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Nutritional and chemical evaluation of dried pomegranate (Punica granatum L.) peels and studying the impact of level of inclusion in ration formulation on productive performance of growing Ossimi lambs

Abstract

Background

By 2050, the world will need to feed an additional 2 billion people and require 70% more meat and milk. The increasing future demand for livestock products, driven by increases in income, population, and urbanization will impose a huge demand on feed resources. A huge quantity of fruit and vegetable wastes and by-products from the fruit and vegetable processing industry are available throughout the world that encourages to using it as a new source feeds in animal ration formulation.

Methods

Twenty-eight male growing Ossimi lambs used to study the impact of inclusion dried pomegranate peels (DPP) on productive performance and economic efficiency. In nutritional and chemical evaluation conducted, amino acids, minerals, vitamins, polyphenolic, and fatty acid compositions were determined. Experimental lambs were allocated to one of four complete feed mixture (CFM) containing 0, 0.50, 1.00, and 2.00% DPP for (CFM1, CFM2, CFM3, and CFM4), respectively throughout the feeding period that continuous for 119 days. Twenty-eight lambs with aged 5–6 months with initial weight of 22.875 ± 0.38 kg were divided into four equal groups, each of 7 lambs and fed one of the four iso-nitrogenous that contains 17.1% CP and iso-energetic that contained 2.76% of ether extract (EE) and gross energy (GE) ranged from 4212 to 4214 kcal/kg DM.

Results

Dried pomegranate peel superior in their contents of crude fiber (CF), ether extract (EE), ash, and lignin in comparison with yellow corn. Meanwhile, yellow corn was superior in CP, nitrogen-free extract (NFE), neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose, cellulose, and GE contents in comparison with dried pomegranate peel. Total essential amino acid value was recorded at 51.30 g/100 g CP; meanwhile, value of non-essential amino acids was recorded at 48.37 g/100 g CP. The DPP contained 342, 120, 150, 68, and 56 mg/100 g of calcium (Ca), phosphorus (P), potassium (K), sodium (Na), and magnesium (Mg), respectively. Meanwhile, it contains 1.08, 0.86, 0.65, 6.11, and 1.07 mg/100 g of zinc (Zn), manganese (Mn), cobber (Cu), iron (Fe), and selenium (Se), respectively. Moreover, vitamins were determined by 0.141, 0.09, 13.26, 4.13, and 0.181 mg/100 g for vit. B1; vit. B2; vit. C; vit. E; and vit. A, respectively. Dried pomegranate peel contains high percentages of unsaturated fatty acids that evaluated by 76.96%, while saturated fatty acids (SFA) recorded 23.04%. DPP contained 1.4404% of total polyphenols. Final weight, total body weight gain, and average daily gain were improved. Dry matter intake was significantly (P < 0.05) decreased when expressed as g/h/d and g/kgW0.75, while feed conversion insignificantly (P > 0.05) improved. Dietary treatment improved relative economical efficiency by 117.1, 130.3, and 109% compared with control one with considered control ration equals 100%. Feed cost (LE/kg gain) was decreased.

Conclusion

From this study, it could be mentioned that dried pomegranate peels can be used safely in animal feeding at level of 1% because this level realized the best growth performance and depressed the price of ration cost and recorded the best relative economical efficiency.

Background

Agro industrial by-products which contain little economical value as edible foods for human consumption have become major sources of dietary nutrients and energy in support of milk and meat production and will continue to do so in the future (Bampidis and Robinson 2006).

Pomegranate fruit has been cultivated around the world in subtropical and tropical regions such as in Iraq, Iran, California, Turkey, Egypt, Italy, India, Chile, and Spain (Sadq et al. 2016).

Pomegranate production amounts could reach to approximately 65,000 tons in Egypt (FAOSTAT-FAO 2010), where the peels (pericarp, rind, or hull) amount to approximately 60% of the pomegranate fruit weight (Lansky and Newman 2007). On the other hand, it is noticed that, about 500 g/kg of the total fruit weight corresponds to the peel, while the rest are the edible parts of pomegranate, consisting of 400 g/kg arils and 100 g/kg seeds (Aviram et al. 2000).

Recently, pomegranate by-products have attracted attention, as they have been found to contain substantial amounts of polyphenols such as ellagic tannins, ellagic acid, punicalagin, and gallic acid (Jami et al. 2012), which have been shown to possess antimicrobial, antioxidant, antiinflammatory, antimitotic, and immunomodulatory properties both in vivo and in vitro (Adams et al. 2006; Jayaprakasha et al. 2006; Rosenblat and Aviram 2006; Kotsampasi et al. 2014).

