Effect of Methanolic Extract of Pomegranate Peel on in vitro Rumen Fermentation Kinetics of Oil Seed Meals

Iranian Journal of Applied Animal Science

مقاله 9، دوره 6، شماره 1، خرداد 2016، صفحه 67-75   

نویسندگان

S. Nezarati¹؛ N. Maheri-Sis^{* 2}

¹Young Researchers and Elite Club, Shabestar Branch, Islamic Azad University, Shabestar, Iran

²Department of Animal Science, Shabestar Branch, Islamic Azad University, Shabestar, Iran

چکیده

This study was carried out to determine the effects of supplementing different levels (0, 0.5 and 1% of buffered rumen fluid) of methanolic extract of pomegranate peel, on rumen fermentation kinetics of four oil seed meals (soybean meal, cotton seed meal, rapeseed meal and sunflower seed meal), using in vitro gas production technique. The samples were incubated in syringes containing rumen liquor taken from three fistulated Iranian Ghezel rams for 2, 4, 6, 8, 12, 24 and 36 h. Results showed that, addition of methanolic extract of pomegranate peel led to significant increase in gas production volume in all incubation times and all of oil seed meals as well. Amount of gas production, also increased by increasing dose of the extract. Also, amounts of a (the gas production from the immediately soluble fraction), b (the gas production from the insoluble fraction) and a + b (the potential gas production) in all of tested oil seed meals increased by increasing pomegranate peel extract doses. Adding pomegranate peel extract resulted in increase volatile fatty acids (VFA) production. Production of VFA increased significantly by the level of the extract supplementation. In conclusion, it is suggested that, adding methanolic extract of pomegranate peel can be lead to higher ruminal fermentation and VFA production in ruminants.

کلیدواژه‌ها

Fermentation؛ gas production؛ methanolic extract؛ oil seed meals؛ pomegranate peel

اصل مقاله

INTRODUCTION

Using cheap, easy available, native and abundant sources of feedstuffs in animal nutrition is one of the most important strategies in developing countries such as Iran. For this purposes, agro-industrial by-products are the first candidate. In addition to nutritional value of these by-products which are usually loosed to the environment (Mirzaei-Aghsaghali and Maheri-Sis, 2008; Delavar et al. 2014), some of them are having functional properties owing to possess secondary metabolites. Plant secondary metabolites may be used in order to modulating rumen microbial population and diversity, volatile fatty acids production and proportion and methane production as well as nitrogen metabolism. Applying essential oils can be a useful strategy to improve efficiency of nutrient utilization by ruminants (Benchaar et al. 2008; Tajodini et al. 2014). Hassanpour et al. (2011) also stated that plant secondary metabolites may exert beneficial effects on protein metabolism and decreasing rumen degradation of dietary protein and increasing absorption of amino acids in the small intestine. Although medicinal plants are used due to their health related characteristics such as antimicrobial, antioxidant, anti-inflammatory, antiparasitic and anticancer properties (Sirohi et al. 2012a; Sirohi et al. 2012b), their production are laborious and expensive. Thus, agro-industrial by-products with same properties are not only being economic but also reducing environmental pollution risk. Pomegranate processing wastes are of the most important by-products with potentially good nutritional value and health benefits in animal production (Ebrahimi et al. 2012; Canbolat et al. 2014; Delavar et al. 2014). Pomegranate (Punica granatum) is an important fruit in Iran. Iran is the main origin of this fruit and has the first rank of quality, varieties, cultivation area, production and export in the world (Kohansal and Rahimi, 2013). Worldwide production of pomegranate is approximately 1.5 million tons and Iran produces 47% of world production (FAO/WHO, 2009). Most of pomegranate produced in the country consumed as fresh or processed for producing juice and sauce. Estimated annual production of pomegranate processing by-products exceeds 120000 tons in Iran, where peels constitute approximately 50-60% of fruits weight (Shabtay et al. 2008; Mirzaei-Aghsaghali et al. 2011). In recent years, due to production of huge amounts of pomegranate by-products in Iran, many researchers in the country, prompted to investigate feeding value and functional capacity of these by-products for ruminant animals (Feizi et al. 2005; Mirzaei-Aghsaghali et al. 2011; Modaresi et al. 2011; Ebrahimi et al. 2012; Taher-Maddah et al. 2012a; Taher-Maddah et al. 2012b; Delavar et al. 2014). Mirzaei-Aghsaghali et al. (2011) reported that metabolizable energy, net energy for lactation, organic matter digestibility and short chain fatty acids production of pomegranate peel are higher than that of pomegranate seeds for ruminants. In addition to higher nutritional value, pomegranate peel also contains higher amounts of polyphenolic compounds, stronger biological activities as well as more antioxidant and antimicrobial capacity than the juice and seeds (Negi and Jayaprakasha, 2003; Olaniyi et al. 2012; Manuel Viuda-Martos et al. 2013). Oliveira et al. (2010) reported that pomegranate extracts contain polyphenolic compounds, which have been shown to possess functional properties such as antioxidant and antimicrobial potency. Most recently, Iranian researchers (Abarghuei et al. 2013; Abarghuei et al. 2014a, Abarghuei et al. 2014b) have used pomegranate peel extract in ruminant nutrition due to its rumen modulator properties. Abarghuei et al. (2013) found that supplementation of pomegranate peel extract decreased protozoa population, NH₃-N concentration, and increased microbial protein together with milk yield and quality of dairy cows. In another study, Abarghuei et al. (2014a) indicated that pomegranate peel extracts successfully manipulate in vitro rumen fermentation products, in particular increased propionate concentration and decreased acetate, proportion of acetate to propionate, ammonia nitrogen production and protozoa population in sheep. Nowadays, oil seed meals are of the main constituents of the dairy and fattening cows’ diets. They are supplying major part of energy and protein needs of the ruminants (Nezarati et al. 2014). Modulating condition of the rumen may be improves efficiency of protein metabolism and volatile fatty acids production. Because of higher price of oil seed meals as well as their direct involving in supplying energy and protein in the ruminants’ diets, we have decided to assess the effect of adding a rumen modulator on their fermentation kinetics. Therefore, the aim of current study was to evaluate the effect of different levels of methanolic extract of pomegranate peel on ruminal gas production parameters and volatile fatty acids production from soybean meal, cotton seed meal, rapeseed meal and sunflower seed meal under in vitro condition.

