Microbiological characterization, nutritional value and digestibility of black oat hay with crotalaria or corn as predecessors

Caracterização microbiológica, valor nutricional e digestibilidade do feno de aveia preta sob efeito do cultivo antecessor crotalária ou milho

ABSTRACT:

The present study assessed the influence of the preceding crop (crotalaria or corn) on the bromatological, digestibility and microbiological aspects of the EMBRAPA 139 black oat hay (Avena strigosa) cultivar. A randomized block design was used, with subplots divided according to time, whereby the preceding crop (crotalaria or corn) was allocated to the main plot and hay storage times (baling, 30, 60 and 90 days of storage) to the subplots, with five repetitions. Crotalaria as predecessor resulted in lower indigestible fiber and higher protein contents in black oat hay, both in acid detergent, as well as higher pH. Corn as the preceding crop provided better hay digestibility due to lower neutral detergent insoluble protein content and higher in vitro neutral detergent fiber digestibility. The preceding crop did not significantly affect the drying time of forage and the microorganism population.

Key words:
forage preservation; grasses; legumes; microbiology; crop rotation

RESUMO:

O presente estudo visou avaliar a influência da cultura antecessora (crotalária ou milho) sob o feno de aveia preta (Avena strigosa) cultivar EMBRAPA 139, nos aspectos bromatológicos, de digestibilidade e microbiológicos. O delineamento experimental foi realizado em blocos casualizados, com parcelas subdivididas no tempo, sendo alocados na parcela principal a cultura antecessora (crotalária ou milho), e nas sub parcelas, o período de armazenamento do feno (enfardamento, 30, 60 e 90 dias de armazenamento), com cinco repetições. A presença da crotalária como cultivo antecessor proporcionou ao feno de aveia preta menores teores de proteína indigestível e maiores teores de fibra, ambas em detergente ácido, bem como pH mais elevado. O feno de aveia preta com o milho como cultivo antecessor proporcionou melhores resultados de digestibilidade na forma de feno, pois menores teores de proteína indigestível em detergente neutro e maior digestibilidade in vitro da fibra em detergente neutro. Já para o período de desidratação da forragem e população de microrganismos, o cultivo antecessor não interferiu significativamente.

Palavras-chave:
conservação de forragem; gramínea; leguminosa; microbiologia; rotação de cultura

INTRODUCTION

Black oat is among the most widely used forage grasses in southern Brazil. Its physiological characteristics are suited to the region and the species exhibits good lodging resistance, excellent flowering and maturation uniformity and average regrowth. Flexibility is one of its primary traits, with applications as forage, green manure for subsequent crops, pre-dried silage and hay (FERRAZZA et al., 2013).

In general, hay has several advantages over silage; for example, it does not undergo fermentation and can be used immediately after production, thereby reducing losses due to its greater aerobic stability and lack of deterioration. It is also easy to store, transport and commercialize, helping to provide income for producers and serving as a long-term quality food (NERES & AMES, 2015).

Hay quality is not only related to dehydration, preparation, and storage, but also to plant care from sowing to cutting. Quality is directly linked to the type of forage and cultivar used, management practices, such as planting technique, fertilization, weed, fungus and insect control, and the regional climate conditions during the growing season (EVANGELISTA et al., 2011).

In addition to these factors, the basis for producing quality hay from this forage are the characteristics of the soil and the nutrients it provides for plants. Well-fertilized loose soil with adequate micro and micromineral levels provides a favorable environment for plant growth, reflecting in higher yields (NERES et al., 2021).

In order to fully meet soil needs, it is important to invest in crop management and fertilization, which ultimately raises production costs. The use of more sustainable alternatives, such as crop rotation, contributes to reducing negative impacts on the soil and investments.

Plants involved in management practices include legumes such as Crotalaria ochroleuca, which has excellent nitrogen (N) fixing capacity and provides good soil decompaction and weed control (SCHEUER & TOMASI, 2011). Corn is also used in crop rotation and exhibits a high degree of rusticity and organic matter accumulation in the soil. In addition to acting as soil temperature and moisture regulators, these plants reduce the risk of erosion through their high carbon to nitrogen ratio (C:N) and slower biomass decomposition (BARRADAS, 2010).

Ths study assessed the influence of the preceding crop (crotalaria or corn) on the bromatological, digestibility and microbiological aspects of the EMBRAPA 139 black oat hay (Avena strigosa) cultivar.