Pomegranate is an important source of bioactive compounds and different parts of it have been used in medicine for many centuries (Hajimahmoodi et al. 2008; Elfalleh et al. 2012; Hajimahmoodi et al. 2013) and the edible parts used pharmaceutical world-wide.

Pomegranate is composed of a rich variety of flavonoids, which comprise approximately 0.2 to 1.0% of the fruit. Approximately 30% of all anthocyanidins found in pomegranate are contained within the peel. These flavonoid-rich pomegranate phenolic fractions are responsible for anticancer activity (Batta 1973; Elango et al. 2011).

Pomegranate peel provides higher yields of phenolics, flavonoids, and proanthocyanidins than the pulp. Flavonoid content was significantly greater in the peel than the pulp (59 vs. 17 mg/g), as were proanthocyanidins (11 vs. 5 mg/g) as reported by (Li et al. 2006).

There are some reports about the presence of tannins, alkaloids, glycosides, flavonoids, and phenolic compounds as antioxidant factors in juice, peel, pulp, and seed fractions of pomegranate (Noda et al. 2002).

Pomegranate (mainly pulp) may play a role in the prevention of cancer and heart disease (Aviram et al. 2002).

Sadq et al. (2016) noted that incorporation pomegranate peels at different levels (0, 1, 2, or 4%) in Karadi lamb rations improved their growth performance.

Also, Shabtay et al. (2008) reported that dietary supplementation with fresh pomegranate peels promoted a significant increase in feed intake, with a positive tendency toward increased body weight gain in bull calves.

So, this work was carried out to investigate the nutritional and chemical evaluation of dried pomegranate peels, in addition to study the impact of inclusion pomegranate peel in growing Ossimi lamb rations at different levels on their performance and economic evaluation.

Methods

The present experiment was carried out at the Sheep and Goats’ Units in El-Bostan area in Nubaria, which belongs to the Animal Production Department, National Research Center, Dokki, Cairo, Egypt.

Twenty-eight male Ossimi lambs, aged 5–6 months old with an average live weight of 22.875 ± 0.38 kg, were divided randomly into four equal groups (seven animals each) to study the effect of incorporation dried pomegranate (Punica granatum L.) peels (DPP) at different levels (0, 0.5, 1.0, and 2.0%) on growth performance and economic evaluation.

Experimental animals were housed in semi-open pens and fed as group feeding for 119 days, the experimental rations received to cover the requirements of total digestible nutrients and protein for growing sheep according to the NRC (1985).

Pomegranate (Punica granatum L.; Punicaceae) peels were obtained from Vitrac company for food industrials. The peel was sun-dried, powdered, and incorporated in experimental complete feed mixture (CFM) at levels of 0.00, 0.50, 1.00, and 2.00% for (CFM1, CFM 2, CFM 3, and CFM 4), respectively.

Daily amounts of experimental CFM were adjusted every 2 weeks according to body weight changes. Total mixed rations were offered twice daily in two equal portions at 800 and 1300 h, while feed residues were daily collected, sun dried, and weekly weighed. Fresh water was freely available at all times in plastic containers. Body weight change was weekly recorded before the morning meal.

Analytical procedures

Representative samples of experimental TMR were analyzed according to AOAC (2005) methods.

Amino acid composition of dried pomegranate peels (DPP) was determined according to the method described by the AOAC (2005) and Millipore Cooperative (1987) using HPLC and the modification of PICO-TAG methods.

Mineral were determined by digested a part of sample in 10 ml of nitric acid overnight on a steam bath and subsequently digested with 70% perchloric acid. Calcium, P, K, Na, Mg, Zn, Mn, Cu, Fe, and Se were analyzed by atomic absorption spectrophotometry using standard procedures of the AOAC (2005). Phosphorus was analyzed using method N-4C according to (Kraul 1966). Meanwhile, selenium was determined with an autoanalyzer fluorometric selenium method described by Brown and Watkinson (1977).

Fatty acid profiles were conducted throughout extracted lipids from DPP by diethyl ether as described by the AOAC (2005). The extracted lipids were converted to methyl esters as described by AOAC (2000) and analyzed for individual fatty acids (C14: 0 to C20: 4) using a gas chromatograph (3400, Varian Inc., Walnut Creek, CA) fitted with a flame ionization detector. Gas chromatography parameters were as follows: the column temperature was 50 °C for 3 min and then increased to 220 °C at 4 °C/min and was held for 15 min. The injector temperature was 200 °C, and the detector temperature was 250 °C. The flow rates of the carrier gases (hydrogen and oxygen) were 30 and 300 ml/min, respectively. Identification and quantification of individual fatty acids were made by using a standard fatty acid methyl ester mixture (2010, Matreya Biochemical LLC, Pleasant Gap, PA).

