.96	4.79	7.03	3.06	4.92
Mean	3.85 b	6.33 a	4.91 a	6.07 a	2.42 b

UR, Urea; PF, Percentage of fruit; DM, dry matter; SEM, standard error of the mean. Means followed by different lowercase in the rows are statistically different according to Tukey’s test p < 0.05.

).

The highest means of silage yield from the melon harvest biomass were seen in the silages with 20%, 5%, and 10% fruit, which were 6.33, 6.07, and 4.91 (t/ha DM).

3.2. Chemical Composition

There was a significant effect of the interaction (p < 0.01) between the different biomass mixtures (increased inclusion of melon in the silage, 0, 5, 10, 20, and 100% AF) and addition of urea (0% and 1.5% urea AF) on the content of soluble carbohydrates (

Table 3. Chemical composition and pH of silages with different mixtures of melon biomass and addition of urea.

UR	Percentage of Fruit (PF)					Mean	SEM	p-Value
	0%	5%	10%	20%	100%			UR	PF	UR × PF
	Dry matter (g/kg)
0%	148.7	158.4	124.7	197.4	88.5	143.5	0.7	0.7	<0.01	0.07
1.5%	158.3	143.2	102.4	202.7	171.4	163.6
Mean	153.5 ab	150.8 ab	149.6 b	200.0 a	129.9 b
	Crude Protein (g/kg DM)
0%	54.3	63.3	58.6	60.8	58.9	59.2 b	0.13	<0.01	0.44	0.33
1.5%	67.3	66.1	73.8	70.4	71.6	69.8 a
Mean	60.80	64.7	66.2	65.6	65.2
	Ash (g/kg DM)
0%	66.3	51.7	55.7	57.2	71.9	60.6 b	0.2	<0.01	0.06	0.25
1.5%	64.3	70.5	71.0	65.9	75.9	69.5 a
Mean	60.7	61.1	63.4	66.1	73.9
	Ether Extract (g/kg DM)
0%	72.5	74.1	81.0	69.5	71.1	73.6 a	0.2	<0.01	0.54	0.38
1.5%	61.3	55.6	64.2	64.5	69.2	62.9 b
Mean	66.9	64.8	72.6	67.0	72.6
	Neutral Detergent Fiber (g/kg DM)
0%	636.7	559.2	667.1	613.4	647.1	624.7	1.6	0.38	0.60	0.08
1.5%	557.7	677.8	618.6	605.7	562.6	604.5
Mean	585.5	618.5	642.8	621.2	604.9
	Acid Detergent Fiber (g/kg DM)
0%	271.3	229.8	327.1	228.2	390.1	289.3	1.58	0.84	<0.01	0.66
1.5%	233.7	268.5	290.1	247.6	429.0	293.8
Mean	252.5 b	249.2 b	308.6 ab	237.9 b	409.5 a
	Water-soluble Carbohydrates (g/kg DM)
0%	208.7 Ab	169.6 Acd	159.0 Ad	200.4 Abc	320.5 Aa	211.6	0.06	<0.01	<0.01	<0.01
1.5%	113.3 Bb	132.7 Bab	121.9 Bb	121.0 Bb	160.6 Ba	129.9
Mean	184.5	151.1	140.5	160.7	217.0
	pH
0%	7.9 Aa	7.7 Ba	7.7 Aa	7.4 Aa	4.3 Bb	7.04	0.8	<0.01	<0.01	<0.01
1.5%	8.1 Aa	8.5 Aa	8.1 Aab	7.3 Ab	5.9 Ac	7.63
Mean	8.0	8.1	7.9	7.4	5.1

UR, Urea; PF, Percentage of fruit; DM, dry matter; SEM, Standard error of the mean. Means followed by different uppercase letters in the column and lowercase in the rows are statistically different according to Tukey’s test p < 0.05.

). In addition, the effect of the different mixtures was observed on the contents of DM (p < 0.01) and ADF (p < 0.01). For CP, Ash, and EE, there was an effect (p < 0.05) of the use of urea. There was no significant effect (p > 0.05) on the NDF content.

The highest mean DM contents of the silages from the melon harvest biomass were found in the silages with 20%, 0%, and 5% fruit, which were 200, 153.5, and 150.8 g/kg. Regarding the CP, the highest mean (69.8 g/kg DM) was found in silages with the addition of urea.

