ORIGINAL

Efficacy of essence oil supplementation to feeds on volatile fatty acid production

La eficacia de los suplementos de aceite de esencia en la producción de ácidos grasos volátiles

Ahmet Tekeli,¹* Ph.D, Gültekin Yιldιz,² Ph.D, Winfried Drochner,³ Ph.D, Herbert Steingass,³ Ph.D.

¹ Yuzuncu Yιl University, Faculty of Agriculture, Department of Animal Science, 65080, Van/Turkey.
² Ankara University, Faculty of Veterinary, Department of Animal Nutrition and Nutritional Diseases, 06110 Ankara/Turkey.
³ Hohenheim University, Department of Animal Nutrition, 70599 Stuttgart/Germany.

*Correspondence: atekeli@yyu.edu.tr

Received: October 2014; Acepted: March 2015.

ABSTRACT

Objective. Determine the effect of some plant extract supplementation to Total Mixed Ration (TMR), concentrate and hay on volatile fatty acid (VFA) production at 8 and 24 hours (h) using in vitro gas production technique in cattle. Material and methods. Three fistulated Holstein dairy cows were used for rumen fluid collection for application of in vitro gas production technique. Four essence oils (T. vulgaris, O. vulgare, S. aromaticum, Z. officinale) were used as plant extracts. Results. Essence oil supplementations to the examined feed groups had significant effect only on C2/C3 VFA level at 8 h in all feed groups (p<0.05). C2/C3 VFA level at 8 h significantly increased in the groups with Oregano 25 ppm supplementation for TMR and concentrate and in the groups with Thymol 25 ppm supplementation for hay. C3 VFA level at 8 h significantly increased in the group that received Syzygium 200 ppm supplementation for hay. Different plant extracts supplemented to TMR, concentrate and hay significantly affected C2, C3, IC4, IC5, C5 and C2/C3 VFA levels at 24 h (p<0.05). Conclusions. The findings of the study indicate that moderate doses of plant extracts result in increased VFA levels in ruminants while higher doses demonstrate the opposite effect.

Key words: Aromatic plants, in vitro, plant extract, ruminant nutrition, volatile fatty acid (Source: USDA).

RESUMEN

Objetivo. Determinar el efecto de los suplementos de algunos extractos de plantas a Ración Total Mezclada (TMR), concentrado y heno sobre los ácidos grasos volátiles (AGV) a las 8 y 24 horas (h) utilizando la técnica de producción de gas in vitro en el ganado bovino. Material y métodos. Tres vacas lecheras Holstein fistuladas se utilizaron para la recogida de fluido ruminal y aplicar la técnica in vitro de producción de gas. Cuatro aceites esenciales (T. vulgaris, O. vulgare, S. aromaticum, Z. officinale) fueron utilizadas como extractos de plantas. Resultados. La suplementación de aceite esenciales a los grupos de alimentos estudiados tuvieron efecto significativo sólo en C2 / nivel C3 VFA a las 8 h en todos los grupos de alimentos (p<0.05). El C2 / nivel C3 VFA a las 8 h aumentó significativamente en los grupos con suplementación de orégano 25 ppm de la RTM y concentrado y en los grupos con suplementación de Timol a 25 ppm para el heno. El C3 nivel de VFA en 8 h aumentó significativamente en el grupo que recibió suplementación de Syzygium a razón de 200 ppm para el heno. Los diferentes extractos de plantas suplementadas con RTM, concentrado y heno afectó significativamente los niveles de C2, C3, IC4, IC5, C5 y C2/C3 VFA a las 24 h (p<0.05). Conclusiones. Los hallasgos del estudio indican que las dosis moderadas de extractos de plantas incrementan los niveles de AGV en rumiantes, mientras que dosis más altas producen el efecto contrario.

Palabras clave: Ácidos grasos volátiles, in vitro, extracto de la planta, nutrición de rumiantes, plantas aromáticas (Fuente: USDA).

INTRODUCTION

Energy and protein losses in rumen fermentation reduce the efficiency of nutrient utilization and cause environmental pollution. Ionophore antibiotics have been successfully used in rumens for many years to reduce energy and protein losses (1). While ionophore antibiotics have been widely used in conventional production systems for the control of rumen fermentation, the European Commission (EC) decided to phase out, and ultimately ban the use of antibiotics (January 1st 2006) (2). After the ban on antibiotic growth promoters, studies have increased on exploring alternatives to antibiotics. One of these possible alternatives are the plant extracts, which are also called as essence oils. Essence oils are plant secondary metabolites responsible for odour and colour of plants (3).