 

MATERIALS AND METHODS

Samples collection and preparation

Samples of the soybean meal, sunflower meal, rapeseed meal and cotton seed meal were obtained from a commercial unit in Tabriz, Iran. Collected samples were milled through a 1 mm sieve for chemical analysis and gas production procedure. Chemical composition of tested oil seed meals have been reported in in our recently published paper (Nezarati et al. 2014).

 

In vitro gas production

The study was carried out to determine the effects of supplementing different levels (0, 0.5 and 1% of buffered rumen fluid) of methanolic extract of pomegranate peel, on rumen fermentation kinetics of oil seed meals using in vitro gas production technique. Rumen fluid required for in vitro incubation obtained from three fistulated Ghezel rams fed twice daily with a diet containing mixture of roughage and concentrate (60:40) before the morning feeding. About 200 mg dry weights of samples (soybean meal, sunflower meal, rapeseed meal and cotton seed meal) were weighed in triplicate into 100 mL calibrated glass syringes following the procedures of Menke and Steingass (1988). The syringes were pre-warmed at 39 ˚C before the injection of 30 ml rumen fluid-buffer mixture (1:2) into each syringe and incubated in an incubator at 39 ˚C. The samples were incubated in syringes containing rumen liquor taken from three fistulated Iranian Ghezel rams for 2, 4, 6, 8, 12, 24 and 36 h. All samples were incubated in triplicate. Three syringes containing only rumen fluid-buffer mixture considered as the blank. The net gas productions of samples were determined by correcting gas volumes for blanks. Net gas production data were fitted to the exponential model outlined by Ørskov and McDonald (1979) and model components (a, b, c) calculated by FITCURVE software version 6 (Chen, 1995):

Y= a + b(1-e ^–ct)

Where:

Y: gas production at time t.

a: gas production from soluble fraction (mL/200 mg DM).

b: gas production from insoluble but fermentable fraction (mL/200 mg DM).

c: gas production rate constant for the insoluble fraction (mL/h).

a + b: potential gas production (mL/200 mg DM).

t: incubation time (h).

e: base for natural logarithms (2.718).

Volatile fatty acids (VFA) were calculated by equation of Makkar (2005): 

VFA (mmol)= 0.0222 GV – 0.00425

Where:

GV: 24 h net gas production volume (mL/200 mg DM).