For ash, the highest mean (69.5 g/kg DM) was found in the silage with the addition of urea. On the other hand, the highest mean EE content was found in the silage without the addition of urea, which was 73.6 g/kg DM.

The silage with 100% melon fruit without the addition of urea, presented a pH value of 4.3. The highest values were observed in silages with 0, 5, 10, and 20% fruit with addition of urea, which were 8.1, 8.5, and 8.1, respectively. The ammonia nitrogen (N-NH₃) contents of the different silages presented higher values in silages with 0, 5, and 10% fruit with the addition of urea (1.4, 1.9, and 1.6% TN, respectively).

3.3. Fermentation Losses

There was the effect on the interaction (p > 0.05) between the different biomass mixtures and the addition of urea on gases, pH, and ammonia nitrogen, but no effect was observed on DMR (

Table 4. Quantification of losses of silages with different mixtures of melon biomass and addition of urea.

UR	Percentage of Fruit (PF)					Mean	SEM	p-Value
	0%	5%	10%	20%	100%			UR	PF	UR × PF
Effluents (kg/t AF)
0%	41.8	39.3	46.9	66.2	54.0	49.6 b	2.2	<0.01	<0.01	0.34
1.5%	56.9	59.2	60.0	66.0	70.2	62.5 a
Mean	49.1 b	49.2 b	53.4 ab	66.1 a	62.1 ab
Gasses (% DM)
0%	0.6 Ab	0.2 Ac	0.3 Abc	0.1 Ac	1.13 Aa	0.5	0.03	<0.01	<0.01	<0.01
1.5%	0.4 Ba	0.2 Ab	0.1 Ab	0.2 Ab	0.1 Bb	0.2
Mean	0.5	0.2	0.2	0.2	0.6
DM Recovery (%)
0%	78.6	85.5	74.6	77.1	81.6	79.5	3.15	0.61	0.34	0.87
1.5%	78.8	82.9	64.2	81.6	78.7	77.2
Mean	78.7	84.2	69.4	79.3	80.1
N-NH3 (%TN)
0%	0.6 Ba	0.5 Ba	0.4 Ba	0.2 Ab	0.3 Ab	0.4	0.07	<0.01	<0.01	<0.01
1.5%	1.4 Aa	1.9 Aa	1.6 Aa	1.3 Ab	1.3 Ab	1.2
Mean	1.0	1.2	1.0	0.3	0.4

UR, Urea; PF, Percentage of fruit; DM, dry matter; SEM, Standard error of the mean. N-NH₃ (% TN) Ammonia nitrogen in relation to the percentage of total nitrogen. Means followed by different uppercase letters in the column and lowercase in the rows are statistically different according to Tukey’s test p < 0.05.

). In addition, an effect (p < 0.01) was found in the amount of fruit (increasing inclusion of melon in the silages, 0, 5, 10, 20, and 100% AF) and the addition of urea (0% and 1.5% urea AF) on effluent losses in the silages produced with the different mixtures of melon biomass.

The highest mean losses through effluent were observed in silages with 20%, 100%, and 10% fruit (66.1, 62.1, and 53.4 kg/t AF). Whereas in silages with the addition of urea, the highest mean value was 62.5 kg/t AF. As for losses through the highest values were seen in silages with 0% and 100% fruit (0.6 and 1.13%) without the addition of urea. In turn, the DM recovery ranged from 74.6 to 81.6, indicating that the use of melon biomass in different mixtures and the addition of urea caused DM losses > 26%.

3.4. Microbiology

In the population analysis of microorganisms in the produced silages (

Table 5. Population of microorganisms of silages with different mixtures of melon biomass and addition of urea.