MATERIALS AND METHODS

The experiment was carried out at the Professor Antônio Carlos dos Santos Pessoa Experimental Station in the municipality of Marechal Cândido Rondon, Paraná state (PR), Brazil (24º31’52’’ S, 54º01’03’’ W, at an altitude of 397 m). According to Koppen’s classification, the local climate is classified as Cfa, subtropical; the average temperature in the coldest month is below 18ºC and in the hottest, above 22 ºC, with hot summers, infrequent frost, and a tendency to heavy rainfall in summer, albeit with no defined dry season (CAVIGLIONE et al., 2000).

The experimental area was divided into two main 560 m² plots, each containing five 112 m² subplots with one hay bale produced per subplot, each corresponding to an experimental unit (repetition). Each main plot contained predecessors to the black oat crop, namely corn or crotalaria (Crotalaria ochroleuca), representing the treatments, with the corn collected for silage production and crotalaria harvested as hay. C. ochroleuca was planted on 11/09/2018 under a no-till system, using10 kg ha^-1 of seeds, at a depth of 2 cm and 0.5 m between rows, and harvested for hay on 02/20/2019. The dry matter (DM) available before cutting was 195.33 g kg^-1 DM and the fresh mass produced 109.26 t ha^-1. No fertilizer was applied to the crotalaria crop.

BREVANT corn hybrid B 2810 was planted on 09/03/2018, also under a no-till system, with 4.7 seed per meter and 0.7 m between rows, and harvested for silage on 01/28/2019. Fresh mass production at cutting was approximately 79.62 t ha^-1. Fertilizer was applied at planting, in the form of 370 kg ha^-1 of NPK 10-15-15 (N-P₂O₅-K₂O), and 150 kg ha^-1 of the same fertilizer was used during development, before emergence of the sixth leaf.

The EMBRAPA 139 black oat cultivar was planted on 05/03/2019, in a no-till system,with 0.17 m between rows, average sowing depth of 2 cm and sowing density of 65 kg of seeds ha^-1. Base dressing was applied at planting, consisting of 200 kg ha^-1 10-20-20 fertilizer (N-P₂O₅-K₂O), in line with the recommendations of the Paraná State Branch of the Brazilian Soil Science Society (PAULETTI & MOTTA, 2019). Weeds were chemically controlled using a commercial metsulfuron-methyl-based herbicide (Ally^®: 6.6g ha1).

The black oat crop was cut for haying in the flowering stage, on 08/05/2019, at 68 days after emergence (DAE), using a disc mower conditioner (^®Khun) equipped with steel fingers for mechanical conditioning, at two heights.

After cutting and conditioning, the forage was left to dry in the field under the sun. Baling was performed on 08/09/2019 at 2 p.m. (102 hours after cutting), preparing rectangular bales with an average weight of 10 kg in all treatments.

For drying curve construction, a randomized block design was used, consisting of two preceding crops (corn or crotalaria), seven black oat sampling times and five repetitions. The sampling times corresponded to drying periods, that is, hours after cutting, namely zero, 5, 22, 29, 54, 78 and 102 hours.

Sampling to determine the drying curves was performed by collecting one sample (whole plant) per plot, with approximately 300 grams per drying time (from zero to 102 hours). The samples were wrapped in paper bags and dried in a forced-air oven at 55 ºC for approximately 72 hours, to determine dry matter content. The sample collected at the last drying time (102 hours) corresponded to the baling sample, and was sent to the laboratory not only to determine dry matter, but for bromatological and digestibility analysis.

The hay bales were stored in a closed brick shed with a concrete floor, arranged on a wooden palette to prevent contact with the ground, until opening. Hay bales were opened after 30, 60 and 90 days of storage. At each opening time, 300g hay samples were collected for bromatological and digestibility analyses, and dried in a forced-air oven at 55 °C for 72 hours to quantify dry matter (DM) content.

After drying, the samples were ground in a Wiley mill with a 1 mm mesh sieve and analyzed in the laboratory to determine mineral matter (MM) and crude protein (CP) content according to the Association of Official Analytical Chemists (AOAC, 1995), neutral detergent fiber (NDF), acid detergent fiber (ADF), neutral detergent insoluble protein (NDIP), acid detergent insoluble protein (ADIP) and lignin (LIG) in line with VAN SOEST et al. (1991), and cellulose (CEL) and hemicellulose (HEM) in accordance with SILVA & QUEIROZ (2006). The pH of the samples was also measured. The analyses were performed at the Animal Nutrition Laboratory of Western Paraná State University (UNIOESTE).