 

Statistical analyses

Data from in vitro gas production test were subjected to analysis of variance as a completely randomized design with four treatments including soybean meal, rapeseed meal, cotton seed meal, sunflower seed meal (three replicates for each treatment) using general linear model (GLM) procedure of SAS (2001). Means were compared by Duncan’s multiple range tests (Steel and Torrie, 1980).

 

RESULTS AND DISCUSSION

Gas production volume

Effect of supplementing methanolic extract of pomegranate peel on in vitro gas production volume of soybean meal, cotton seed meal, rapeseed meal and sunflower seed meal at different incubation times have been illustrated in Tables 1 to 4. Results showed that, addition of methanolic extract of pomegranate peel led to significant increase in gas production in all incubation times and all of oil seed meals as well. In other hand, gas production, also increased by increasing dose of the extract. This is an unexpected result; because we had hypothesized that pomegranate peel extract should decrease gas production due to modifying microbial fermentation. Our results were partly in line with Pashachalandari et al. (2014) and Niasati et al. (2014). Pashachalandari et al. (2014) demonstrated that gas production volume obtained from canola meal initially (till 12 h incubation) decreased and then (24-96 h incubation) increased when supplemented by different levels of methanolic extract of the nettle (Urtica dioica). Niasati et al. (2014) also obtained same results when added methanolic extract of Viscum album on soybean meal. But they could not interpret gas production enhancement from oil seed meals treated by medicinal plants extracts. Our previous in vitro studies by some medicinal plants extracts led to various results (mostly decrease) in gas production from practical diets and oil seed meals (Halimi Shabestari et al. 2011; Mirzadeh Ahari et al. 2011; Salamat Azar et al. 2011; Salamatazar et al. 2011; Rezaei et al. 2011; Salamat Azar et al. 2012; Salamatazar et al. 2012). Salamat Azar et al. (2011) showed that in vitro addition of Zataria multiflora water extract on soybean meal at the level of 0.15 mL/30 mL buffered rumen fluid, could not affect gas production amounts in any incubation times. While in other study, Salamatazar et al. (2011), found that supplementation of sunflower meal by the thyme methanolic extract, at the same level significantly decreased in vitro gas production volume in all incubation times. Kilic et al. (2011) indicate that essential oils, doses, and essential oils × dose interactions significantly affected in vitro gas production. They have concluded that gas production kinetics may be affected differently by various essential oils. In their study, in vitro gas production was decreased by essential oils of oregano (Origanum vulgare), garlic (Allium sativum) and anise (Pimpinella anisum), unaffected by black seed (Nigella sativa), laurel (Laurus nobilis) and cinnamon (Cinnamomum verum) and increased by cumin (Cumminum cyminum). Maleki Baladi et al. (2014) reported that effect of tannin extract obtained from pomegranate pomace on in vitro gas production volume of soybean meal at different incubation times is dose dependent. Treating soybean meal by 0, 1.5 and 3% pomegranate pomace extract produced approximately same amount of gas; while addition of 4.5 and 6% extract resulted in significant reduction in gas production volume. In another study, Abarghuei et al. (2014a) have evaluated different levels (0, 15 and 30 mg total phenolic compounds per g dry matter) of pomegranate peel extracted by either water or solvent mixture on in vitro gas production and ruminal fermentation patterns and found that gas production at 24 h incubation time, was not significantly affected by the type and levels of pomegranate peel extract. In spite of the fact that decreased in vitro gas production by some essential oils may indicate more efficient utilization of energy due to the controlled loss of energy as methane; it may not easily to interpret gas production enhancement when some medicinal plants derived materials added to the rumen liquor. It is notable that, some other researchers (Kilic et al. 2011; Niasati et al. 2014; Pashachalandari et al. 2014) have also mentioned to the variable effects of medicinal plants extracts on ruminal in vitro gas production kinetics.

 

Table 1 Effect of addition of methanolic extract of pomegranate peel on in vitro gas production volume (mL/200 mg DM) of cotton seed meal at different incubation times (h)

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 2 Effect of addition of methanolic extract of pomegranate peel on in vitro gas production volume (mL/200 mg DM) of soybean meal at different incubation times (h)

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 3 Effect of addition of methanolic extract of pomegranate peel on in vitro gas production volume (mL200 mg DM) of rapeseed meal at different incubation times (h)

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 4 Effect of addition of methanolic extract of pomegranate peel on in vitro gas production volume (mL/200 mg DM) of sunflower meal at different incubation times (h)