The concentrations of essence oil within plants vary according to stage of growth, stress, plant health and environmental factors such as light, temperature and moisture etc. (4). Essence oils are found throughout the plants, including roots, the bark, flowers, petals, leaves, fruit bodies and stems. Essence oils are the volatile components responsible for the characteristic aroma of spices. As active components in many spices and preservatives, essence oils have historically been used to inhibit bacterial growth (5). All essence oils have aromatic properties. They have also shown biological activities, such as antioxidative (6), antifungal (7) and antimicrobial activities (8), Furthermore they have been found to have psychological and physical effects, which is why they have found their application in aromatherapy.

One of the alternative to antibiotic feed supplements are plant extracts. In this study, Oreganum vulgare, Thymus vulgaris, Zingiber officinale, Syszygium aromaticum essence oils were supplemented to TMR, concentrate and hay to explore their effects on the levels of volatile fatty acid (VFA) concentrations at 8 and 24 h in in vitro conditions.

MATERIAL AND METHODS

Animals and feeds. Three fistulated Holstain dairy cows were used for rumen fluid collection for application of in vitro gas production technique. For each feed (TMR, Concentrate and Hay), ruminal fluid samples were collected twice daily on different days. Four essence oils (T. vulgaris, O. vulgare, S. aromaticum, Z. officinale) were used as plant extracts. T. vulgaris, S. aromaticum, and Z. officinale essence oils were obtained from Manisa Province (Turkey) and O. vulgare essence oil from Mersin Province (Turkey). All plant extracts were extracted with distilated water. For each extract, different doses were tested to determine harmful and usable doses. Incubation was run for each regulation in 8 and 24 h time periods. Total mixed ration (TMR), concentrate and hay were used as substrates. In the first phase of the experiment, gasification quantity was measured. This step was iterated for the effects of each plant extract and each dose on substrates (TMR, concentrate, hay). After gasification measurements, harmful extract doses and critical time periods were identified. In the second phase of the experiment, VFA levels were measured.

The group with only rumen liquor was assigned as the negative control group and the group with rumen fluid and feed samples was assigned as the positive control group. The groups with rumen fluid, feed samples and thymol, oregano, zingiber and syzygium essence oils were the treatment groups. The compositions of TMR, concentrate and hay used in the experiment are presented respectively in table 1. The effects of Syzygim aromaticum, Zingiber officinale, Thymus vulgaris, Oreganum vulagare supplementation to TMR, concentrate and hay on VFA production were assessed in the present experiment by incubation of in buffered rumen fluid using an in vitro gas production technique (HFT).

Sample preparation for VFA. Subsequent to the preparation of samples on experiment day, 1:11 with an internal Standard (4-Methylvalerianic acid and formic acid 1:101) was prepared in an hour for measurement of acetic, propionic, i-butiric and n-butiric acid as well as i-valerianic and n-valerianic acid. The samples were frozen at -20°C. Before determination of the fatty acids, the samples were thawed at room temperature and centrifuged in 400 g for ten minutes and afterwards, bright parts were taken for measurements.

Analysis. Concentrations of VFAs were analysed in pooled samples of each replicate, using a gas chromatograph (GC 14B, Shimadzu, Japan) equipped with a flame ionisation detector, and samples were treated with an Internal Standard (4-Methylvalerianic acid and formic acid 1:101) according to Geissler et al (9). A total of 6 parallel analyses were performed for each sample.

Statistical analysis. The descriptive statistics for the examined parameters were expressed in terms of average and standard errors. Factorial Analysis of Variance (Factorial ANOVA) was conducted to determine any differences between the means of supplements and feed types with respect to the examined parameters. Additionally, Repeated Measures ANOVA was performed to determine any differences with respect to feeds and durations (hours). Following analyses of variance, Tukey test was conducted to determine varying means (10). Statistical significance level was set at 5% and calculations were performed by SPSS (11) statistical software package.

RESULTS

The descriptive statistics of some VFAs at 8 and 24 h according to supplement and feed groups and their comparative results according to supplement groups are given in tables 2 and 3.