During storage, the average weekly temperature (°C) and relative humidity (RH%) in the shed and in the hay were monitored using a datalogger (Figure 1).

Figure 1
Average weekly temperature (°C) and relative humidity (RH%) in the environment (inside the shed) and black oat hay bales at 90 days of storage.

In vitro dry matter digestibility (IVDMD) was performed using the technique described by TILLEY & TERRY (1963), adapted to the artificial rumen (in vitroincubator, Tecnal^® TE-150), in line with HOLDEN (1999). To that end, the samples were sent to the Animal Nutrition Laboratory of the State University of the Central West (UNICENTRO), in Guarapuava, PR. For rumen fluid collection with a rumen cannula, two castrated male Jerseycattle grazing on Urochloa sp. were used. The ground black oat hay was divided into 0.25 g samples, placed in filter bags (TNT - 100 g cm², cut and sealed to a size of 5.0 x 5.0 cm) and incubated in jars containing rumen fluid and buffer solution. The material was incubated for 48 hours under constant rotation and controlled temperature (39 °C). IVDMD was calculated based on the difference between the amount incubated and the residue after incubation. In vitro organic matter digestibility (IVOMD) was determined by burning the residue of the incubated material obtained after IVDMD analysis in a muffle furnace at a 600 °C for 4 hours, and calculating the difference between the incubation residue and the ash. In vitro neutral detergent fiber digestibility (IVNDFD) was calculated using the methodology described by GOERING & VAN SOEST (1975), incubating the samples for 48 hours at 39 °C followed by NDF analysis. To that end, two 6-year-old steers with rumen fistulas and an average live weight of 700 kg were used. The animals are housed at the UNICENTRO Beef Cattle Teaching Unit in Guarapuava (PR) and their use was authorized by the Animal Ethics Committee (CEUA/UNICENTRO) in notice no. 005/2021, issued on February 5, 2021.

For microbiological analyses, the microorganisms on black oat plants, in the soil, and on the straw left by crotalaria or corn, were identified (Table 1) by collecting five 25g of plant, soil and straw samples from the two treatments for quantification of the microbiological population at the UNIOESTE Microbiology and Biochemical Laboratory.

Table 1
Microorganism population (log CFU g-1) in fresh black oat plants, the soil and straw in the two experimental areas.

Preceding crop	-----------------------------------------Microorganism population (log CFU g-1)-------------------------------------------
	Lactic acid bacteria	Enterobacteria		Clostridium	Mesophilic aerobes
---------------------------------------------------------------------------------Soil-------------------------------------------------------------------------------
Corn	2.93	3.75		4.79	4.98
Crotalaria	2.60	3.45		4.73	5.10
Straw
Corn	2.92	3.93		5.12	6.35
Crotalaria	3.18	4.15		5.75	6.09
------------------------------------------------------------------Fresh black oat plants-------------------------------------------------------------------------
Corn	2.95	2.96		5.09	5.71
Crotalaria	*	2.46		4.86	4.54

• ^*
  No colony forming units found; CFU - Colony forming unit.

The bacterial and fungal populations were determined in duplicate using culture techniques described by SILVA et al. (2017) and the following media: Lactobacillus MRS Broth for lactic acid bacteria counting, by incubating the plates at 30 °C for 48 hours; Violet Red Bile Agar for enterobacterial counting, with incubation at 36 °C for 24 hours; Reinforced Clostridial Agar for Clostridium counting, under 24-hour incubation in a CO₂ incubator at 36 °C; Potato Dextrose Agar for fungal and yeast counts, incubated at room temperature for seven days; and Plate Count Agar for an aerobic plate count, with incubation in an oven (EletrolabEL202) at 37 °C for 24 to 48 hours.

After incubation the colonies were counted using a Quebec colony counter, in Petri dishes containing 30 to 300 CFUs (colony forming units), with results expressed in log CFU g-¹d. In conjunction with fungal and yeast counts, the genera of the fungi in the corn and crotalaria straw were identified (Figure 2).