UR	Percentage of Fruit (PF)					Mean	SEM	p-Value
	0%	5%	10%	20%	100%			UR	PF	UR × PF
	Lactic acid bacteria (CFU/g)
0%	6.9 Ab	6.9 Ab	7.0 Ab	6.9 Bb	7.9 Aa	7.1	0.07	0.94	<0.01	<0.01
1.5%	7.2 Aab	6.6 Ab	7.3 Aa	7.5 Aa	7.5 Bab	7.2
Mean	7.0	6.8	7.2	7.2	7.5
	Yeasts (CFU/g)
0%	0.0 Ab	0.0 Ab	0.0 Ab	3.2 Aa	5.6 Aa	4.2	0.02	0.09	0.05	<0.01
1.5%	0.0 Bb	0.0 Bb	0.0 Bb	2.3 Aa	3.8 Ba	5.8
Mean	0.0	0.0	0.0	2.3	4.35
	Molds (CFU/g)
0%	5.0	5.8	5.2	5.5	5.2	5.3	0.02	0.98	0.13	0.73
1.5%	4.7	5.6	4.8	5.8	5.7	5.3
Mean	4.8	5.7	5.0	5.6	5.5
	Enterobacteria (CFU/g)
0%	3.5	3.7	4.1	3.8	4.1	3.8	0.08	0.39	0.10	0.78
1.5%	3.6	3.4	3.9	3.9	3.4	3.6
Mean	3.6	3.6	4.0	3.8	3.7

UR, Urea; PF, Percentage of fruit; DM, dry matter; SEM, Standard error of the mean. Means followed by different uppercase letters in the column and lowercase in the rows are statistically different according to Tukey’s test p < 0.05.

), there was an effect of the interaction (p < 0.05) between the different mixtures of melon biomass and the addition of urea on the population of LAB and yeasts.

The silages with 20% fruit with the addition of urea and 100% fruit without urea showed the largest LAB populations, 7.5 and 7.9 log CFU/g, respectively. While the largest yeasts populations were observed in the silages with 100% fruit without urea (5.6 log CFU/g).

The mean values of mold populations ranged from 4.8 to 5.5 log CFU/g, while enterobacteria ranged from 3.0 to 4.0 log CFU/g (Table 5), with no significant effect (p > 0.05).

3.5. Volatile Fatty Acids

Regarding the volatile fatty acids content, there was an effect of the interaction (p < 0.05) between the different biomass mixtures (increased inclusion of melon in the silages, 0, 5, 10, 20, and 100% AF) and the addition of urea (0% and 1.5% urea AF) on the contents of lactic and acetic acid, and an effect (p < 0.01) of the amount of fruit on propionic and butyric acid (

Table 6. Volatile fatty acids (VFA) contents of silages with different mixtures of melon biomass and addition of urea.

UR	Percentage of Fruit (PF)					Mean	SEM	p-Value
	0%	5%	10%	20%	100%			UR	PF	UR × PF
Lactic acid (g/kg DM)
0%	0.77 Aa	0.23 Ab	0.22 Bb	0.22 Bb	0.94 Ba	0.46	0.05	<0.01	<0.01	<0.01
1.5%	0.76 Ab	0.35 Ab	0.59 Ac	0.84 Ab	3.05 Aa	1.12
Mean	0.76	0.41	0.28	0.50	1.99
Acetic acid (g/kg DM)
0%	6.87 Ab	3.43 Bc	9.10 Aa	10.0 Ba	7.62 Ab	7.40	0.13	0.10	<0.01	<0.01
1.5%	5.07 Bd	5.14 Ad	7.70 Bb	11.12 Aa	6.39 Bc	7.08
Mean	5.97	4.29	8.40	10.56	7.00
Propionic acid (g/kg DM)
0%	3.01	1.01	3.01	3.27	2.06	2.47	0.19	0.32	<0.01	0.19
1.5%	1.99	1.64	2.08	3.97	1.13	2.16
Mean	2.50 b	1.33 c	2.54 b	3.62 a	1.59 c
Butyric acid (g/kg DM)
0%	1.67	0.81	1.66	1.40	1.05	1.32	0.07	0.92	<0.01	0.24
1.5%	1.44	0.92	1.13	2.24	0.96	1.33
Mean	1.55 a	0.86 b	1.39 a	0.95 b	1.00 b

UR, Urea; PF, Percentage of fruit; DM, dry matter; SEMS, standard error of the mean.. Means followed by different uppercase letters in the column and lowercase in the rows are statistically different according to Tukey’s test p < 0.05.

).

Silages with 20% and 100% fruit and the addition of urea showed the highest lactic acid contents, 0.84 and 3.05g/kg DM, respectively.