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Most of workers referred this variation to the varied antioxidant, antibacterial, antiprotozoal, antifungal and antiviral potency of medicinal plants (Calsamiglia et al. 2007; Benchaar et al. 2008; Shabtay et al. 2012). Some others also suggested that the dissimilar results obtained from different functional plant materials on rumen fermentation pattern may be depends on rumen conditions (e.g. pH), chemical and physical nature of the tested feedstuffs, animal species, rumen microbial population and diversity, history of feeds offered to the experimental animals, duration of the experiment period, adaptation time and dose of the utilized medicinal plants as well as preparation methods such as extracting solvent and procedure (Kilic et al. 2011; Sirohi et al. 2012a; Sirohi et al. 2012b; Tajodini et al. 2014).

 

Gas production parameters

Effect of methanolic extract of pomegranate peel on in vitro gas production parameters (a, b, c, a+b) of soybean meal, cotton seed meal, rapeseed meal and sunflower seed meal have been illustrated in Tables 5 to 8. There are significant differences for a (the gas production from the immediately soluble fraction), b (the gas production from the insoluble fraction) and a + b (the potential gas production) between treated and untreated oil seed meals. Amounts of a, b and a + b in all of tested oil seed meals increased by pomegranate peel extract treating; higher dose of extract addition, have led to higher amounts of a, b and a + b. Since, gas production parameters (a, b and a+b), are subsequent of gas production volume in gas test technique, it was predictable that, increase in gas volume have been conduced to increase in gas production parameters. In spite of harmonic increase in a, b and a + b, the parameter c (the gas production rate constant for the insoluble fraction), had different behavior dependent on type of tested oil seed meals. It is unaffected by pomegranate peel extract supplementation, in case of rapeseed meal, and decreased in other oil seed meals. However, it was reduced usually by increasing extract dose. Salamatazar et al. (2011) reported that adding thyme methanolic extract at the levels of 0.15 and 0.3 mL/30 mL buffered rumen fluid, decreased all of the in vitro gas production parameters (a, b, c, a+b) of sunflower meal. While similar to our findings, Niasati et al. (2014) and Pashachalandari et al. (2014) showed that in vitro gas production parameters (b, c, a+b) of soybean meal and canola meal increased when rumen liquor supplemented with methanolic extract of Urtica dioica and Viscum album. However, they have reported that parameter c was unaffected or decreased by adding above mentioned extracts, they did not find any interpretation for their results. Maleki Baladi et al. (2014) evaluated the effects of adding 1.5, 3, 4.5 and 6% tannin extracted from pomegranate pomace on in vitro gas production parameters of soybean meal. They have reported that a + b of soybean meal was significantly reduced, when it was treated with 4.5% and 6% of tannin extract; but not by 1.5 and 3%. In their study, amount of parameter c remained unaffected by extract of pomegranate pomace. In contrast, Abarghuei et al. (2014a) informed that parameter c significantly reduced by adding pomegranate peel extract, however they are showed that asymptotic gas production (a+b) did not affected by type or level of pomegranate peel extract. Although, various results obtained from supplementing medicinal plants extracts in different in vitro studies, but results of current study surprisingly unexpected. In spite of the fact that it can be as a result of gas produced from either alcohol residues or the fermentable main constituents in the methanolic extract of pomegranate peel, we cannot interpret and justify our findings and it is remain unknown. Further knowledge and studies required for recognizing and interpreting the current results.

 

Volatile fatty acids (VFA) production

Estimated in vitro VFA production has been shown in Tables 5 to 8. As it is shown, VFA productions from all of the tested oil seed meals significantly affected by supplementing pomegranate peel extract. Adding pomegranate peel extract has led to increase VFA production. Production of VFA increased significantly by the level of the extract supplementation. It is well known that reduction in gas production associated with increase in VFA production is one of the important desires of ruminant nutritionists. Since VFA contributes to at least 65 to 75% of the total metabolizable energy supply for ruminants; so decreasing gas production (including methane) will be resulted in improved rumen energy efficiency as well as reducing environmental problems. Albeit approaching this goal is not easy, because gas production quantitatively and qualitatively is a result of VFA production. Enhancement of VFA production is a main index of higher digestibility and energetic value (Maheri-Sis et al. 2008; Mirzaei-Aghsaghali et al. 2011). Castillejos et al. (2006) stated that many of essential oil compounds have important antimicrobial activity and decreased total VFA concentration, although at appropriate doses, these compounds also modified rumen microbial fermentation without decreasing total VFA concentration. Benchaar et al. (2008) also stated that effects of secondary metabolites on total VFA concentration and VFA pattern is variable among studies, depending on the dosage and the source of component. Thus exploring and investigating natural compounds and their suitable dose in order to achieving later characteristics may be a useful vector in ruminant nutrition science. Previously, we have found that estimated VFA production of canola meal decreased by in vitro ruminal supplementation of Thymus vulgar (Salamatazar et al. 2012). Kilic et al. (2011) deduced that higher dose of thymol and carvacrol decreased and Eugenol increased ruminal VFA production.