Figure 2
Yeast and fungal genera present in the corn and crotalaria straw in both experimental areas before black hay cutting.

For microbiological analysis of the hay, 25g samples were collected at the three bale openings, after 30, 60 and 90 days of storage. Analyses were carried out in duplicate using culture techniques described by SILVA et al. (2017) and the following media: Reinforced Clostridial Agar for Clostridium counting, incubating the plates in a CO₂ incubator for 24 hours at 36 °C; Potato Dextrose Agar for fungal and yeast counts, with incubation at room temperature for seven days; and Plate Count Agar for an aerobic plate count, with incubation in an oven (EletrolabEL202) at 37 °C for 24 to 48 hours.

After incubation the colonies were counted using a Quebec colony counter, in Petri dishes containing 30 to 300 CFUs (colony forming units), with results expressed in log CFU g-¹d.

The data were submitted to normality testing via the Shapiro-Wilk test, followed by analysis of variance (ANOVA) considering a randomized block design, where factor 1 was the preceding crop (corn or crotalaria) and factor 2 the storage time (baling, and 30, 60 and 90 days of storage), with five repetitions. For the in vitro digestibility and microbiological analyses of hay, four repetitions were used. Interactions between factors were decomposed. Polynomial regression was performed to analyze the effect of storage time. All the statistical procedures were carried out in R software, version 4.0.2.

RESULTS AND DISCUSSION

Analysis of the description of the microorganisms (log CFU g^-1) present in the fresh black oat plants, soil and straw from the experimental plots (Table 1) revealed similarities between the predecessors (corn and crotalaria), except for lactic acid bacteria, which were only found in fresh plants preceded by corn (2.95 log UFC g^-1); however, the high organic matter content of hay and limited development of these microorganisms prevents them from having a negative effect. The difference between the presence of lactic acid bacteria in fresh corn and crotalaria plants, and the similarity between CFUs in the soil and straw indicate that this factor will not affect hay quality.

The drying curve for black oat hay showed no effect for the preceding crop or interaction with the drying time after cutting. Dry matter content exhibited quadratic behavior during drying for both predecessors (Figure 3).

Figure 3
Quadratic behavior of black hay drying at different times after cutting, under the effect of the preceding crop (corn or crotalaria).

At baling, the dry matter content of black oat (Table 2) in both treatments was within the desired range, with 876.5 and 877.6 g kg^-1DM for corn and crotalaria as the preceding crops, respectively. The total drying time was 102 hours (4 days and 6 hours), which does not exceed the maximum of seven days recommended to produce quality hay for animal feed (PASQUALOTTO et al., 2015).

Table 2
Mean values of the bromatological data dry matter (DM), mineral matter (MM), organic matter (OM), crude protein (CP), neutral detergent indigestible fiber (NDIF) and acid detergent indigestible fiber (ADIF) of black oat hay under four storage times, associated with different preceding crops (corn or crotalaria).

	DM, g kg-1DM	MM, g kg-1DM	OM, g kg-1DM	CP, g- kg-1DM	NDIP, g kg-1CP	ADIP, g kg-1CP
-------------------------------------------------------------------------Preceding crop--------------------------------------------------------------------------
Crotalaria	877.6	65.7	934.3	131.4	287.8	58.2
Corn	876.5	67.7	932.3	132.9	255.1	71.5
---------------------------------------------------------------------Hay storage time in days------------------------------------------------------------------
0	823.1	66.4	933.6	137.5	251.6	78.8
30	892.3	66.6	933.4	133.3	277.7	74.9
60	903.1	66.2	933.8	130.9	285.9	58.6
90	892.9	67.4	932.6	126.0	273.1	42.2
----------------------------------------------------------------------Probability (P < 0.05)---------------------------------------------------------------------
C	0.902	0.113	0.113	0.500	<0.001	0.0027
T	<0.001	0.771	0.771	0.003	0.0037	<0.001
C*T	0.080	0.552	0.552	0.064	<0.001	0.0014
---------------------------------------------------------------------------Coefficient----------------------------------------------------------------------------
Intercept	824.856	NDS	NDS	137.3981	251.4272	83.0802
Linear	2.806	NDS	NDS	-0.1235	1.2096	-0.4267
Quadratic	-0.023	NDS	NDS	-	-0.0108	-
R2	0.8857	NDS	NDS	0.2938	0.1597	0.5829
P value Reg	<0.001	NDS	NDS	<0.001	0.042	<0.001

• C-preceding crop effect; T- storage time effect; C^*T- preceding crop-storage time interaction; (-) variable with no quadratic effect; NDS -no difference for storage time.