The highest concentrations of acetic acid were found in the silages with 20% fruit with the addition of urea and 10% fruit without urea, which were 11.12 and 9.10 g/kg DM, respectively. Regarding propionic acid, the highest mean concentration was observed in the silage with 20% fruit, which was 3.62 g/kg DM. As for the butyric acid, the highest values were found in the silages with 0% and 10% fruit, which were 1.55 and 1.32 g/kg DM.

3.6. Aerobic Stability

There was an effect of the interaction (p < 0.05) between the different biomass mixtures (increasing inclusion of melon in the silages, 0, 5, 10, 20, and 100% AF) and the addition of urea (0% and 1.5% urea AF) on the break of aerobic stability and internal temperature of the silages (

Table 7. Aerobic stability of silages with different mixtures of melon biomass and addition of urea, after silo opening.

UR	Percentage of Fruit (PF)					Mean	SEM	p-Value
	0%	5%	10%	20%	100%			UR	PF	UR× PF
Hours
0%	56.0 Aa	56.0 Aa	56.0 Aa	56.0 Aa	48.0 Bb	54.4	0	<0.01	<0.01	<0.01
1.5%	56.0 Aa	56.0 Aa	56.0 Aa	56.0 Aa	56.0 Aa	56.0
Mean	56.0	56.0	56.0	56.0	52.0
Internal temperature (°C)
0%	33.5 Aa	31.1 Aab	27.8 Abc	27.1 Ac	33.5 Aa	30.6	0.2	0.09	0.05	<0.01
1.5%	27.8 Ba	29.0 Aa	28.5 Aa	27.5 Aa	27.5 Ba	28.0
Mean	30.6	30.0	28.1	27.3	30.5
pH
0%	7.6	7.5	6.8	6.3	4.3	6.5 b	0.1	<0.01	<0.01	0.15
1.5%	8.2	8.9	8.4	7.7	6.7	8.0 a
Mean	7.9 a	8.2 a	7.2 b	7.0 b	5.1 c

UR, Urea; PF, Percentage of fruit; DM, dry matter; SEM, Standard error of the mean. Means followed by different uppercase letters in the column and lowercase in the rows are statistically different according to Tukey’s test p < 0.05.

).

The break of aerobic stability occurred for the silages with 100% fruit without urea at 48 h and for the other silages it happened after 56 h. The highest values of internal temperature were recorded in the silages with 0% and 100% fruit without urea, which showed 33.5 °C in the break of aerobic stability. The highest mean pH value (7.6) was observed in the silages with 0% and 5% fruit, and with the addition of urea the highest pH value was 8.0.

4. Discussion

Regarding the silage yield from the melon harvest biomass, the highest mean values that were observed in the silages with 20%, 5%, and 10% fruit were mainly due to the environment and the management adopted for the crop during its cycle. In the case of melon, the ground cover helps to improve fruit quality by preventing direct contact of the fruit with the soil [26].

The DM contents of the silages 20% melon fruit biomass were increased by urea (Table 3). The DM content of the plant is important in the ensiling process because it is considered one of the determining factors of the type of fermentation that will develop inside the silo, besides the content of soluble carbohydrates. The DM contents found in the present study are below the range that can be considered ideal for adequate silage, which should be between 260 to 380 g/kg, according to Dias et al. [27].

For CP, the highest mean contents that were found in silages with the addition of urea is due to the fact that urea consists of non-protein nitrogen which enables the increase in silages, especially of melon. Thus, there was a possibility of retention of nitrogen in the silage through ureolytic activity, which transforms urea into ammonia and, consequently, retains it in the material as described by Grizotto et al. [28].

Regarding the ash, the highest value that was observed with the addition of urea is attributed to the formation of organic salts during ensiling. According to Ryley et al. [29], the partial binding of ammonia produced from the degradation of urea with organic acids occurs for the formation of organic salts during anaerobic fermentation in the melon biomass.

As for the ether EE, the highest value that was observed in the silages without urea is related mainly to the seeds of the melon fruit that contains a high amount of fat. According to Andreatta et al. [30], this vegetable stores its energy in the grain in the form of oil. EE values should be around 50 g/kg DM, and values higher than that limit animal intake, indicating that it is not recommended to provide the melon fruit silage as the only source of roughage for ruminants, since higher values were found in the present study [31]. For Pinedo et al. [32], excess fat in the diet can also reduce DM intake and nutrient passage.

