Improve Quality Of Alfalfa Silage Ensiled With Orange Pulp And Bacterial Additive

This study evaluated the effects of Lactobacillus buchneri (LAB) and orange pulp (OP) supplementation on chemical composition, in vitro and in situ degradability, dry matter (DM), pH and aerobic stability of alfalfa silage. Treatments were 1) alfalfa without any additive (control), 2) alfalfa + OP (70% alfalfa mixed with 30% fresh OP, LAB0), 3) treatment 2 with 1.5 g LAB/ton (LAB1), 4) treatment 2 with 3 g LAB/ton (LAB2) and 5) treatment 2 with 4.5 g LAB/ton (LAB3). Treatments were replicated three times in laboratory mini silos for 90 d in a closed barn with a ambient temperature (15 to 18 oC). As a result of this study, the experimental treatments did not affect on DM of silage at the end of the ensiling period. Control treatment had the highest pH (4.5) among the experimental treatments ( P<0.05). Supplementation alfalfa with OP resulted in significant increase in water soluble carbohydrate (WSC) and total volatile fatty acids (VFAs) ( P<0.05). Treating alfalfa silage with OP and LAB improved aerobic stabilities of treatments significantly, as compared to LAB0. Supplementation of silage with OP and bacterial inoculant increased DM digestibility. These findings indicated that LAB can improve the aerobic stability and quality of silage in laboratory silos and also, OP and LAB might improve silage quality and cause better silage management in the farm. Research Article Article History Received : 11.01.2020 Accepted : 04.06.2020


INTRODUCTION
Improving rumen condition and production in livestock production farms is dependent on improving the conditions for digestion of the fiber (Jalc et al. 2009;Hashemzadeh et al. 2011,). Alfalfa in dry condition is more used than silage, which in some cases accounts for more than half of the ration (Delavar et al. 2004;Kowsar et al. 2008;Soltani et al. 2009, Hashemzadeh et al. 2011). However, alfalfa silage is commonly used in dairy ration to benefit production (Benchaar et al., 2007). Especially, silage of legumes compared to dried herbs milk they have higher feeding value for their cows (Mut et al., 2020). Recent 30 to 40 years, the addition of carbohydrate and bacterial additives to improve the quality of the silage has been studied. Citrus pulp, tomato pomace, apple pomace and pistachio pulp are among the byproducts of the agricultural and processing industries that are introduced as a potential source of livestock feeding (Alnaimy et. al., 2017;Ayaşan and İnci, 2019). These materials are produced in the respective factories depending on the season of production of fruit and are largely disposed of without contaminating the environment (Karimi et al. 2017).
Applying technically appropriate silage making procedure is prerequisite to obtain a high-quality silage. In addition, variability of silage micro flora, their interaction and growth in silo are main factors affecting silage quality Exposure of silage to the air at the time of feeding causes deterioration of the silage. Yeasts that are able to metabolise lactic acid are the first cause of deterioration, which causes a rise in pH (Kleinschmit and Kung 2006;Jalc et al. 2010), which is also a stimulant for the growth of other harmful microorganisms in the silage (Sadegni et al. 2012;Anonim 2016). These unwanted reduces livestock production due to food depreciation or poisoning. It This study evaluated the effects of Lactobacillus. buchneri 40788 inoculants on the dry matter (DM) content pH, aerobic stability and microbial population in alfalfa with fresh orange pulp silage.

MATERIALS and METHODS
Whole fourth cut alfalfa was harvested in the prebloomed stage (Ahar town, Iran, late september of 2016) at DM of 22 % and with orange pulp chapped manually with a theoretical size of 2.5 centimeters and wilted at room temperature for 24 h. Chemical composition of wilted alfalfa and OP before ensiling are given in Table 1. Based on mini silos capasity, the amount of 1750 g of alfalfa was blended with 750 g of orange pulp and after adding levels of 0, 1.5 × 10 8 cfu/g, 3 × 10 8 cfu/g and 4.5 × 10 8 cfu/g of the bacterial additive (Lactobacillus boucheri MT40788) was ensiled for 90 days. Distilled water used to dissolve bacterial supplements was added to the control treatment, which was 2.5 kg of alfalfa forage before the ensiling. The laboratory silos were made of UPVC tubes with a height of 90 cm and a diameter of 10 cm with a volume of 2.5 kg. Treatments were 1) alfalfa without any additive (control), 2) alfalfa + OP (70% alfalfa mixed with 30% fresh OP, LAB0), 3) treatment 2 with 1.5 g LAB/ton (LAB1), 4) treatment 2 with 3 g LAB/ton (LAB2) and 5) treatment 2 with 4.5 g LAB/ton (LAB3).
At the end of 90 days ensiling, silos were opened and immediately pH, DM, microbial culture and soluble carbohydrate samples were measured and the remaining samples were stored in the refrigerated room at -20 o C for subsequent analysis. Acid detergent fibre (ADF) and neutral detergent fibre (NDF) contents were determined with the method described by Van Soest et al. (1991), Crude ash (CA) and crude protein (CP) contents of the resultant silages were determined by the method of AOAC (2000). Water-soluble carbohydrates were determined according to the method described by Dubois et al. (1956). Concentration of VFA of silages were measured according to the method of Filya (2003).
The aerobic stability was measured using the method described by Adesogan et al. (2004). 400 g of each sample was transferred into containers, and a thermometer was placed in the center of each silage mass and two thermometers in two different points of the room (ambient temperature 12° C). Silage and ambient temperature were recorded manually every two hours until heating occurred. When the silage mass temperature reached 2 °C above the ambient temperature, silage was considered to be spoiled (Ranjit and Kung 2000).

