The effect of different inclusion levels of polyethylene glycol as a silage additive on ensilage characteristics of pomegranate peel and in vitro rumen fermentation

This study was conducted to evaluate the effects of ensiling pomegranate peel (PP) with different levels of polyethylene glycol (PEG) on its chemical composition, tannin content, in vitro gas production and fermentation characteristics. Fresh PP was chopped and ensiled in mini silos made of polyvinyl chloride tubing. Five levels of PEG were studied: 0 (control), 5, 10, 15, and 20% of fresh PP (dry matter basis). Total phenolics, total tannins, crude ash, crude protein, neutral detergent fiber and acid detergent fiber content and pH decreased with increasing PEG levels, whereas dry matter and non-fiber carbohydrates content, non-tannin phenols, lactic acid and ammonia concentrations and buffering capacity increased. The water soluble carbohydrates and ether extract concentrations were not influenced by the addition of PEG. The partitioning factor and efficiency of microbial biomass production were quadratically decreased (p=0.020 and p=0.032, respectively) as PEG inclusion increased, but the in vitro apparent dry matter disappearance did not differ among treatments. Compared to control, the in vitro true disappearance and in vitro fiber digestibility had a tendency to be higher in silages treated with PEG (p=0.081 and p=0.069, respectively). The metabolizable energy content and total volatile fatty acids concentration increased quadratically by PEG inclusion. The asymptotic gas production and rate of gas production were higher in PEG-treated silages. Overall, ensiling PP with PEG can improve the fermentation characteristics of this byproduct. Additional key words: Punica granatum; agro-industrial by-products; tannin; microbial biomass; lactic acid; partitioning factor Abbreviations used: ADF (acid detergent fiber); CP (crude protein); DM (dry matter); dNDF (in vitro fiber digestibility); EE (ether extract); EMBP (efficiency of microbial biomass production); GP (gas production); ivTD (in vitro true disappearance); L (linear); MBP (microbial biomass production); ME (metabolizable energy); NDF (neutral detergent fiber); NFC (non-fibrous carbohydrates); PEG (polyethylene glycol); PF (partitioning factor); PP (pomegranate peel); Q (quadratic); TVFA (total volatile fatty acids). Citation: Hatami, A.; Alipour, D.; Hozhabri, F.; Tabatabaei, M. (2015). Short communication: The effect of different inclusion levels of polyethylene glycol as a silage additive on ensilage characteristics of pomegranate peel and in vitro rumen fermentation. Spanish Journal of Agricultural Research, Volume 13, Issue 2, e06SC01, 6 pages. http://dx.doi.org/10.5424/sjar/2015132-6463 Received: 30 Jun 2014. Accepted: 06 Mar 2015 Copyright © 2015 INIA. This is an open access article distributed under the Creative Commons Attribution License (CC by 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: The authors received no specific funding for this work Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Daryoush Alipour: alipourd@basu.ac.ir; daryoush.alipour@gmail.com Pomegranate (Punica granatum), native to Persia (Iran), is an edible fruit cultivated in subtropical and tropical regions around the world with different microclimatic zones such as in the United States, Turkey, Egypt, Italy, India, Chile and Spain. Pomegranate juice has nutritional and medical benefits and, due to the increasing consumer awareness of its potential health benefits, the global production and consumption of pomegranate has greatly expanded in recent years. The edible parts of pomegranate are used extensively in the form of juice, concentrate, canned Ali Hatami, Daryoush Alipour, Fardin Hozhabri and Meisam Tabatabaei Spanish Journal of Agricultural Research June 2015 • Volume 13 • Issue 2 • e06SC01 2 The in vitro gas test method was performed according to Menke & Steingass (1988). Rumen liquor samples were obtained from three rumen-canulated sheep fed twice daily a diet containing alfalfa hay (650 g/kg) plus concentrate mixture (350 g/kg) prior to their morning feeding. Samples were pooled and placed in a prewarmed CO2-filled flask and transported immediately to the laboratory. Rumen liquor was mixed, squeezed through four layers of cheesecloth and added to the buffer solution (1:2, v/v), which was kept in a water