After cutting, water is initially lost via the stomata, followed by the cuticle, the former being an important pathway in haymaking due to the speed at which water loss occurs. When the plant reaches a water content of 45%, the stomata close, thus initiating slower water lossvia cuticular transpiration (EVANGELISTA et al., 2011). In the present study, stomatal water loss began before 22 hours post-cuttingand then slowed until reaching the ideal DM for baling.

Plasmolysis occurs in the final stage of dehydration, whereby the cell membrane loses its selective permeability, resulting in rapid water loss. In this phase, drying is less influenced by management strategies and more sensitive to climate conditions (EVANGELISTA et al., 2011). It is likely that the high temperatures in Marechal Cândido Rondon during black oat drying and baling contributed to the rapid water loss from the plant (Figure 1).

The variables NDIP and ADIP showed a significant difference for the preceding crop effect, with the highest values obtained for crotalaria and corn, respectively. There was no significant effect (P < 0.05) for storage time for MM and OM, whereas storage time behavior was quadratic for DM and NDIP and decreasing linear for CP and ADIP (Table 2, Figure 2).

There was a significant interaction effect between storage time and preceding crop (P < 0.05) for NDIP and ADIP, with no statistical difference for the remaining variables (Table 2).

The increase in DM after cutting in relation to baling and storage is normal because it is a natural part of plant dehydration. The variations in DM content from 60 to 90 days of storage can be explained by the change in climate conditions. Hay is hygroscopic, meaning it can lose and absorb moisture from the environment, influenced by relative humidity (RH%) (AMES et al., 2015).This was confirmed by the increase in environmental RH% in the last two weeks of assessment before the final collection, at 90 days of storage (Figure 1).

SILVA (2011) studied different cutting heights and storage times for black oat hay and obtained different results for CP, which increased during storage, with values of 118.6 g kg^-1 DM at baling and 149.8 g kg^-1 DM after 30 days of storage. PASQUALOTTO et al. (2015) analyzed Tifton 85 hay under two conditioning intensities and different storage times and observed quadratic behavior for CP, which increased during storage. However, similar to our findings, the highest CP content was recorded at baling (Table 2).

Interaction analysis indicated quadratic behavior for NDIP for both preceding crops (Figure 4), while ADIP showed linear decreasing and quadratic behavior for corn and crotalaria, respectively (Figure 5).

Figure 4
Quadratic behavior of black oat hay neutral detergent insoluble protein in relation to the preceding crop (crotalaria or corn) and storage time.

Figure 5
Behavior of black oat hay acid detergent indigestible fiber in relation to the preceding crop (crotalaria or corn) and storage time.

Heat is one of the factors that negatively affects the N content linked to NDF, causing protein coagulation and denaturation. Intense heat can cause Maillard reactions that further compromise protein availability, incorporating it into ADF. In addition, reducing sugars and moisture predispose nitrogen binding to indigestible fiber (BERCHIELLI et al., 2011). None of these interferences were detected in the present study, where environmental conditions were controlled, and plants were not exposed to heat stress during drying or baling.

Although, NDIP was higher with crotalaria as predecessor, the values recorded were within the normal range. SILVA (2011) studied black oat hay under different cutting heights and storage times and found higher average values than those obtained here, with398.4 and 429.5 g kg^-1 CP at baling and 30 days of storage, respectively. The treatment with crotalaria as the preceding crop obtained the highest NDIP, likely due to the larger NDF content recorded at 60 and 90 days of storage (Tables 2 and 3).

Table 3
Mean values of the bromatological data neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin (LIG), hemicellulose (HEM), cellulose (CEL) and pH of black oat hay under four storage times, associated with different preceding crops (corn or crotalaria).