As seen in table 2, compared to negative control groups, essence oil supplementations had significant effect on C2, C3, IC4, C4 IC5, C5 and C2/C3 VFA level at 8 h (p<0.05). For TMR, Concentrate and hay group, C2, C3, and C4 VFA levels increased at 8 h in the treatment groups with essence oil supplementations compared to negative control group (p<0.05). On the contrary, for TMR, C4 VFA level decreased in the group with Syzygium 200 supplementation with respect to positive control group (p<0.05). For concentrate group, C4 VFA level decreased at 8 h in the group with Syzygium 200 supplementation compared to positive control groups (p<0.05). For hay group, C4 VFA level increased at 8 h in the other treatment groups with respect to negative control group (p<0.05).

Table 3 indicates that essence oil supplementations had significant effect on C2, C3, IC4, IC5, C5 and C2/C3 VFA levels at 24 h (p<0.05). C3 volatile fatty acid concentration significantly increased in TMR and hay groups with 200 ppm of Zingiber and Syzygium supplementations (p<0.05). 200 ppm of Syzygium supplementation significantly decreased propionic acid concentration for concentrate (p<0.05). In all feed groups, IC4 VFA levels significantly decreased with Thymol 25, Oregano 25 and Syzygium 200 essence oil supplementations compared to positive and negative control groups (p<0.05). IC5 VFA levels also significantly decreased in all feed groups with Thymol 25 and Syzygium 200 essence oil supplementations compared to positive and negative control groups (p<0.05). C5 VFA level significantly decreased in TMR group supplemented with Thymol 25, in concentrate group supplemented with Syzygium 200 and in hay groups supplemented with Thymol 25, Oregano 25 and Syzygium 200 essence oils (p<0.05). C2/C3 VFA level at 24 h significantly increased with 25 ppm dose of Thymol and Oregano extracts and significantly decreased with 200 ppm dose of Zingiber and Syzygium for all feed groups (TMR, concentrate and hay).

DISCUSSION

Effects of essence oils on VFA. As a result of the microbial activity in the rumen of ruminants, carbohydrates such as cellulose are broken down and converted to VFAs, carbon dioxide, methane, ammonia and microbial cells (12). VFAs are the main energy source of ruminants. About 70% of the metabolizable energy in ruminants is generated by the VFAs produced in the rumen (13). If reduction of VFA concentrations in rumen at in vitro conditions occurs also at in vivo conditions, this situation may adversely affect nutrition efficiency (14). In this study, the effects of thymol, oregano, zingiber and syzygium supplementations to TMR, concentrate and hay at 8 and 24 h were examined in in vitro conditions.

As seen in table 2, compared to positive and negative control groups, essence oil supplementations did not have any statistically significant effect at 8 h on isobutyric acid, isovaleric acid and valaric acid concentrations in the examined feed groups. Beauchemin and McGinn (15) reported no effects of 750 mg/d essence oil on total VFA production 4 h post feeding in cattle. Benchaar et al. (16) demonstrated no effects of thymol, eugenol, vanilin and limonen essence oil combination (2g/ day) on total VFA and individual VFA concentrations in cows at 8 h. In the study of Chaves et al (17) on Holstein cows, carvacrol and cinnamaldeyhde supplementation at cattle ration did not have any effect on total VFA and individual VFA concentrations at 6 h. Kamalak et al (18) suggested that thymol has no effect on molar proportions of acetate, propionate, butyrate, isobutyrate, valerate and isovalerate. The findings of these researchers support the findings of this study which showed that essence oils have no significant effect on isobutyric, isovaleric and valeric acid VFAs at 8h.

As seen in table 2, essence oil supplementations had statistically significant effect at 8 h on butyric acid concentration (p<0.05) according to feed. Compared to positive control groups, Syzygium 200 ppm supplementation resulted a significant decrease in butyric acid levels for TMR and concentrate (p<0.05). Öztürk et al (19) revealed that 150 mg of olive leaf extract reduced butyrate production. The findings of these researchers are in agreement with the findings obtained in this study. Contrary to the findings of this study, Castillejos et al (20) reported that 500 mg/l of thymol and eugenol supplementation to 60:40 roughage:concentrate ration significantly increased butyric acid concentration. Bencharr et al (14) demonstrated that carvacrol and eugenol increased butyrate proportions. García et al (21) collected rumen liquors in in vitro conditions from goats fed on 70:30 barley:alfalfa ration and found that 250mg/l of carvacrol supplementation increased butyrate proportions after 48 h incubation. Chaves et al (22) observed that carvacrol or cinnamaldehyde supplementation to barley or corn grain-based lamb rations did not have any significant effect on butyrate proportions compared to negative control group. Sirohi et al (23) observed no effect of Myristica fragrans extract on butyrate proportions. As indicated by the literature, while some essence oils increase butyric acid concentrations, some essence oils decrease these concentrations and some essence oils have no effect on these concentrations. The varying results obtained from studies can be attributed to the ration compositions and types of plant extracts used in the studies as well as their extraction methods and doses.