bath at 30 °C with CO2 saturation. Ensiled samples (200mg) were weighed in triplicate and placed in glass syringes to which 30 ml of buffered-rumen liquor was dispensed. Then, the glass syringes were immediately placed into a thermostatically-controlled water bath (39 ± 0.5 °C). Three syringes containing only bufferedrumen fluid (no silage sample) were also included in the experiment and the mean GP value for these syringes was used as the blank value. Cumulative GP was recorded after incubation for 0, 2, 4, 6, 8, 12, 16, 20, 24, 48, 72, 96, 120 and 144 h. All incubation experiments were done in three runs. The kinetic parameters of GP were fitted using the exponential model proposed by France et al. (2000) as follows: G = A (1−e−ct) where G (mL) denotes the cumulative GP at time t; A (mL/g DM) is the asymptotic GP, c (h−1) is the fractional rate of GP. Since the lag time was equal to zero in all cases, it was removed from original equation. In separate runs of GP, fermentation parameters and substrate digestibility were measured in syringes containing 500 mg substrates and 40 mL buffered rumen fluid. Gas production after 24 hours of incubation (GP24), the in vitro apparent dry matter disappearance, in vitro true dry matter disappearance (ivTD), in vitro fiber digestibility (dNDF), partitioning factor (PF) and ammonia concentration in acidified rumen fluid (1 mL 6 N HCl per 15 mL of filtrate) were measured as described by Taghavi-Nezhad et al. (2011). In vitro microbial biomass production (MBP) and efficiency of microbial biomass production (EMBP) were estimated from concomitant gas volume and truly disappeared dry matter (ivTD) measurements (Blümmel, 2000) as: MBP = ivTD − (gas volume × SF) EMBP = ivTD – (gas volume × SF) ivTD where SF is the stoichiometric factor, that has a value of 2.20 for forages. A number of studies have suggested the application of PP in animal nutrition. However, its high content of tannin has been reported to cause some problems when fed to animals (Makkar, 2003; Shabtay et al., 2008). To ameliorate the negative effects of dietary tannins, several methods such as using polyethylene glycol (PEG) and ensiling are proposed (Makkar, 2003). Since PP is produced within a short time period (during the harvest season), ensiling seems to be an efficient way to inactivate PP tannins. Little information is available on simultaneous application of PEG and ensiling in tannin-containing by-products such as PP. Therefore, the aim of the current experiment was to assess the effect of different inclusion levels of PEG on ensilage characteristics and in vitro gas production (GP) parameters of PP. Fresh PP was collected from the Sahar factory (Hamadan, Iran) and chopped to a cut length of 20-30 mm using a forage chopper. The chopped PP (32.4% dry matter (DM)) was then ensiled in mini silos made of polyvinyl chloride tubing (15 cm diameter and 70 cm height; capacity 12 kg) and the filled silos were sealed with plastic lids. Five levels of PEG (MW6000; Shazand Petrochemical CO, Arak, Iran) were added to fresh PP before ensiling: 0 (control), 5, 10, 15, and 20% of fresh PP (DM basis). Four replications were used for each treatment. All mini-silos were kept in laboratory for 70 days. After opening the mini-silos, subsamples were taken and stored at –20 °C until further analysis. The DM, crude protein (CP), ether extract (EE), crude ash (AOAC, 1990), neutral detergent fiber (NDF; without a heat stable amylase and ash included) and acid detergent fiber (ADF) content were measured (Van Soest et al., 1991). Non-fibrous carbohydrates (NFC) content was calculated using the following equation (NRC, 2001): NFC% = 100 – (% NDF + % CP + % EE + %Ash) Total phenolics, non-tannin phenols and total tannins were determined as described by Makkar (2000). Total volatile fatty acids (TVFA) content of sample extracts were determined by steam distillation (Barnett & Reid, 1957), lactic acid concentration as described by Taylor (1996) and water-soluble carbohydrates content using the water extraction–anthrone method (MAFF, 1982). The pH of silages was analyzed according to Faithfull (2002) using a portable digital pH meter (pH315i, WTW 82362 Weilheim, Germany). Ammonia was measured in the extract by the phenol-hypochlorite assay (Broderick & Kang, 1980) and the buffering capacity of silage was determined according to Moharrery (2007).

