doi:10.14720/aas.2020.115.2.1483 original research article / izvirni znanstveni članek Comparison study of flaxseed, cinnamon and lemon seed essential oils additives on quality and fermentation characteristics of lucerne silage Maghsoud BEsHARATI 1, Valiollah PALANGI 2 3, Masomeh NIAZIFAR 1, Zabihollah NEMATI 1 Received February 04, 2020; accepted May 5, 2020. Delo je prispelo 04. februarja 2020, sprejeto 05. maja 2020. Comparison study of flaxseed, cinnamon and lemon seed essential oils additives on quality and fermentation characteristics of lucerne silage Abstract: This experiment was performed to investigate the effects of some essential oils on chemical properties and aerobic stability of lucerne silage. Treatments included lucerne silage without additives (control), lucerne silage with 60 mg cinnamon essential oil/kg, lucerne silage with 60 mg flaxseed essential oil/kg, lucerne silage with 60 mg lemon seed essential oil/kg, lucerne silage with 180 mg blend of essential oils (60 mg cinnamon + 60 mg flaxseed + 60 mg lemon seed essential oils/ kg). Adding essential oils to lucerne silage reduced silage pH (p < 0.001) compared to control. The highest level of total volatile fatty acids (tVFA) was found when lemon seed essential oil and the lowest level when flaxseed essential oil was used. The lucerne silages treated with essential oils had the highest crude protein contents (p < 0.01). Untreated lucerne silage had the highest level of gas production compared to lucerne silage treated with lemon seed and flaxseed essential oils (p < 0.01). The essential oil additives increased the aerobic stability of the silage. It can be concluded that the use of essential oil additive in the preparation of high quality lucerne silage, can improves the quality and nutritive value of silages. Key words: lucerne silage; essential oils; medicinal plants; nutritive value Primerjava učinkov eteričnih olj lanenih semen, cimeta in limoninega semena na kemično sestavo in fermentacijske značilnosti silaže lucerne Izvleček: Namen poskusa je bil preučiti učinke nekaterih eteričnih olj na kemijske lastnosti in aerobno stabilnost silaže lucerne. obravnavanja so obsegala silažo lucerne brez dodatkov (kontrola), silažo lucerne z dodatkom cimetovega eteričnega olja, silažo lucerne z dodatkom eteričnega olja iz lanenega semena in silažo lucerne z dodatkom eteričnega olja iz semen limone (60 mg kg-1) in silažo lucerne z dodatkom 180 mg mešanice eteričnih olj na kg silaže (60 mg cimetovega + 60 mg lanenega + 60 mg limoninega eteričnega olja na kg silaže). Dodajanje eteričnihj olj je znižalo pH silaže (p < 0,001) v primerjavi s kontrolo. Največja vsebnost celokupnih hlapnih ma-ščobnih kislin (tVFA) je bila izmerjena pri dodatku eteričnega olja semen limone in najmanjša pri dodatku eteričnega olja iz lanenega semena. silaža lucerne, ki je bila obdelana z eteričnimi olji je imela največjo vsebnost surovih beljakovin (p < 0,01). Neobdelana silaža lucerne je imela največjo proizvodnjo plina v primejavo s silažo obdelano z eteričnimi olji iz semen limone in lana (p < 0,01). Dodatek eteričnih olj je povečal aerobno stabilnost silaže. Zaključimo lahko, da dodatek eteričnih olj pri pripravi kvalitetne silaže lucerne izboljša njeno kakovost in hranilno vrednost. Ključne besede: silaža iz lucerne; eterična olja; zdravilne rastline; hranilna vrednostnutritive value 1 University of Tabriz, Ahar Faculty of Agriculture and Natural Resources, Department of Animal science, Iran 2 ataturk University, agricultural Faculty, Department of animal science, Erzurum, turkey 3 Corresponding author, e-mail: dizajparvane@yahoo.com Acta agriculturae Slovenica, 115/2, 429-436, Ljubljana 2020 115-2 vsebina.indd 455 23. 06. 