The highest contents of ADF that were observed in silages with 100% and 10% fruit, had a lower amount of hemicellulose, and these results may come from the seeds in the fruit that present a higher proportion of less digestible fractions such as lignin and cellulose. High levels of ADF lead to the unavailability of potentially degradable structural carbohydrates, since a component of the cell wall, the lignin, forms a barrier that prevents microbial adhesion and enzymatic hydrolysis of cellulose and hemicellulose, consequently decreasing the digestibility of fiber, quality, and use of the feed according to Epifanio et al. [33].

The highest soluble carbohydrate contents that were found in the silages with 100% fruit (Table 3) are attributed to the melon fruit that has high concentrations of soluble carbohydrates, which favors the growth of yeasts due to the excess of substrates for their development, since according to several studies, it is known that contents of 6 to 8% of soluble carbohydrates are sufficient for an adequate microbial fermentation [34]. However, the high amount of soluble sugars can result in higher levels of lactic acid and residual soluble carbohydrates, and there may be a predominance of yeasts, which results in alcoholic fermentation [7], which in turn, can result in losses of DM and low aerobic stability of the silage.

The highest losses through effluent were found in the silages produced with 20%, 100%, and 10% biomass of melon fruit (Table 4). The amount of effluent is directly related to the moisture content of the material, which in this case, came from the melon fruit and the urea, where the respiratory activity of the plant and growth of microorganisms contribute to the release of effluent (water released from the mass). According to Paziani et al. [35], besides the contamination of the environment, there will be a reduction in the silage quality, carrying important nutrients such as sugars, proteins, minerals, vitamins, and organic acids.

The highest losses through gas were found in the silages with 0% and 100% fruit with the addition of urea (Table 4). This result probably occurred due to the presence of gas-producing microorganisms, such as enterobacteria and clostridia bacteria that act mainly in the presence of moisture that normally grow in media with higher pH. According to Ramos et al. [36], the production of gases during ensiling directly interferes with the loss of DM, because the loss of soluble carbohydrates in the form of gases has as consequence the production of water, reducing the DM content which promotes its loss, a fact that was observed in the study.

Regarding the rate of recovery of DM, these results can be considered good (74.6 and 81.6), and are related to the low production of gases and effluents during the fermentation process of the silage, indicating inhibition of heterofermentative bacteria and proteolytic bacteria considering that the production of CO₂ by yeasts during fermentation is the main responsible for reducing the recovery of DM, [37] which was also observed in this study.

The pH value of the silage with 100% fruit (4.3) without urea was considered close to the ideal for a good-quality silage, which according to McDonald et al. [38], ranges from 3.8 to 4.2, so that there is no compromise in the fermentative processes and avoids proteolysis. The high pH in silages with 0, 5, and 10% fruit with the addition of urea, may have occurred due to the buffering power of the ammonia and the plant (29.1 mg NaOH equivalent/100 g DM) (Table 1) on the acetic fermentation, besides retarding possible secondary fermentations after the opening the silo. According to Rabelo et al. [39], it is explicit that the increase in buffer capacity promoted by urea, which possibly hindered the pH drop observed in the silages of the present study.

Regarding the N-NH₃, silages with 0, 5, 10% fruit with addition of urea had increased contents, but the values obtained are below the recommended for good-quality silage which is <10% N-NH₃ [40]. This response can be explained by the addition of an ammonia source. Melo et al. [41] states that this occurs because with the degradation of urea, there is a release of ammonia, which may also have helped to increase the content of non-protein nitrogen [7]. Furthermore, the hydrolysis reaction of urea increases the concentration of ammonia in the ensiled mass.

The largest populations of lactic acid bacteria that were found in silages with 100% fruit are related to the higher content of soluble carbohydrates for the multiplication of these bacteria and the addition of urea (Table 5). The quantification of the lactic acid bacteria population is important because the production of lactic acid by these microorganisms promotes the reduction of pH causing the acidification of the medium. The purpose of using urea in silages is related to the fermentation capacity of the ensiled material besides its inhibitory action on undesirable microorganisms such as yeasts and molds since it raises the pH level [42]. The LAB populations were larger than the minimum limit of 5 log CFU/g, which is recommended by Pahlow et al. [43] and Muck et al. [44] for good fermentation and good-quality silages.