Pomegranate (Punica granatum), native to Persia (Iran), is an edible fruit cultivated in subtropical and tropical regions around the world with different microclimatic zones such as in the United States, Turkey, Egypt, Italy, India, Chile and Spain.Pomegranate juice has nutritional and medical benefits and, due to the increasing consumer awareness of its potential health benefits, the global production and consumption of pomegranate has greatly expanded in recent years.The edible parts of pomegranate are used extensively in the form of juice, concentrate, canned 2 The in vitro gas test method was performed according to Menke & Steingass (1988).Rumen liquor samples were obtained from three rumen-canulated sheep fed twice daily a diet containing alfalfa hay (650 g/kg) plus concentrate mixture (350 g/kg) prior to their morning feeding.Samples were pooled and placed in a prewarmed CO 2 -filled flask and transported immediately to the laboratory.Rumen liquor was mixed, squeezed through four layers of cheesecloth and added to the buffer solution (1:2, v/v), which was kept in a water bath at 30 °C with CO 2 saturation.Ensiled samples (200mg) were weighed in triplicate and placed in glass syringes to which 30 ml of buffered-rumen liquor was dispensed.Then, the glass syringes were immediately placed into a thermostatically-controlled water bath (39 ± 0.5 °C).Three syringes containing only bufferedrumen fluid (no silage sample) were also included in the experiment and the mean GP value for these syringes was used as the blank value.
Cumulative GP was recorded after incubation for 0, 2, 4, 6, 8, 12, 16, 20, 24, 48, 72, 96, 120 and 144 h.All incubation experiments were done in three runs.The kinetic parameters of GP were fitted using the exponential model proposed by France et al. (2000) as follows: where G (mL) denotes the cumulative GP at time t; A (mL/g DM) is the asymptotic GP, c (h −1 ) is the fractional rate of GP.Since the lag time was equal to zero in all cases, it was removed from original equation.
In separate runs of GP, fermentation parameters and substrate digestibility were measured in syringes containing 500 mg substrates and 40 mL buffered rumen fluid.Gas production after 24 hours of incubation (GP 24 ), the in vitro apparent dry matter disappearance, in vitro true dry matter disappearance (ivTD), in vitro fiber digestibility (dNDF), partitioning factor (PF) and ammonia concentration in acidified rumen fluid (1 mL 6 N HCl per 15 mL of filtrate) were measured as described by Taghavi-Nezhad et al. (2011).
In vitro microbial biomass production (MBP) and efficiency of microbial biomass production (EMBP) were estimated from concomitant gas volume and truly disappeared dry matter (ivTD) measurements (Blümmel, 2000) as: where SF is the stoichiometric factor, that has a value of 2.20 for forages.
A number of studies have suggested the application of PP in animal nutrition.However, its high content of tannin has been reported to cause some problems when fed to animals (Makkar, 2003;Shabtay et al., 2008).To ameliorate the negative effects of dietary tannins, several methods such as using polyethylene glycol (PEG) and ensiling are proposed (Makkar, 2003).Since PP is produced within a short time period (during the harvest season), ensiling seems to be an efficient way to inactivate PP tannins.Little information is available on simultaneous application of PEG and ensiling in tannin-containing by-products such as PP.Therefore, the aim of the current experiment was to assess the effect of different inclusion levels of PEG on ensilage characteristics and in vitro gas production (GP) parameters of PP.
Fresh PP was collected from the Sahar factory (Hamadan, Iran) and chopped to a cut length of 20-30 mm using a forage chopper.The chopped PP (32.4% dry matter (DM)) was then ensiled in mini silos made of polyvinyl chloride tubing (15 cm diameter and 70 cm height; capacity 12 kg) and the filled silos were sealed with plastic lids.Five levels of PEG (MW6000; Shazand Petrochemical CO, Arak, Iran) were added to fresh PP before ensiling: 0 (control), 5, 10, 15, and 20% of fresh PP (DM basis).Four replications were used for each treatment.All mini-silos were kept in laboratory for 70 days.