2020 07:25:36 M. BESHARATI et al. 1 INTRODUCTION Lucerne is one of the most nutritious forage crops for ruminants. In areas with adverse climates, and in areas where there is not enough time to dry the late harvested lucerne the lucerne is usually ensiled. Lucerne is often difficult to ensile because of its high protein, high buffering capacity and low water-soluble carbohydrate contents (WsC) (Khadem et al., 2009). The use of silage additives could improve the silage quality and its nutritive value. As an option for silage additives the essential oils can be used. Essential oils are natural secondary metabolites that are responsible for providing plants and spices with their characteristic essence and color (Beauchemin, 2008). These non-nutritive and biologically active compounds accumulate in various plant tissues and are extracted by steam-based distillation. Essential oils and their compounds are known to be active against a wide variety of micro-organisms, including Gram-negative bacteria, Gram-positive bacteria and fungi. Although the microbial effect of plant essential oils is recognized, there is limited research about these substances to be used as silage additives. For example, Fraser et al. (2007) observed a reduction in ammonia nitrogen (NH -N) concentration in culture medium by using cinnamon essential oil. Mcintosh et al. (2003) also used a commercial blend of essential oils and observed inhibition of hyper-ammonia producing bacteria. Moreover, some of former studies demonstrated the potential of essential oils to alter rumen fermentation by reducing the proportion of acetate to propionate and also by inhibition of methano-genesis (benchear et al., 2007). The aim of this study was to determine the potential use of flaxseed, cinnamon and lemon seed essential oils as silage additives on chemical composition and nutritive value of lucerne silage. cedure, oven-dried at 39 °C for 48 h and ground to pass a 1 mm-screen. Essential oils content of each plant was obtained with hydro-distillation of grinded samples using clevenger apparatus (Jahani-Azizabadi et al., 2014). The obtained essential oils were stored in refrigerator (4 °C) until they were used in the experiment. 2.2 experimental treatments and silage preparation The chemical composition of ensiling material is presented in Table 1. The fourth cut of lucerne was dehydrated for 24 hours. Then it was chopped at 3-5 cm length and ensiled in laboratory scale mini pvc silos (3 ± 0.25 kg) for 60 days. Treatments included lucerne silage without additives (control), lucerne silage with 60 mg cinnamon essential oil/kg (C60), lucerne silage with 60 mg flaxseed essential oil/kg (F60), lucerne silage with 60 mg lemon seed essential oil/kg (L60), lucerne silage with 180 mg blend of essential oils (60 mg cinnamon + 60 mg flaxseed + 60 mg lemon seed essential oils/kg; M60). All additives were dissolved in 120 mg kg-1 aqueous ethanol solution (Chaves et al., 2012) and sprayed onto the chopped forages. The same amount of the aqueous ethanol solution was also added to the control. Three silos for each treatment were made and stored at ambient temperature (28 °C to 33 °C). All silos were opened after 60 days of ensiling and the contents were used for the determination of the silage chemical composition, nutritive value and aerobic stability. Analyses of silage composition and nutritive value were done on the contents of individual silo and averaged for use in the statistical analyses. 2.3 chemical COMPOSITION 2 MATERIALS AND METHODS 2.1 essential oils preparation Cinnamon, flaxseed and lemon seeds, used in this study, were purchased from local markets in Ahar and Tabriz cities. The samples obtained were cut or crushed into small pieces according to Palangi et al. (2012) pro- After the opening of silages, the pH, dry matter (DM) and soluble carbohydrate (WsC) of the samples were determined. DM content of the silages was determined by oven drying of lucerne samples (65 °C for 48 h). DM, ash (CA), ether extract (EE) and crude protein (CP) contents were determined by the procedures given by AoAC (2002). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) concentrations were deter- Table 1: Chemical composition of lucerne before ensiling (% DM) Item Chemical composition ADF NDF wsc CA CP pH DM Lucerne 17 ± 1.40 24.8 ± 1.058 3.74 ± 0.087 11.6 ± 0.028 19.6 ± 0.427 6.14 ± 0.011 22.2 ± 0.975 DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; WCs: water soluble carbohydrates, CA: Crude Ash. 456 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 456 23. 06. 2020 07:25:39 Comparison study of flaxseed, cinnamon and lemon seed essential oils additives on quality and fermentation characteristics of lucerne silage mined according to Van Soest et al. (1991) procedures without the use of sodium sulphite. NDF was analyzed without amylase and contains the ash. Aqueous extract was prepared from ensiled samples by mixing 20 g of forage with 180 ml of deionized water and homogenizing this mix for 1 min. Then, silage pH was determined using a portable pH meter. Ammonia-N (NH3-N) concentration of acidified silage extracts were determined using Kjeldahl method. Phenol sulfuric acid method was used to measure WSC contents (Dubios et al., 1956). The distillation method described by Markham (1942) was used to measure total volatile fatty acids (tVFA) in silages. One ml of 25 % meta-phosphoric acid (v/w) was added to 5 ml of filtered extract to calculate the volatile fatty acids. For the determination of lactic acid (LA) contents, the method of Borshchevskaya et al. (2016) was used. double layer of sterile cheesecloth was placed over each container to prevent drying and contamination but allow penetration of air. Silage and ambient temperature were recorded manually every two hours until heating occurred. 2.6 STATISTICAL ANALYSIS Obtained data from this study were subjected to analysis as a completely randomized design by the GLM procedure of SAS (SAS, 2002) and Duncan's multiple range test was used for the comparison of means. Significance was declared at p < 0.01. 3 RESULTS AND DISCUSSION 2.4 In vitro GAS PRODUCTION 3.1 CHEMICAL COMPOSITION Ruminal fluid was collected approximately 2 h after morning feeding from two fistulated sheep. Gas production was measured by Fedorak and Hrudy (1983) method. Approximately 300 mg of dried and ground (2 mm) samples were weighed and placed into serum bottles. Rumen fluid buffered with McDougall (1948)'s buffer (20 ml) was pipetted into each serum bottle. The me-tabolizable energy (ME; MJ kg-1 DM) content of samples was calculated using equation of Getachew et al. (2004) equation. The short chain fatty acid (SCFA) and organic matter digestibility (OMD) for feeds were calculated using equations of Menke et al. (1979) equations. Gas production parameters were calculated using the following mathematical model in the SAS package program according to the model reported by 0rskov and McDonald (1979). P = a + b (1-e-c(t)) where 'P' is the disappearance at time 't', 'a' quickly degradable fraction (or washing loss), 'b' denotes slowly degradable fraction and 'c' is constant rate of degradation of 'b' (Palangi and Macit, 2019). 2.5 AEROBIC STABILITY Aerobic stability of silages represents the time (hours) during which the temperature of silage do not increase more than 2 oC above ambient temperature (Moran et al., 1996). Aerobic stability was determined on all treatments. About 400 g of each silage was transferred into separate 1l containers. The containers were implanted with thermocouples to monitor temperature. A Effect of essential oil additives on chemical composition of lucerne silage are shown in Table 2. The highest DM contents were observed for F60 and C60 silages which were significantly higher than other silages (p < 0.05). This effect could be due the consequence of limited development of specific microorganisms and therefore smaller loss of nutrients (Selwet, 