In situ trail
Two rumen-cannulated sheeps (18±2 months old, 45±3 kg) were used to study the in situ rumen degradation of the silage. The animals were offered 400 g lucerne, 200 g barley and 200 g soybean meal twice a day at 09:00 h and 17:00 h (NRC, 1980). Animals had free access to water. Dried and ground (1 mm screen) silage samples (three replications) were weighed into polyester nylon bags (Vanzant et al. 1998 In vitro trial The amount of in vitro DM degradation of treatments was measured in bottles (in 3 replications) using the techniquıe described by Fedorak and Hurdy (1983) . The amount of DM disappearance of treatments was recorded at 2, 4, 8, 12 and 24 h post-incubation.

Statistical analysis
The results of the experiment were analyzed by oneway analysis of variance and treatment means were compared by the Duncan multible range test (SAS, 2002).

RESULTS
Bacterial innoculation and OP effects on nutritional value of the resultant alfalfa silages are given in Table 2. The results showed that the addition of OP to alfalfa silage reduced pH compared to control (P<0.05).
Bacterial additives were also reduced pH in comparison to control (P<0.05). This decrease in pH value was intensified by increasing the use of additives, but no significant difference was observed between 4 different levels tested. The addition of OP to the alfalfa silages increased soluble carbohydrates compared to the control (P<0.05). The DM of silages was not affected by the bacterial additives or OP. Total volatile fatty acids in the treatment of alfalfa with OP without bacterial additive increased compared to control (P<0.05; Table 2). Although OP did not affect the NDF contents of the resultant silages, OP significantly increased ADF in LB0 compared to the control (P<0.05). Bacterial additive consumption in all 3 levels decreased ADF compare to LB0 that this reduction was significant (P<0.05). Addition of OP and bacterial additives reduced CP contents of silages compared to the control (P<0.05).
The effects of inoculation and OP on aerobic stability of wilted alfalfa silages are given in Figure 1. Additional OP to alfalfa silage decreased aerobic stability compared to control and other treatments. The effect of noculation and OP supplementation on bacterial populations of the resultant silages are shown in Table 3. The addition of OP to wilted alfalfa did not affect yeast and fungi population but the OP silage increased the population of lactobacilli and total bacterial numbers compared to the control treatment (P<0.01). Higher bacterial additives level were significantly decreased the population of yeasts and fungi. This reduction increased with increasing intakes of additives and significant differences were observed among different bacterial treatments (P<0.01).
Effect of treatments on in situ DM disappearance on incubation times are shown in Table 4 and Figure 2. Adding OP to alfalfa silage significantly increased the dry matter degradation at all incubation times except for 0 h (P<0/05). Rapidly degraded fraction (a) for LB3 and LB2 was higher than the other treatments, Treatments; alfalfa with no additive (CON), alfalfa (70%) with fresh orange pulp (30%) and treated with LAB for final application rate 0, 1.5, 3 and 4.5 g LAB inoculant/ton (LB0, LB1, LB2 and LB3, respectively).
Within a row, means followed by different letters differ (P<0.05).
which it shows these treatments had high level of nonstructural carbohydrates (Table 5). All treatments except control had greatest amount of fraction b (insoluble but fermentable component; P<0.05).
Effect of treatments on in vitro DM degradation on incubation times are shown in Table 6 and Figure 3. After 24 h incubation LB0 and control treatments had the highest and lowest DM degradability among treatments, respectively (P<0.05).