After opening the mini-silos, subsamples were taken and stored at -20 °C until further analysis.The DM, crude protein (CP), ether extract (EE), crude ash (AOAC, 1990), neutral detergent fiber (NDF; without a heat stable amylase and ash included) and acid detergent fiber (ADF) content were measured (Van Soest et al., 1991).Non-fibrous carbohydrates (NFC) content was calculated using the following equation (NRC, 2001): Total phenolics, non-tannin phenols and total tannins were determined as described by Makkar (2000).Total volatile fatty acids (TVFA) content of sample extracts were determined by steam distillation (Barnett & Reid, 1957), lactic acid concentration as described by Taylor (1996) and water-soluble carbohydrates content using the water extraction-anthrone method (MAFF, 1982).The pH of silages was analyzed according to Faithfull (2002) using a portable digital pH meter (pH315i, WTW 82362 Weilheim, Germany).Ammonia was measured in the extract by the phenol-hypochlorite assay (Broderick & Kang, 1980) and the buffering capacity of silage was determined according to Moharrery (2007).
3 Short communication: Effect of PEG on ensilage characteristics of pomegranate peel the dose of PEG added were assessed using orthogonal polynomial contrasts to test for linear (L), quadratic (Q) and cubic effects of the level of PEG.Probability levels less than p < 0.05 were considered significant, whereas 0.05 < p < 0.10 was considered a trend.
The chemical composition of the different silages is presented in Table 1.The crude ash, CP, NDF and ADF concentrations decreased linearly (p < 0.001, p = 0.001, p = 0.016, and p = 0.019, respectively) with increasing PEG levels, whereas EE concentration was not influenced by the addition of PEG.The inclusion of PEG increased DM concentration as well as the NFC concentration.The amount of total phenolics and total tannins decreased linearly (p = 0.004 and p = 0.007, respectively), whereas a linear increment (p = 0.028) was seen in non-tannin phenols due to the addition of PEG.The lactic acid concentration and buffering capacity values increased, whereas pH value was lowered by including PEG in the PP silages (Table 2).The ammonia concentrations in the PEG-treated silages The metabolizable energy (ME) content of silages was calculated using equation proposed by Menke et al. (1979).
The data were analyzed according to a completely randomized design using the GLM procedure of SAS (2004).Data of each of the three days within the same levels of PEG were averaged before statistical analysis.Mean values of each individual level of PEG (three replicates for each) were used as the experimental unit.The model used for analysis was: where Yij is the value of each individual observation for the dependent variable, μ is the overall mean, βiis the effect of i level of PEG (i = 0, 5, 10, 15, 20) and εij is the random residual error.The effect of addition of PEG was evaluated with a contrast testing the difference between the control and all treatments receiving the PEG preparation (control vs. PEG).The effects of PEG: polyethylene glycol (% of dry matter); DM: dry matter (g/kg fresh weight), CP: crude protein (g/kg DM), NDF: neutral detergent fiber(g/kg DM), ADF: acid detergent fiber (g/kg DM), EE: ether extract (g/kg DM), NFC: non fiber carbohydrates (g/kg DM), TP: total phenols (g of tannic acid equivalents /kg DM), TT: total tannins (g of tannic acid equivalents /kg DM); NTP: non-tannin phenols (g of tannic acid equivalents /kg DM).The GP in silages treated with PEG was higher than that of the control silage.Increases in GP due to the inclusion of PEG to tanniferous feeds have been previously reported by Baba et al. (2002).Also, inactivation of tannins through PEG binding enhances availability of nutrients resulting in increased microbial activity and GP (Makkar, 2005).The higher ME, TVFA and ammonia values in the silages treated with PEG was in agreement with the findings of Alipour & Rouzbehan (2007), who reported that addition of PEG (750 mg per 375 mg substrate in gas syringes) in grape pomace silage resulted in a higher ME, organic matter digestibility and ammonia concentrations than in the control silage.It should be taken into account that the extent of the limited ability of PEG to completely inhibit the negative effects of tannins on in vitro ruminal fermentation seems to depend on both the type of tannin and the microbial species in the rumen inoculum donor (Frutos et al., 2004).Baba et al. (2002) and Alipour & Rouzbehan (2007) reported that PF values of tanniniferous feeds were above the theoretical range of 2.75-4.41for feedstuffs (Blümmel et al., 1997).However in the present study, the PF values of all treatments were within the theoretical range (3.85-4.33).This could be due to the fact that PEG decreased the PF value in the PP silages.Similarly, Alipour & Rouzbehan (2007) and Baba et al. (2002) reported decreased PF value due to PEG tended to be higher (p = 0.089) than that in the control silage, but addition of PEG had no effect on watersoluble carbohydrates content.