2009). The CP concentrations of M60 and C60 silages were significantly higher than in other silages (p < 0.01). Degradation of protein in silage is a consequence of proteolytic microorganisms, such as Clostridia and/or enterobacteria (McDonald et al., 1991). Inhibitory effects of essential oils on growth of some microorganisms such as Clostridia reported in previous studies (Ismaiel and Pierson, 1990). Addition of essential oils to lucerne increased the protein content significantly after 60 d ensiling. These results were in agreement with findings of Soycan-Önen^ et al. (2017) and Chaves et al. (2012). The results of current study show that the addition of M60 did not affect DM contents of silages, what was not in agreement with the findings of Soycan-Önen^ et al. (2017). The pH of silages supplemented with essential oils was significantly lower than control group (p < 0.01). The obtained results of this study were in agreement with the findings of Kung et al. (2000). In addition, the pH values of all silages were lower than those obtained by Bolsen et al. (1996). In the study by Soycan-Önen^ et al. (2017), the pH values of 4.40 and 4.47 were determined in the silages prepared by addition of oregano and cinnamon essential oil to field peas. Higher pH values in majority of the treated silages could be the result of the reduced activity of Lactobacillus bacteria, as LA could decrease the pH of the silage (Kung and Ranjit, 2001). However, in some of Acta agriculturae Slovenica, 115/2 - 2020 457 115-2 vsebina.indd 457 23. 06. 2020 07:25:39 M. BESHARATI et al. Table 2: Effect of essential oils on chemical properties of lucerne silage after 60 d of silage (% DM) Treatments1 Chemical composition2 DM NDF ADF WSC tVFA nh3-n CA CP LA PH EE Control 24.44c 49.07a 22.67b 4.09b 12.63b 84.93a 11.40c 11.62d 69.38d 4.65a 4.27c C60 25.68b 42.43bc 25.67a 4.78a 11.65c 79.80b 11.66b 12.29b 80.29a 3.66b 3.94c F60 26.82a 43.17b 15.34d 4.05b 10.36d 83.53a 10.80d 12.22c 71.22c 3.73b 4.07c L60 24.50c 48.03a 19.34c 4.63a 14.90a 85.16a 12.31a 12.37b 76.23b 3.61b 4.73a M60 24.23c 39.60c 18.67c 4.24b 12.77b 83.53a 11.70b 12.54a 70.82c 3.79b 4.53ab SEM 0.308 1.039 0.615 0.066 0.049 0.660 0.518 0.043 0.244 0.055 0.123 p-value 0.0005 0.0003 <.0001 <.0001 <.0001 0.0013 <.0001 <.0001 <.0001 <.0001 0.0055 Treatment-control: Lucerne silage without additives, C60: lucerne silage with 60 mg cinnamon essential oil kg-1, F60: lucerne silage with 60 mg flaxseed essential oil kg-1, L60: lucerne silage with 60 mg lemon seed essential oil kg-1, M60: lucerne silage with 180 mg blend of essential oils (60 mg cinnamon essential oil + 60 mg flaxseed essential oil + 60 mg lemon seed essential oil kg-1). Chemical composition2: DM, dry matter; CP, crude protein; EE, ether extract; CA, crude ash; NDF, neutral detergent fiber; ADF, acid detergent fiber; NH3-N: ammonia nitrogen (% of total nitrogen), tVFA: total volatile fatty acid (mmol), LA: lactic acid. WSC: water soluble carbohydrate. Means within same column with different superscripts differ (p < 0.05). the previous studies, essential oils had no inhibitory effect on lactic acid producing bacteria (Kung et al., 2008). NH3-N content significantly decreased in C60 silage (p <3 0.01; Table 2). The treatments F60, L60 and M60 had no effect on NH3-N concentration, which is in agreement with the findings of Kung et al. (2008). In the study of Hodjatpanah et al. (2016), essential oils of cinnamon added to ensiling material in amounts of 120 and 240 mg kg-1, had no effect on NH3-N in corn silage, whereas essential oils of oregano and thyme in same amounts and peppermint essential oil in amount of 120 mg kg-1 level decreased NH3-N content. The amounts of tVFAs determined in silages in this experiment was different by the addition of essential oil (Table 2). The highest amount of tVFA was found in L60 and the lowest in F60. Essential oils