 

Table 5In vitro gas production parameters and estimated VFA production of cotton seed meal affected by methanolic extract of pomegranate peel

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

a: the gas production from the immediately soluble fraction; b: the gas production from the insoluble fraction; a + b: the potential gas production and c: the gas production rate constant for the insoluble fraction b (/h).

VFA: volatile fatty acids.

 

Table 6 In vitro gas production parameters and estimated VFA production of soybean meal affected by methanolic extract of pomegranate peel

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

a: the gas production from the immediately soluble fraction; b: the gas production from the insoluble fraction; a + b: the potential gas production and c: the gas production rate constant for the insoluble fraction b (/h).

VFA: volatile fatty acids.

 

Table 7In vitro gas production parameters and estimated VFA production of rapeseed meal affected by methanolic extract of pomegranate peel

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

a: the gas production from the immediately soluble fraction; b: the gas production from the insoluble fraction; a + b: the potential gas production and c: the gas production rate constant for the insoluble fraction b (/h).

VFA: volatile fatty acids.

 

Table 8 In vitro gas production parameters and estimated VFA production of sunflower meal affected by methanolic extract of pomegranate peel

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

a: the gas production from the immediately soluble fraction; b: the gas production from the insoluble fraction; a + b: the potential gas production and c: the gas production rate constant for the insoluble fraction b (/h).

VFA: volatile fatty acids.

 

Tajodini et al. (2014) reviewed that these differing results may be partially explained by the experimental conditions of studies, including type of diets, animal and plant species, type and concentration of active substances and adaptation as well as pH values of rumen fluid.In the study conducted by Maleki Baladi et al. (2014), short chain fatty acids and subsequently metabolizable energy and net energy for lactation of soybean meal have significantly reduced when treated by 4.5% and 6%, but not by lower doses of pomegranate pomace extracted tannin. However, Abarghuei et al. (2013) declared that concentrations of total VFA and molar proportions of individual VFA were not influenced by supplementation of pomegranate peel extract in the diet, they have suggested that addition of pomegranate peel extract has reduced protozoa population, NH₃-N concentration, and increased microbial protein. In another study, Abarghuei et al. (2014a) concluded that inclusion of water or solvent extracts of pomegranate peel kindly manipulate rumen fermentation parameters, particularly increased propionate and decreased acetate and NH₃-N concentration as well as protozoa population. Refat et al. (2015) also found that supplementing high grain diets with pomegranate peel extracts in Rusitec system, results in decreasing total and branched-chain VFA and ammonia nitrogen concentration as well. Effect of natural rumen modifier compounds on VSAs production and their proportions may be varied owing to their antimicrobial and antioxidant capacity (Benchaar et al. 2008; Tajodini et al. 2014). Pomegranate peel due to higher and diverse phenolic compounds such as tannins is a vigorous antioxidant, antiprotozoal and antibacterial by-product. Olaniyi et al. (2012) showed that pomegranate methanolic peel extracts have strong broad-spectrum activity against Gram-positive and Gram-negative bacteria. However, Castillejos et al. (2006) cleared that Gram-positive bacteria are generally more sensitive to essential oils than Gram-negative bacteria; so that pomegranate peel extracts may have ionophores like properties on rumen metabolism without their application anxieties. Jami et al. (2012) evaluated the effects of pomegranate peel extract addition to the diet of lactating cows at the levels of 1, 2 or 4% on in vivo digestibility as well as rumen bacterial population, and stated that when specific bacteria were examined, some of them did not exhibit any significant change (e.g., Prevotella spp.), some of species involved in soluble sugar utilization, such as Succinivibrio dextrinosolvens, Eubacterium ruminantium and Streptococcus bovis increased and some others which are mainly known as cellulose degraders (i.e. Fibrobacter succinogenes and Ruminococcus albus) significantly decreased. However, cellulose digestibility was not decreased and neutral detergent fibre (NDF) digestibility even increased in the cows fed with highest level (4%) of pomegranate peel extract. In other hand, Abarghuei et al. (2013) and Abarghuei et al. (2014a) have emphasized on antiprotozoal capacity of pomegranate peel, which in turn it means that, this by-product may exert anti-methanogenic effects in the rumen and subsequently altering energy efficiency and environmental safety. Alternatively, since protozoa populations are bacteria predator, reducing protozoa community may be resulted in higher growth of bacterial cells and causing to higher VFA and microbial protein production. From antioxidant point of view, phenolic compounds of pomegranate peel may acts as free radical scavenger in rumen environment and by this way leads to enhance rumen health and microbial efficiency.