The GP parameters of the different silages are shown in Table 3.Using PEG during ensiling increased the asymptotic GP value (p = 0.025) and the GP rate (p = 0.036).The ME and TVFA contents were quadratically increased by PEG addition (p = 0.022 and p = 0.024, respectively), and ivTD and dNDF in PEGtreated silages tended to be higher than in the control (p = 0.081 and p = 0.069, respectively).Inclusion of PEG increased ammonia concentrations (L, p < 0.001; Q, p < 0.003).The GP 24 was increased quadratically (p = 0.024) by increasing the PEG inclusion level.On the other hand, the PF and EMBP values were decreased quadratically (p = 0.020 and p = 0.032, respectively) by PEG inclusion.However, compared to the control silage, addition of PEG lowered MBP (p = 0.035).
The reduction observed in CP, ash, NDF or ADF content was due to the dilution effect of the added PEG to PP.The higher CP level and lower ammonia concentration in the control group could be explained by the lower CP degradation in the presence of tannins.
The higher lactic acid concentrations, as well as the lower pH values in PEG-treated silages, may be due to the inactivation of tannins by PEG, through the formation of tannin-PEG complexes.In addition, PEG treatment has been found capable of reversing the already formed tannin-substrates complexes, thus making substrates available for fermentation (Mangan, 1988).Salawu et al. (1999) reported that lactic acid concentration in perennial ryegrass silages treated with 0.5 and 5.0% of condensed tannins (on a DM basis) was reduced in comparison with the control.It is note- addition.Decreased of EMBP and MBP due to the inclusion of PEG were observed.These findings were similar to those of Bento et al. (2005) who also argued that EMBP was lower owing to the addition of PEG in comparison with without PEG addition.It should be noted that addition of PEG can affect the presence of some species of bacteria (Belenguer et al., 2011) and therefore the rumen fermentation characteristics.It seems that the inclusion of PEG directed the nutrients toward the production of volatile fatty acids rather than to microbial biomass.Makkar et al. (1998) demonstrated that treating tannin-containing feeds with PEG increased the incorporation of 15 N into microbial protein but reduced EMBP.
Ensiling was found a suitable method for storing and using PP as animal feed in order to reduce the cost of diet and to prevent environmental pollution.Although the ME content of PP is low, it can be used to feed animals near to maintenance ME level, especially for sheep during feed scarcity.Further research is needed to investigate the effect of ensiled PP on productive performance of ruminants, such as milk or meat production, and also on their product quality.

Table 1 .
Chemical composition of pomegranate peel silages treated with different amounts of PEG

Table 2 .
Fermentation ensiling characteristics of pomegranate peel silages treated with different amounts of PEG Rozes & Peres (1998)ycol (% of dry matter); WSC: water soluble carbohydrates; TVFA: total volatile fatty acids; BC: buffering capacity. 4 worthy that tannins may impair the growth of microorganisms responsible for silage fermentation.Rozes & Peres (1998)observed that the growth of Lactobacillus plantarum (a bacterium producing lactate during ensilage) was inhibited by high concentrations of tannins (1 g/L).

Table 3 .
In vitro rumen fermentation parameters of pomegranate peel silages treated with different amounts of PEG Nutritional effects of tannins in animal feeds.Nutr Res Rev 1: 209-231.http://dx.doi.org/10.1079/NRR19880015 Markham R, 1942.A steam distillation apparatus suitable for micro-Kjeldahl analysis.Biochem J 36:790.Menke KH, 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.