Vitamins including B1 (Thiamin), B2 (riboflavin), and C (L-Ascorbic) of DPP were evaluated according to the method described in the AOAC (2005) using high-performance liquid chromatography (HPLC), while both vitamin E (α-Tochoferol) and vitamin A (Retinol) were determined using high-pressure liquid chromatography (HPLC) method that described by (Leth and Sondergaro 1983).

The phenolic compounds of DPP were extracted according to the method of Singleton et al. (1999) in which a known weight (0.5 g) of dried DPP was extracted with 50 ml methanol for 3 h. Identification of individual phenolic compounds of investigated DPP was performed on a HPLC.

Economic evaluation

Economic evaluation was done using the relationship between feed costs (local market price of ingredients) and sheep live body weight gain. Economic evaluation was calculated as follows:

The cost for 1-kg gain = total cost {Egyptian pound (LE)} of feed intake/total gain (kilogram).

Statistical analysis

Collected data of feed intake, live body weight, average daily gain, and feed conversion were subjected to statistical analysis as one-way analysis of variance using the general linear model procedure of SPSS (2008). Duncan’s Multiple Range Test (1955) was used to separate means when the dietary treatment effect was significant according to the following model:

Yij = μ + Ti+ eij. Where Yij = observation, μ = overall mean, Ti = effect of tested complete feed mixture (CFM) containing different levels of dried pomegranate peels (DPP) for i = 1–4, 1 = CFM contained 0% DPP, 2 = CFM contained 0.50% DPP, 3 = CFM contained 1.00% DPP, and 4 = CFM contained 2.00% DPP.

eij = the experimental error.

Results

Nutritional and chemical evaluation of dried pomegranate peels

Chemical analysis

Data of Table 1 cleared that dried pomegranate peel (DPP) superior in their contents of CF, EE, ash, and lignin compared with yellow corn. Meanwhile, yellow corn was superior in CP, NFE, NDF, ADF, hemicellulose, cellulose, and gross energy contents in comparison with dried pomegranate peels. The present results mentioned that chemical composition of DPP near from yellow corn, so it can be using DPP as unconventional source of energy in sheep ration formulation. The corresponding values were (8.85 vs. 6.52 CP); (4.80 vs. 10.50 CF); (3.30 vs. 3.46 EE); (80.48 vs. 76.09 NFE); (2.57 vs. 3.43 ash); (37.14 vs. 29.36 NDF); (23.65 vs. 19.22 ADF); (2.18 vs. 3.90 ADL); (13.49 vs. 10.14 hemicellulose); (21.47 vs. 15.32 cellulose); and (4349 vs. 4287 kcal/kg dry matter for gross energy) for yellow corn and dried pomegranate peel, respectively.

Table 1 Chemical analysis of yellow corn and dried pomegranate peel

Amino acid composition

Amino acid composition of dried pomegranate peels (g/100 g CP) that illustrated in (Table 2) cleared that DPP protein contained a much higher content of essential amino acids (arginin, histidine, leucine, lysine, phenylalanine, and valine). The corresponding values were 8.23, 7.56; 7.16, 7.23, 7.14, and 5.33 (g/100 g CP) for the same amino acids, respectively. On the other hand, both isoleucine and methionine recorded the moderate values (3.51 and 3.02 g/100 g CP, respectively); meanwhile, thronine showed the lowest value (2.12 g/100 g CP). Essential amino acids recorded (51.30 g/100 g CP) of total amino acid in DPP, while non-essential amino acids (alanine, aspartic, cystine, glutamic, glycine, praline, serine, and tyrosine) recorded (48.37 g/100 g CP).

Table 2 Amino acid composition of dried pomegranate peels (g/100 g CP)

Minerals content

Determination of mineral content of dried pomegranate peel (DPP) that presented in Table 3 showed that DPP contained all tested minerals. The DPP contained the most determined minerals at adequate concentration and the predominant minerals in DPP were found to be Ca, P, K, Na, and Mg at levels of 342, 120, 150, 68, and 56 mg/100 g DM, respectively. In addition, the DPP contained a considerable content of Zn, Mn, Cu, Fe, and Se at levels of 1.08, 0.86, 0.65, 6.11, and 1.07 mg/100 g DM, respectively. So, in general, the present results can be concluded that DPP were characterized with their richness with the most determined nutritious minerals and they are considered a good source of macro- and micro-elements. Therefore, they should be utilized in food fortification.

Table 3 Mineral content of dried pomegranate peels (mg/100 g)

Vitamin contents

Data of Table 4 included the vitamins that determined in DPP that composed of vit. B1 (Thiamine), vit. B2 (Riboflavin), vit. C (L-Ascorbic acid), vit. E (α-Tochoferol), and vit. A (Retinol). The corresponding values of vitamins determined above were 0.141; 0.09, 13.26, 4.13, and 0.181 mg/100 g DM of DPP.