 

CONCLUSION

In conclusion, it is suggested that, adding methanolic extract of pomegranate peel to the rumen can be lead to higher ruminal fermentation and VFA production from oil seed meals for ruminants. We cannot suggest a clear mechanism for this result. However, in further studies, it should be considered an additional “control” containing rumen fluid-buffer plus extract without tested feedstuffs.

 

ACKNOWLEDGEMENT

This article prepared from MS thesis in animal science, Islamic Azad University, Shabestar Branch (thesis supervisor: Dr. N. Maheri-Sis). We would like to acknowledge Dr. R. Salamatdoust and Dr. A. Aghajanzadeh-Golshani for their valuable assistance in all stages of the work. The authors are grateful to the Animal Research Center and laboratories of Islamic Azad University, Shabestar Branch.

مراجع

Abarghuei M.J., Rouzbehan Y., Salem A.Z.M. and Zamiri M.J. (2013). Nutrient digestion, ruminal fermentation and performance of dairy cows fed pomegranate peel extract. Livest. Sci. 157, 452-461.

Abarghuei M.J., Rouzbehan Y. and Salem A.F. (2014a). The influence of pomegranate-peel extracts on in vitro gas production kinetics of rumen inoculum of sheep. Turkish J. Vet. Anim. Sci. 38, 212-219.

Abarghuei M.J., Rouzbehan Y., Salem A.Z.M. and Zamiri M.J. (2014b). Nitrogen balance, blood metabolites and milk fatty acid composition of dairy cows fed pomegranate-peel extract. Livest. Sci. 164, 72-80.

Benchaar C., Calsamiglia S., Chaves A.V., Fraser G.R., Colombatto D., McAllister T.A. and Beauchemin K.A. (2008). A review of plant-derived essential oils in ruminant nutrition and production. Anim. Feed Sci. Technol. 145, 209-228.

Calsamiglia S., Busquet M., Cardozo P.W., Castillejos L. and Ferret A. (2007). Invited review: essential oils as modifiers of rumen microbial fermentation. J. Dairy Sci. 90, 2580-2595.

Canbolat Ö., kamalak A. and Kara H. (2014). The effects of urea supplementation on pomegranate pulp (Punica granatum) silage fermentation, aerobic stability and in vitro gas production. Ankara Üniv. Vet. Fak. Derg. 61, 217-223.

Castillejos L., Calsamiglia S. and Ferret A. (2006). Effect of essential oils active compounds on rumen microbial fermentation and nutrient flow in in vitro systems. J. Dairy Sci. 89, 2649-2658.

Chen X.B. (1995). "Fitcurve" Macro. IFRU, the Macaulay Institute, Aberdeen, UK.

Delavar M.H., Tahmasbi A.M., Danesh-Mesgaran M. and Valizadeh R. (2014). In vitro rumen fermentation and gas production: influence of different by-product feedstuffs. Ann. Res. Rev. Biol. 4(7), 1121-1128.

Ebrahimi B., Taghizadeh A., Mehmannavaz Y. and Palangi V. (2012). Evaluation of pomegranate pomace using in situ and gas production techniques. J. Environ. Sci. Eng. A. 1, 951-955.

FAO/WHO. (2009). Project document for a regional standard for Pomegranate. FAO/WHO Coordinating Committee for the Near East. Tunis, Tunisia.

Feizi R., Ghodratnama A., Zahedifar M., Danesh Mesgaran M. and Raisianzadeh M. (2005). Apparent digestibility of pomegranate seed fed to sheep. J. Dairy Sci. 88(1), 194-203.

Halimi Shabestari A., Salamatdoustnobar R., Maheri-Sis N., Gorbani A., Mirzadeh Ahari K., Noshadi A., Samadi H. and Salimi Nezhad J. (2011). Evaluation effects of clove methanol extract on methane production in the in vitro condition. Pakistan J. Nutr. 10(12), 1154-1157.