For yeasts, the highest value was observed in the silage with 100% fruit. This result might be due to the fact that the fruit has a higher concentration of soluble sugars (257.8 g/kg DM) (Table 1), which can favor the growth of yeasts and fungi. The reduction in yeast populations reflects a positive result of the use of urea (1.5%), because its inclusion in the ensiling process of melon with a high content of soluble carbohydrates is an alternative that aims to better control the pH, preventing its reduction and development of undesirable microorganisms, such as yeasts and filamentous fungi, because of its antimicrobial activity [45], consequently reducing the production of ethanol and the losses of DM and soluble carbohydrates. In the literature, the recommended values are below 5 log CFU/g [46].

The silages of melon biomass with 20% and 100% fruit with the addition of urea showed higher contents of lactic acid, indicating that there is an increase in the population of lactic acid bacteria with the increase of fruit and addition of urea (Table 6). The presence of lactic acid in silages is desirable, since this acid is responsible for the rapid drop in pH to values that prevent a favorable environment for the development of undesirable microorganisms. The lactic acid content (%DM) for silage to be considered of very good quality according to Roth et al. [47] should be between 4 and 6%.

The high acetic acid content, with 10% fruit and the addition of urea in the silage of melon biomass is related to higher values of final pH in the silages, corresponding mainly to the prolonged action of heterofermentative LAB, [48] in addition to antifungal principles. According to [40], the concentration of acetic acid in silages varies from 1.0 to 10.0% DM. In the present study, the values were close to those described by the abovementioned authors.

Propionate, as well as acetic acid, has antifungal principles, thus playing an important role in controlling yeasts allied with urea in the silages of melon biomass [49].

The highest mean values that were observed for butyric acid in the silages with 0% and 10% fruit, may be related to the buffer capacity (29.1 mg NaOH equivalent/100 g DM) (Table 1), which prevented the rapid pH drop. The butyric acid content found in the silage indicates the presence of undesirable microorganisms, such as bacteria of the genus Clostridium that perform the fermentation of lactic acid into butyric acid; therefore, high levels of butyric acid compromise the quality of the silage [50].

Regarding the break of aerobic stability of the silage with 100% fruit, the results found might be due to the fact that the melon fruit is rich in soluble carbohydrates (Table 7). This result can be explained by several factors, among them, the growth of microorganisms such as larger amounts of yeast that compete for lactic acid turning into acetic acid. The content of soluble carbohydrates favors the break of aerobic stability in silages [51].

Regarding the addition of urea, all treatments broke the stability with 56 h, and urea acts in the ensiled mass by raising the pH value of the medium, reducing the potential development of yeast populations. Ammonia is toxic to secondary microorganisms (yeasts and fungi), thus promoting greater aerobic stability and proving to be effective in reducing silage deterioration [7,52], which occurs in silages with the addition of urea.

Regarding the internal temperature in the melon silages with 0% and 100% fruit without urea, (Table 5), the results found can be explained by the fact that ammonia is toxic to secondary microorganisms (yeasts and fungi), thus promoting greater aerobic stability, proving to be effective in reducing the deterioration of silage [7]. Araújo et al. [53] reported that silages treated with ammonia reach a lower temperature, and a lower DM loss than silage not treated with the additive. According to Andrade et al. [54] the temperature increase is attributed to the onset of metabolic activities of opportunistic microorganisms, where they produce heat and consume residual carbohydrates.

The highest pH values were found in the silages with 0% and 5% fruit with the addition of urea. This high pH level can be explained by the high buffering power of the plant associated with the effect of urea. According to Furtado et al. [55], the aerobic fermentation that occurs in silages after opening the silo is performed by microorganisms that use organic acids as main substrates, such as lactic acid, soluble sugars, and ethanol. Possibly, the pH elevation is due to the volatilization of organic acids, and the high temperatures observed for these silages, in addition to the consumption of organic acids by spoilage microorganisms [7].

5. Conclusions

The silages made with the melon biomass and the addition of 20% and 100% of fruit showed differences regarding the fermentative pattern, chemical and bromatological composition, and aerobic stability, making these mixtures of biomass the most indicated for silage production.

The addition of urea reduced the number of yeast populations, which is important for the increase in aerobic stability of the melon fruit, being an option for farmers in the melon-producing regions.