obtained from cinnamon and flaxseed decreased tVFA concentration in comparison to control (p < 0.01). It is possible that the decrease of NDF and ADF contents is affected by pH. Soycan-Onen^ et al. (2017) determined that LA content decreased in field pea silages prepared with the addition of oregano, cinnamon, and oregano+cinnamon essential oils. However, in this study it was determined that there was an increase in LA contents. While F60 and M60 reveal a decreasing effect on LA amount through inhibiting beneficial microorganisms, C60 caused increase in lactic acid amount by promoting beneficial microorganisms activity. 3.2 GAS PRODUCTION Relative to the control, total produced gas from L60 and F60 decreased (p < 0.01) and M60 produced more gas (p < 0.01) after 120 h of incubation (Table 3). The chemical composition of silage can influence on the rumen microbial fermentation patterns (Navarro-Villa et al., 2013). Furthermore, some of essential oils have a good potential to alter rumen microbial fermentation and specially reducing rumen methanogenesis (Jahani-Azizabadi et al., 2014) and ammonia producing bacteria in the rumen (Mcintosh et al., 2003). Effects of essential oils on rumen microbial populations are dose-dependent (Macheboeuf et al., 2008). Effects of essential oils on NH3-N concentrations were only noticed during the first 120 h of incubation. At 120 h, treatments M60, F60 and L60 had higher (p < 0.01) ammonia concentration than the control (Table 4). The cumulative curve of gas production parameters in different lucerne silage treatments is shown in Figure 1. At the end of incubation, the highest volume of gas produced was obtained with M60 with 148.54 ml g-1 DM and the lowest was for treatment F60 with 123.43 ml g-1 DM. The results of this experiment are in agreement with the findings of Hodjatpanah et al. (2016) and Chavez et al. (2012). Aminipour et al. (2017) used thyme essential oils as an additive to alter the fermentation characteristics of lucerne silage in ruminants and showed that thyme essential oil reduced the amount of gas produced in comparison with control silage. Fraser et al. (2007) used cinnamon essential oil which reduced the amount of gas produced after 24 h of incubation. Thestudy of Busquet et al. (2005) showed that the use of garlic essential oil reduced the amount of gas production after 17 hours of incubation and that the increasing levels of garlic essential oil levels decreased the in vitro gas production. The mean gas produced from the potential degrad-able (b) was significantly different between the experi- 458 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 458 23. 06. 2020 07:25:40 Comparison study of flaxseed, cinnamon and lemon seed essential oils additives on quality and fermentation characteristics of lucerne silage Table 3: The effect of different essential oils on gas production of lucerne silage (ml g-1 DM) Treatments1 Incubation times (h) 2 4 6 8 12 16 24 36 48 72 96 120 Control 17.61b 32.40b 40.06b 51.72b 65.18b 82.28b 103.98b 111.98b 123.10b 130.90b 134.42b 135.52b C60 15.51bc 30.83bc 38.50bc 45.83c 55.94c 70.79c 89.02c 104.75c 115.54c 122.13c 126.47c 128.70c F60 14.18c 27.50c 35.03c 45.22c 56.01c 69.19c 88.70c 99.56c 110.29c 118.94c 121.80c 123.43c L60 13.59c 27.03c 37.50bc 50.50b 60.74bc 73.11c 89.16c 102.22c 112.14c 117.74c 122.87c 124.97c M60 20.24a 40.02a 50.01a 62.48a 76.33a 91.63a 110.93a 126.12a 137.12a 143.79a 147.74a 148.54a SEM 0.802 1.392 1.344 1.490 1.561 1.845 2.101 1.981 2.445 2.434 2.397 2.411 Treatment-control: Lucerne silage without additives, C60: lucerne silage with 60 mg cinnamon essential oil/kg, F60: lucerne silage with 60 mg flaxseed essential oil kg-1, L60: lucerne silage with 60 mg lemon seed essential oil kg-1, M60: lucerne silage with 180 mg blend of essential oils (60 mg cinnamon essential oil + 60 mg flaxseed essential oil + 60 mg lemon seed