	NDF, g kg-1DM	ADF, g kg-1DM	LIG, g kg-1DM	HEM, g kg-1DM	CEL, g kg-1DM		pH
Preceding crop
Crotalaria	553.7	273.2	23.1	280.5	250.1		5.96
Corn	539.1	262.0	20.9	277.2	241.1		5.81
Hay storage time in days
0	536.4	262.0	20.1	274.4	241.9		5.86
30	542.8	266.5	21.8	276.3	244.7		5.89
60	550.5	271.8	23.1	278.7	248.7		5.94
90	558.3	271.6	23.3	286.7	248.4		5.87
Probability (P < 0.05)
C	<0.001	0.002	<0.001	0.333	0.014		<0.001
T	<0.001	0.198	<0.001	0.047	0.544		0.068
C*T	0.0465	0.972	0.942	0.676	0.984		<0.001
Coefficient
Intercept	535.9256	NDS	20.4943	273.0977	NDS		NDS
Linear	0.2452	NDS	0.0346	0.1339	NDS		NDS
R2	0.4591	NDS	0.3053	0.1844	NDS		NDS
P value Reg	<0.001	NDS	<0.001	0.0057	NDS		NDS

• C-preceding crop effect; T- storage time effect; C^*T- preceding crop-storage time interaction; NDS - no difference for storage time.

In research on hay production, WUNSCH et al. (2007) reported an increase in NDIP every month for four months during hay storage. In our study, this behavior was only observed for crotalaria as predecessor, with increasing values despite the quadratic behavior observed, whereas NDIP began to decline after 33 days of storage with corn as the preceding crop.

Because ADIP in hay is unavailable to animals, levels ideally should not increase during storage. In Tifton 85 hay, NERES et al. (2011) found that ADIP content was higher at cutting than at baling or after 30 days of storage, corroborating the present study, where ADIP declined in both treatments during storage.

The ideal ADIP in g kg^-1 of CP is between 40 and 70, meaning that 930 to 960 g kg^-1 of crude protein is available for digestion (BERCHIELLI et al., 2011). Based on this premise, the values obtained for crotalaria as predecessor, regardless of storage time, are within the expected range, not exceeding 70 g kg^-1CP, while values for corn at baling and 30 days of storage were greater than 83 g kg^-1CP. It is believed that the high nitrogen fixing capacity of C. ochroleuca and good nutrient availability in the soil improved protein digestibility in black oat hay.

The preceding crops showed a significant effect for the variables NDF, ADF, LIG, CEL and pH, with higher values for crotalaria. In regard to the effect of storage time, increasing linear behavior was observed for NDF, LIG and HEM, with no interference for the remaining variables (Table 3).

In black oat hay submitted to different cutting heights and storage times, SILVA (2011) recorded higher NDF values (766.6 g kg^-1 DM) than those observed here, but with increasing linear behavior. The same author reported quadratic behavior for ADF, with values peaking at baling (479.5 g kg^-1 DM). However, average ADF (448.06 g kg^-1 DM) was far higher than that obtained here for corn (262.0 g kg^-1 DM) or crotalaria (273.2 g kg^-1 DM) (Table 3). These differences may be related to climate conditions and plant development stages.

MARIANI et al. (2012) studied the performance of winter cereals on the residue of perennial forage and summer crops and found no significant difference for NDF and ADF in black oat as a succession crop to a legume or grass species. However, NDF and ADF were higher with soybean as predecessor (460 and 237 g kg^-1 DM, respectively) when compared with corn (427 and 220 g kg^-1 MS, respectively). Similar behavior was observed in our study, whereby fiber content was lower with corn as the preceding crop.

Lignin is a phenolic compound present in the bond that forms the fibrous fraction of forage. In addition to its structural role in the plant, lignin is a major limiting factor in forage digestibility (BERCHIELLI et al., 2011). SILVA (2011) reported an LIG content of 68.3 g kg^-1 DM in black oat hay, higher than that recorded here. The author also observed quadratic behavior for LIG, with peak levels reached at baling, unlike the present study.

Hemicellulose (HEM) and cellulose (CEL) levels are related to NDF and ADF content, and the gradual increase in these two variables also raised the concentration of their components (HEM, CEL and LIG). Hemicellulose is a chain of amorphous polysaccharides with a far lower degree of polymerization than cellulose, contributing to its faster degradation in the rumen when compared to cellulose. HEM content is directly linked to the digestibility and use of fiber in forage (BERCHIELLI et al., 2011).

In black oat hay submitted to different cutting heights and storage times, SILVA (2011) obtained higher HEM (318.6 g kg^-1 DM) and CEL values (336.2 g kg^-1 DM) than those observed here. The same author reported increasing linear behavior for CEL and quadratic for HEM, attributing the high concentrations to the increase in ADF and LIG during storage.