Cardozo et al (24) reported that high doses (300 mg/l) of garlic, cinnamon, yucca, anise, oregano, capsicum essence oils and cinnamaldehyde, anethole, eugenol plant secondary metabolites used in their study significantly decreased total VFA concentrations at 24 h. In the study of Busquet et al (25), 3.000 mg/l doses of carvacrol, clove bud, eugenol and oregano oil supplementation to 50:50 roughage:concentrate rations significantly decreased total VFA concentrations at 24 h. Castillejos et al (20) demonstrated that 5.000 mg/l doses of thymol and eugenol supplementation to 60:40 roughage:concentrate rations significantly decreased total VFA concentrations at 24 h. Petersen et al (26) observed that Larrea plant extract significantly decreased total VFA concentrations in 100% timothy and 50:50 alfalfa-corn rations at 48 h. Canpolat et al. (27) reported that varying doses of carvacrol, peppermint oil and orange oil supplementations to rumen liquors collected from rams fed on 60:40 alfalfa grass-concentrate ration significantly decreased total VFA, butyric acid and acetic acid/propionic acid concentrations. In our studies, for all feed groups, Thymol 25, Oregano 25, Syzygium 200 ppm supplementations significantly reduced isobuytric acid concentration at 24 h. Further, Thymol 25 and Syzygium 200 ppm supplementations significantly reduced isovaleric VFA concentration at 24 h compared to positive and negative control groups (p<0.05). Castillejos et al (20) observed that 500 mg/l of thymol and eugenol supplementations significantly decreased isovalerate and isobutytrate concentrations at 24 h. The findings of these researchers support the findings of this study.

Similarly, at 24 h, propionic acid concentration significantly decreased in the TMR, concentrate and hay groups with 25 ppm of Thymol supplementations compared to positive and negative control groups (p<0.05). Isovaleric concentration significantly decreased particularly in Thymol 25 and Syzygium 200 ppm supplemented in TMR, concentrate and hay group compared to positive and negative control groups (p<0.05). Acetic acid/propionic acid concentrations decreased significantly in Zingiber 200 ppm and Syzygium 200 supplemented TMR, concentrate and hay groups compared to positive and negative control groups. The study findings on VFA concentrations for feed groups at 24 h are in agreement with the findings of the researchers mentioned above. Decreased VFA concentrations in rumen liquor can be attributed to reduced activities of ruminal microorganisms due to the antimicrobial effect of essence oils.

In the present study, Syzygium 200 ppm supplementation increased propionic acid production at 8 h for hay. Propionic acid production at 24 h increased with Zingiber 200 ppm and Syzgium 200 ppm supplementations for TMR and hay, and with Zingiber 200 ppm supplementation for concentrate. Acetate:propionate proportions at 8 h significantly increased with Oregano 25 ppm supplementation for TMR and concentrate and with Thymol 25 ppm supplementation for hay. The same proportion at 24 h significantly increased with Thymol 25 and Oregano 25 ppm supplementations for all feed groups. Busquet et al (25) reported that different doses of anethol, anise oil, carvone and tea tree oil  decreased the proportion of acetate and propionate, which suggests that these compounds may not be nutritionally beneficial to dairy cattle. Demirtaş et al (28) reported that 250 mg of rosemary (Rosmarinus officinalis L. ) and sage (Salvia officinalis L. ) supplementations to 50:50 roughage:concentrate rations did not have any significant effect on propionate production. However, rosemary and sage extracts were found to decrease acetate:propionate proportions compared to control fermenters without supplementations. Similarly, acetate:propionate proportions tended to decrease with increased doses of essence oils. Bodas et al. (29) demonstrated that among the essence oils tested, only Rheum nobile was found to increase propionate:acetate proportion.

In conclusion essence oils can be used as alternatives to chemical supplements used for rumen regulators. Being natural, these substances are environment-friendly. The essence oils of Thymol, Oregano, Zingiber and Syzygium used in the present study have the potential of improving rumen efficiency.

Acknowledgements

The authors are grateful to Hohenheim University (Germany) for its financial and technical support.

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