Hassanpour S., Maheri-Sis N., Eshratkhah B. and Baghbani Mehmandar F. (2011). Plants and secondary metabolites (Tannins): a review. Int. J. Forest. Soil. Erosion. 1(1), 47-53.

Jami E., Shabtay A., Nikbachat M., Yosef E., Miron J. and MizrahiI. (2012). Effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population. J. Dairy Sci. 95, 5996-6005.

KilicU., Boga M., Gorgulu M. and Şahan Z. (2011). The effects of different compounds in some essential oils on in vitro gas production. J. Anim. Feed Sci. 20, 626-636.

Kohansal M.R. and Rahimi M. (2013). Investigating factors marketing pomegranate in Fars. Int. J. Agron. Plant. Prod. 4(10), 2759-2763.

Maheri-Sis N., Chamani M., Sadeghi A.A., Mirza-Aghazadeh A. and Aghajanzadeh-Golshani A. (2008). Nutritional evaluation of kabuli and desi type chickpeas (Cicer arientinum) for ruminants using in vitro gas production technique. African J. Biotechnol. 7(16), 2946-2951.

Makkar H.P.S. (2005). In vitro gas methods for evaluation of feeds containing phytochemicals. Anim. Feed Sci. Technol. 123, 291-302.

Maleki Baladi R., Moghaddaszadeh-Ahrabi S. and Afrouziyeh M. (2014). Influence of the addition of different levels of tannin extracted from pomegranate pomace, on some nutritive value of soybean meal. European J. Exp. Biol. 4(2), 148-154.

Manuel Viuda-Martos J.A., Pérez-álvarez E.S. and Fernández-López J. (2013). In vitro antioxidant properties of pomegranate (Punica granatum) peel powder extract obtained as coproduct in the juice extraction process. J. Food Proc. Pres. 37(5), 772-776.

Menke K.H. and Steingass H. (1988). Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28, 7-55.

Mirzadeh Ahari K., Salamatdoustnobar R., Maheri-Sis N., Gorbani A., Halimi Shabestari A., Noshadi A., Samadi H. and Salimi Nezhad J. (2011). Effects of the thyme extract on the ruminal methane production. Pakistan J. Nutr. 10(12), 1146-1148.

Mirzaei-Aghsaghali A. and Maheri-Sis N. (2008). Nutritive value of some agro-industrial by-products for ruminants: a review. World J. Zool. 3(2), 40-46.

Mirzaei-Aghsaghali A., Maheri-Sis N., Mansouri H., Razeghi M.E., Mirza-Aghazadeh A., Cheraghi H. and Aghajanzadeh-Golshani A. (2011). Evaluating potential nutritive value of pomegranate processing by-products for ruminants using in vitro gas production technique. ARPN J. Agric. Biol. Sci. 6(6), 45-51.

Modaresi J., Fathi Nasri M.H., Rashidi L., Dayani O. and Kebreab E. (2011). Effects of supplementation with pomegranate seed pulp on concentrations of conjugated linoleic acid and punicic acid in goat milk. J. Dairy Sci. 94, 4075-4080.

Negi P.S. and Jayaprakasha G.K. (2003). Antioxidant and antibacterial activities of Punica granatum peel extracts. J. Food Sci. 68(4), 1473-1477.

Nezarati S., Maheri-Sis N., Salamatdoust-Nobar R., Aghajanzadeh-Golshani A. (2014). In vitro fermentation characteristics and nutritive value of Iranian oil seed meals for ruminants. Greener J. Biol. Sci. 4(2), 53-58.

Niasati M., Palizdar M.H., Pourelmi M.R. and Pasha Chalandari H. (2014). Effect of methanolic extract of Viscum album on in vitro fermentation and digestibility of soybean meal. Res. Opin. Anim. Vet. Sci. 4(7), 411-415.

Olaniyi A.F., Nokwanda P.M. and Umezuruike L.O. (2012). Antibacterial, antioxidant and tyrosinase-inhibition activities of pomegranate fruit peel methanolic extract. BMC Comp. Altern. Med. 12, 1-11.

Oliveira R.A., Narciso C.D., Bisinotto R.S., Perdomo M.C., Ballou M.A. and Dreher M. (2010). Effects of feeding polyphenols from pomegranate extract on health, growth, nutrient digestion, and immunocompetence of calves. J. Dairy Sci. 93, 4280-4291.