The linear increase in the fibrous components of the hay during storage suggested a concentration effect, due to the consumption of soluble carbohydrates by the microorganisms in the hay, especially those of the genus Clostridium, which were present despite the small number of CFUs (NEUMANN et al., 2007; BERTO & MÜHLBACH, 1997).

Silage has a low pH (3.5 to 4.5) due to the fermentation process and acid production, while the pH of fresh or preserved forage (hay) is approximately 6.0. Unwanted fermentation in hay due to excess moisture and heat causes loss of sugars and the formation of volatile nitrogen bases, which tend to increase pH (WEISS et al., 2003). However, this was not observed in the present study. Despite the significant difference between treatments, all the pHs remained close to ideal (5.5 to 6.03) (Table 3), albeit with quadratic behavior for crotalaria and linear for corn (Figure 6).

Figure 6
Behavior of black oat hay pH in relation to the preceding crop (crotalaria or corn) and storage time.

Analysis of the in vitro digestibility data in table 4 shows a significant effect for the preceding crop (P < 0.05) for in vitro neutral detergent fiber digestibility (IVNDFD), with greater digestibility for corn than crotalaria. In vitro dry matter (IVDMD) and organic matter digestibility (IVOMD) and IVNDFD exhibited decreasing linear behavior during storage, with reductions of 0.3186, 0.3657 and 0.5304 percentage points per day (Table 4).

Table 4
Mean values for in vitro dry matter (IVDMD), organic matter (IVOMD) and neutral detergent fiber digestibility (IVNDFD) of black oat hay under four storage times, associated with different preceding crops (corn or crotalaria).

	In vitro DM digestibility g kg-1DM	In vitro OM digestibility, g kg-1DM	In vitro NDF digestibility, g kg-1DM
------------------------------------------------------------------------Preceding crop---------------------------------------------------------------------------
Crotalaria	790.9	789.3	658.6
Corn	793.1	788.3	679.3
Hay storage time in days
0	804.3	799.8	688.7
30	797.3	797.7	679.7
60	785.4	784.3	653.4
90	776.5	768.5	644.2
---------------------------------------------------------------------Probability (P < 0.05) ---------------------------------------------------------------------
C	0.489	0.807	0.046
T	<0.001	<0.001	0.019
C*T	0.005	0.014	0.886
----------------------------------------------------------------------------Coefficient---------------------------------------------------------------------------
Intercept	805.4	804.3	691.3
Linear	-0.3186	-0.3657	-0.5304
R2	100	89.52	100
P value Reg	<0.001	<0.001	<0.001

• C-preceding crop effect; T- storage time effect; C^*T- preceding crop-storage time interaction.

Interaction was observed between the preceding crop and storage time for NDF and pH (Table 3), with increasing linear behavior for NDF for both predecessors (Figure 7), and in the case of pH, quadratic for crotalaria and increasing linear for corn (Figure 6).

Figure 7
Increasing linear behavior of black oat hay neutral detergent indigestible fiber in relation to the preceding crop (crotalaria or corn) and storage time.

There was also interaction between predecessors and storage time for IVDMD and IVOMD (Table 4), with decreasing linear behavior for both variables in corn and respective reductions of 0.5064 and 0.5859 percentage points per storage day. For crotalaria, there was no effect on the response variable, with no significant effect for storage times (Figures 8 and 9).

Figure 8
Behavior of in vitro dry matter digestibility of black oat hay in relation to the preceding crop (crotalaria or corn) and storage time.

Figure 9
Behavior of in vitro organic matter digestibility of black oat hay in relation to the preceding crop (crotalaria or corn) and storage time.

Analysis of interference by the preceding crop alone indicated a significant difference (P < 0.05) for IVNDFD, with greater digestibility for corn at 679.3 g kg^-1 DM, and 658.6 g kg^-1 DM for crotalaria. This difference can be explained by the high NDF, ADF and LIG contents obtained with crotalaria as predecessor (Table 3). Although, the climate conditions were similar for drying, baling and storage, it is believed that hay preceded by crotalaria experienced greater soluble carbohydrate loss, thus increasing the fiber content and digestibility of NDF (TAFFAREL et al., 2014).