DISCUSSION
The results showed that the addition OP to alfalfa silage reduce pH compared to control (P<0.05). The results obtained in the current study were consistent with the results of Touqir et al. (2007)       This is probably due to the use of citrus pulp in that experiment was much lower than our test. The results of studies by Silva (2002), who was ensiled Tanzania forage along with citrus pulp for 60 days also conformed. Silva (2002) reported that the addition of 120 g per kg of dry matter from the peels to feed increased 3 percent is soluble carbohydrate. Asadi et al. (2002)   0.082 0.575 0.0005 † Treatments; CON: alfalfa with no additive, alfalfa (70%) with fresh orange pulp (30%) and treated with LAB for final application rate 0, 1.5, 3 and 4.5 g LAB inoculant/ton (LB0, LB1, LB2 and LB3, respectively). Within a column, means followed by different letters differ (P<0.05). KSU J. Agric Nat 23 (6) and Koç et al. (2020). Evaluated 14 types of bacterial additive on the first cutting of alfalfa hay, reported that all additives increased VFA. The increase in total volatile fatty acids can be attributed with high populations of LAB and the availability of carbohydrate source to produce organic acids such as lactic acid and acetic acid in the treatments compared to the control.
Bacterial additives at all 3 levels compared to control treatment did not cause a change in the NDF. Ranjit and Kung (2000), Asadi et al. (2002), Kizilsimsek et al. (2007), Hashemzadeh et al. (2011) and Besharati et al., (2017 reported similar results with the results of the current study. In Filya et al. (2007) studied, at 6 additives an increase in the NDF, 5 additives without change and 3 additives reported a decrease in the NDF compared to control. In the second stage of this study on the second cutting alfalfa forage 10 additives decreased, 2 Additives increased and 2 additives unchanged compared to the control.
Orange pulp significantly increased ADF in LB0 compared to the control (P<0.05). Bacterial additive consumption in all 3 levels decreased ADF compare to LB0 significantly (P<0.05). In Ranjit and Kung (2000) experiment consumption, low level of L. Buchneri was reduced ADF. In a study conducted by Kung and Ranjit (2001) L. Buchneri additives used at 3 levels on barley hay, which reduces numerically ADF in 100-day silage. Hashemzadeh et al. (2011), , Adesogan et al. (2004, Khorvash et al. (2006) ; Kizilsimsek et al. (2007), Besharati et al., (2017, in a different experimental reported reduction in the ADF that affected by bacterial inoculant. Addition of OP and bacterial additives caused to reduce crude protein (CP) compared to the control (P<0.05). This is possibly due to low crude protein content in OP. Hashemzadeh et al. (2011) in their study on the effect of adding molasses to alfalfa reported a reduction in crude protein content with increasing levels of molasses. Bacterial additives at low levels control did not cause a change in the amount of crude protein compared to LB0, but at higher levels of additives reduced crude protein than the LB0 treatment (P<0.05). Addition of OP and bacterial additives caused to improve silage quality index compared to the control. Uher et al., (2019) bacterial inoculant treatments had lower values of ADF, NDF and pH and significantly lower values butyric acid, alcohols and NH3-N/total nitrogen compared to control silage and showed that bacterial inoculant increases silage quality compared to control silage.
Additional OP to alfalfa silage decreased aerobic stability compared to control and other treatments.
The aerobic stability in all L. Buchneri additives were used increased compare to control and LB0 treatment that this rate of increase in aerobic stability, improve by increasing the amount of L. Buchneri (LB1,LB2 and LB3,144,192 and 200,respectively). numerical increase in population of yeasts and fungi that the increase was not significant compared to the control treatment (P>0.01) but the OP silage increased the population of lactobacilli and total bacterial numbers compared to the control treatment (P<0.01). This can be explained by an increase in carbohydrate resources available for growth all microbial species present in the silage. Bacterial additives in their higher level were significantly decreased the population of yeasts and fungi, that this reduction, increased with increasing intakes of additives and significant differences were observed among different bacterial treatments (P<0.01). Also, bacterial additives caused to increase the population of lactobacilli and total bacterial numbers in the silage compare to treatment LAB0. This increase has a direct and significant relation with higher amounts of bacterial additives that used (P<0.01). Schmidt et al. (2009) Kizilşimşek et al. (2020) showed that bacterial inoculation decreased the pH level, increased the production of lactic acid, whereas decreased the acetic acid, propionic acid and butyric acid in silage content. L. brevis strain as well as wilting can be used successfully in alfalfa silage production when crude protein and lactic acid production in silage were taken into consideration. Dumlu Gül et al. (2015) suggested that in order to make successful silage from alfalfa, addition of molasses + rolled barley, cutting alfalfa in late flowering and wilting after cutting can be useful.
Adding OP to alfalfa silage did not caused any effect on DM digestibility at 0 h of the test, but from 2 pm to 96 hours significantly increased digestibility compared to control treatment (Table 4) (P<0.05). The increase in digestion, similar to other studies that used different sources of carbohydrates, due to higher soluble carbohydrate content of this treatment seems reasonable. Overall degradation due to the break in the silage treated with molasses Ligno celluloses links between structural composition and amount of soluble carbohydrates in this category silage (Rowghani and Zamiri, 2009). Bacterial additives at a low level until 12 h demonstrated no change in digestion compare to LB0, but from 12 h to 96 h was significantly reduced digestibility (Table 4) (P<0/05). At higher levels of additives from 6 h to the end of trial except 8 h, significant reductions were observed in digestibility compare to LB0 (Table 4)  Rapidly degraded fraction (a) for LB3 and LB2 was higher than the other treatments, which showeed treatments had high level of nonstructural carbohydrates (  (2020), positively affected fermentation properties and feed quality parameters of treatments compared to control.

CONCLUSION
Results of this study ndicated that LAB can improve the aerobic stability and silage quality in laboratory silos and also, OP and LAB might improve silage quality and cause a better management of alfalfa silage in farms.

Ethical statement
None of the animals were harmed during the collection of in situ trial and in vitro trials.

Conflict of interest
We have declared no conflict of interest.