Ørskov E.R. and McDonaldI. (1979). The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J. Agric. Sci. 92, 499-503.

Pashachalandari H., Palizdar M.H., Mohammadian-Tabrizi H.R. and Niasati M. (2014). Effect of methanolic extract of nettle (Urtica dioica) on in vitro fermentation and gas production of canola meal. Res. Opin. Anim. Vet. Sci. 4(7), 416-419.

Refat B., AneleU., He Z.X., Bassiony S.M., Abdel-Rahman G.A. and Yang W.Z. (2015). Effect of sainfoin hay and pomegranate peel extracts on in vitro fermentation and protein degradation using the Rusitec technique. Canadian J. Anim. Sci.

Rezaei N., Salamat doust-Nobar R., Maheri Sis N., Salamatazar M., Namvari M., Goli S. and Aminipour H. (2011). Evaluation effect of some plant extracts on degradability of soybean meal with gas product technique. Ann. Biol. Res. 2(4), 224-228.

SAS Institute. (1991). SAS^®/STAT Software, Release 6.0. SAS Institute, Inc., Cary, NC. USA.

Salamat Azar M., Najafyar S., Rezaei N. and Aminipour H. (2011). Zataria multiflora extract affect in vitro gas producing techniques rumen microbial fermentation. Ann. Biol. Res. 2(5), 632-636.

Salamat Azar M., Salamat Doust-Nober R., Maheri Sis N., Aghdam Shahryar H. and Asadi Y. (2012). Effects of zataria multiflora extract as rumen modifiers using in vitro gas production technique. Curr. Res. J. Biol. Sci. 4(4), 350-354.

Salamatazar M., Salamatdoust-nobar R. and Maheri Sis N. (2012). Evaluation of the effects of Thymus vulgar on degradability kinetics of canola meal for ruminant using in vitro gas production technique. J. Cell. Anim. Biol. 6(11), 164-168.

Salamatazar M., Salamatdoustnobar R., Asadi Y., Maheri Sis N., Najafyar S., Khodaparast B., Aminipour H., Rezayi N. and Maleki M. (2011). Effect of thyme methanolic extract on degradability kinetics of sunflower meal. Ann. Biol. Res. 2(4), 104-109.

Shabtay A., Eitam H., Tadmor Y., Orlov A., Meir A., Weinberg P., Weinberg Z.G., Chen Y., Brosh A., Izhaki I. and Kerem Z. (2008). Nutritive and antioxidative potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed. J. Agric. Food Chem. 56, 10063-10070.

Shabtay A., Nikbachat M., Zenou A., Yosef E., Arkin O., Sneer O., Shwimmer A., Yaari A., Budman E., Agmon G. and Mirona J. (2012). Effects of adding a concentrated pomegranate extract to the ration of lactating cows on performance and udder health parameters. Anim. Feed Sci. Technol. 175, 24-32.

Sirohi S.K., Goel N. and Pandey P. (2012a). Efficacy of different methanolic plant extracts on anti-methanogenesis, rumen fermentation and gas production kinetics in vitro. Open. Vet. J. 2, 72-77.

Sirohi S.K., Mehta M., Goel N. and Pandey P. (2012b). Effect of herbal plants oil addition in total mixed diets on anti-methanogenic activity, rumen fermentation and gas production kinetics in vitro. J. Nat. Prod. Plant Resour. 2(1), 73-80.

Steel R.G.D. and Torrie J.H. (1980). Principles and Procedures of Statistics: A Biometrical Approach. McGraw Hill, New York, USA.

Taher-Maddah M., Maheri-Sis N., Salamatdoustnobar R. and Ahmadzadeh A. (2012a). Comparing nutritive value of ensiled and dried pomegranate peels for ruminants using in vitro gas production technique. Ann. Biol. Res. 3(4), 1942-1946.

Taher-Maddah M., Maheri-Sis N., Salamatdoustnobar R. and Ahmadzadeh A. (2012b). Estimating fermentation characteristics and nutritive value of ensiled and dried pomegranate seeds for ruminants using in vitro gas production technique. Open. Vet. J. 2, 40-45.

Tajodini M., Moghbeli P., Saeedi H.R. and Effati M. (2014). The effect of medicinal plants as a feed additive in ruminant nutrition. Iranian J. Appl. Anim. Sci. 4(4), 681-686.