essential oil kg-1). Means within same column with different superscripts differ (p < 0.05). Figure 1: The effect of essential oils on gas production at different incubation times of lucerne silage mental treatments. The treatment M60 had the highest b and c amounts among the treatments (p < 0.01). Lucerne silage prepared with addition of essential oils lucerne had a significant effect on tVFA and NH3-N. Treatments M60 and L60 had the highest tVFA and NH3-N among treatments, respectively. Treatments F60 and C60 increased the amounts of tVFA and NH3-N in comparison with control, respectively. Hart et al. (2008) using medium containing a set of rumen microorganisms showed that limonene, thymol, vanillin, guaiacol and oregano extract reduced rumen NH3-N concentration. Brochers (1965) showed that the addition of thymol to rumen fluid resulted in the accumulation of amino acids and a decrease in NH3-N concentration. He suggested that the thymol prevents the deamination of amino acids by rumen bacteria. It seems that since the plant essential oils have inhibitory effects on proteolysis and deamina-tion, their inhibitory effects on proteolytic activities may reduce the degradation of the silage protein and consequently decrease the ammonia nitrogen content. The re- duction in NH3-N concentration has been attributed to the antimicrobial activity of essential oils. This property limits the fermentation process and reduces the breakdown of protein into ammonia. It has also been suggested that effective compounds in essential oils are able to bind to proteins, which reduces nitrogen loss. 3.3 AEROBIC STABILITY All silages treated with essential oils improved the aerobic stability compared with control, of which control obtained 77 h, while M60, C60, L60, and F60 obtained 112, > 99.33, > 96.66, >92 h, respectively. Higher aerobic stability of treated silages was in agreement with the findings of Chaves et al. (2012). In their experiment, silages treated with oregano or cinnamon leaf essential oils at 120 mg kg-1 remained stable for two weeks. Exposure to air in silos may result in silage deterioration. The increase in temperature is the result of the metabolism of organic Acta agriculturae Slovenica, 115/2 - 2020 459 459 115-2 vsebina.indd 457 23. 06. 2020 07:25:39 M. BESHARATI et al. Table 4: The effect of experimental treatments on gas production parameters of lucerne silage Treatments1 Items2 pH nel SCFA ME OMD DOMD tVFA nh3-n b c Control 6.60* 1.26a 0.152a 3.22a 27.23a 24.01* 8.62b 47.22c 133.74b 0.0592b C60 6.63* 1.17bc 0.128bc 3.08bc 26.55* 23.46bc 7.14c 29.36d 126.78bc 0.0526d F60 6.60* 1.20ab 0.137ab 3.14ab 26.90a 24.26a 3.00d 47.91c 121.62c 0.0539dc L60 6.50b 1.10c 0.1142c 3.00c 26.05b 23.01c 3.26d 51.10a 121.92c 0.0579bc M60 6.65a 1.20ab 0.1360ab 3.13ab 26.92a 23.74* 12.92a 49.71b 145.34a 0.0645a SEM 0.042 0.026 0.006 0.036 0.240 0.202 0.438 0.467 2.488 0.002 p-value 0.1641 0.0041 0.0041 0.0047 0.0244 0.0016 <.0001 <.0001 <.0001 0.0006 Treatment-control: Lucerne silage without additives, C60: lucerne silage with 60 mg cinnamon essential oil kg-1, F60: lucerne silage with 60 mg flaxseed essential oil kg-1, L60: lucerne silage with 60 mg lemon seed essential oil kg-1, M60: lucerne silage with 180 mg blend of essential oils (60 mg cinnamon essential oil + 60 mg flaxseed essential oil + 60 mg lemon seed essential oil kg-1). ME: metabolizable energy (MJ/Kg DM); SCFA: short chain fatty acid (mmol 0.2 g-1 DM); DOMD: digestible organic matter in dry matter (%); NEL: net energy lactation (MJ kg-1 DM); tVFA: total volatile fatty acids (mmol l-1); NH3-N: ammonia nitrogen (mg l-1); OMD: organic matter digestibility (%); b: gas production of the potentially degradable (insoluble); c: fraction after 24 h incubation (ml 200 mg-1 DM). Means within same column with different superscripts differ (p < 0.05). — — F I f-i m -f is. 'j-j r- r- oo oo C- c^. O-,