The three digestibility variables displayed decreasing linear behavior during storage. This was also reported by AMES et al. (2015), who studied Tifton 85 hay and found higher IVDMD at baling than after 30 days of storage. SCHMOELLER et al. (2019) assessed different environmental conditions for Tifton 85 hay and observed no significant difference for IVDMD after 90 days of storage.

In regard to microorganism growth (Table 5), the preceding crop did not affect the presence of mesophilic aerobes, Clostridium, fungi and yeast in black oat hay. Storage time affected Clostridium, with a larger amount of these microorganisms observed at 30 days of storage than at other assessment times, but no interaction between the effects evaluated.

Table 5
Microorganism population (log CFU g^-1) in black oat hay at three storage times associated with different preceding crops (corn or crotalaria).

	Mesophilic aerobes, log CFU g-1	Clostridium, log CFU g-1	Fungi, log CFU g-1	Yeast, log CFU g-1
------------------------------------------------------------------------Preceding crop---------------------------------------------------------------------------
Crotalaria	4.67	5.31	3.51	2.26
Corn	4.33	4.96	3.14	2.10
--------------------------------------------------------------------Hay storage time in days-------------------------------------------------------------------
30	4.68	6.27a	3.41	2.26
60	4.51	4.48b	3.38	2.28
90	4.24	3.97b	3.17	2.00
-----------------------------------------------------------------------Probability (P < 0.05)--------------------------------------------------------------------
C	0.284	0.467	0.08	0.31
T	0.36	0.00	0.42	0.27
C*T	0.877	0.60	0.72	0.7

• C-preceding crop effect; T- storage time effect; C^*T- preceding crop-storage time interaction; Means followed by different lowercase letters differ significantly according to Tukey’s test (P > 0.05); CFU - colony forming unit.

The high DM content of hay (above 80%) hampers bacterial growth since most bacteria require a certain degree of moisture for their growth and development. Excess heat and moisture content greater than 25% can cause unwanted fermentation in hay and promote fungal and bacterial growth (SCHMOELLER et al., 2019). Since the drying, baling, storage, and temperature conditions (Figure 1) were ideal for hay production and maintenance, bacterial growth was insufficient to compromise its quality.

BARON & GREER (1988) assessed six different preservatives for alfalfa hay and reported greater microorganism growth in controls without preservatives, with the following values for total fungi (fungi + yeasts), aerobic and anaerobic bacteria: 5.6, 8.3 and 6.7 log CFU g^-1, respectively. The authors also observed an increase in total fungi and stability for aerobic and anaerobic growth (BARON & GREER, 1988). This differs from the present study, where both bacterial and fungal growth declined during storage.

The considerable presence of fungi and yeast in hay is evident to the naked eye by the change in color and characteristic odor. Baling at a moisture content below 20% favors the growth of microorganisms (bacteria, fungi and yeast) which, in addition to comprising the physical appearance of hay, affects its nutritional value and digestibility. High temperatures combined with moisture can cause non enzymatic and fermentative reactions, compromising hay quality and altering pH (AMES et al., 2015). There were no changes in physical appearance visible to the naked eye for the hay produced in the present study.

In research with Tifton 85 bermudagrass and wrangler grass hay stored under different environmental conditions, SCHMOELLER et al. (2019) reported similar values for fungi (3.75 log CFU g^-1) and yeast (1.75 log CFU g^-1). AMES et al. (2015) studied Tifton 85 hay and observed smaller fungal populations than those found in the present study, with an average of 1.97 log CFU g^-1 after 30 days of storage.

CONCLUSION

EMBRAPA 139 black oat hay preceded by corn showed greater acid detergent fiber digestibility.C. ochroleuca as predecessor resulted in higher fiber content when compared with corn. The preceding crop did not interfere in forage drying time or the microorganism population.

ACKNOWLEDGEMENTS

To the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil, for granting a scholarship - financial code 001. Process number 88887.634844/2021-00.

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• CR-2023-0435.R1

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

• Notice no. 0 0 5 202 1 CEUA/UNICENTRO.

Edited by

Editors: Leandro Souza da Silva (0000-0002-1636-6643)

Denise Montagner (0000-0003-2688-8063)

Publication Dates

• Publication in this collection
  25 Mar 2024
• Date of issue
  2024

History

• Received
  09 Aug 2023
• Accepted
  12 Dec 2023
• Reviewed
  16 Feb 2024