Table 4 Vitamin content (mg/100 g) of dried pomegranate peel

Fatty acid profile

Data of fatty acid profile that illustrated in Table 5 cleared that DPP contains high percentages of unsaturated fatty acids {ω-9 cis-9 Oleic (C18: 1); ω-6 cis-9,12 linoleic (C18: 2); ω-3 cis-9, 12, 15α-linolenic acid (C18: 3); ω-3 cis-4, 7, 10, 13, 16, 19 eicosapentaenoic acid (C20: 5); and ω-3 cis-4, 7, 10, 13, 16, 19 docosahexaenoic acids}. The corresponding values were 10.95, 13.89, 6.03, 12.23, and 17.22%, respectively. Total unsaturated fatty acids (TUFA) recorded 76.96%, while saturated fatty acids (SFA) recorded 23.04%. On the other hand, ω3 fatty acid value was 35.48%; meanwhile, ω6 fatty acid value was 20.72% and ω9 fatty acid value was 17.64%.

Table 5 Fatty acid profile of dried pomegranate peel

Polyphenolic fraction contents

Data of polyphenolic fraction content of DPP is presented in Table 6. The obtained results showed that DPP contained a high content (1440.40 mg/100 g DDP) that equals (1.4404%) of total polyphenols. The major polyphenolic fractions in DPP were catechins, phenol, gallic acid, caffeic acid, ellagic acid, p-coumaric acid, and resocenol which were found at the level of 892, 254.36, 128, 55.23, 52.03, 14.22, and 14.09 mg/100 g DM of DPP, respectively. On the other hand, DPP also contained adequate amounts from protocatechol, p-hydroxy benzoic acid, vanilline, and ferulic acid at concentrations of 4.62, 10.33, 4.17, and 6.11 mg/100 g DM of DPP, respectively.

Table 6 Polyphenolic fractions content (mg/100 g) of dried pomegranate peel

Composition and chemical analysis of complete feed mixture

Data of Table 7 cleared that different experiment complete feed mixture was formulated as iso-caloric and iso-nitrogenous. Crude protein contents were ranged from 17.07 to 17.12%; ether extract content ranged from 2.75 to 2.76%; and gross energy ranged from 4212 to 4214 kcal/kg DM; meanwhile, crude fiber varied from 10.90 to 11.01%; nitrogen-free extract ranged from 61.00 to 61.08%; and ash content ranged from 8.14 to 8.16%.

Table 7 Composition and chemical analysis of complete feed mixture

Growth performance

The present results of growth performance are presented in Table 8 showed that inclusion dried pomegranate peel (DPP) at different levels (0.5, 1.0, and 2.0%) in complete feed mixture (CFM2 to CFM4) improved final weight, total body weight gain, and average daily gain. These values were improved by (6.52, 10.87 and 2.17% for final weight); (10.64, 18.09 and 3.19% for total body weight gain); and (10.38, 17.97 and 3.29% for average daily gain) for CFM2, CFM3, and CFM4, respectively in comparison with the control (CFM1). The best values were noticed by (CFM3 that contained 1% DPP).

Table 8 Growth performance of experimental groups

Economic evaluation

The profitability of using agro-industrial by-products in ration formulation of livestock depends on upon the costing of tested materials, ingredients used in ration formulations, and the growth performance obtained.

Data of Table 9 cleared that incorporation DPP at different levels in ration formulation slightly decreased price of 1 kg of CFM; however, dietary treatment improved relative economical efficiency by 117.1, 130.3, and 109% compared with control one with considered the relative economical efficiency of control ration equals 100%. On the other hand, feed cost (LE/kg gain) was decreased with inclusion DPP in the CFM. The corresponding values of feed cost were 22.98, 20.64, 18.88, and 20.83 (LE/kg gain) for CFM1, CFM2, CFM3, and CFM4, respectively.

Table 9 Economic evaluation of experimental groups

Discussion

The present results of chemical analysis of dried pomegranate peel that illustrated in Table 1 were in harmony with those noted by Shabtay et al. (2008) and Kushwaha et al. (2013).

Our results obtained in Table 2 cleared that DPP is good quality source of protein that can be used in sheep ration formulation. Added to that, these results in agreement with those noticed by (Rowayshed et al. 2013) who noted that pomegranate fruit by-products (peels and seeds, powder) contained a much higher exceptionally content of lysine, isoleucine, and amino acid-containing sulphur (Methionine and cysteine), which are usually deficient in the most food stuffs, than the reference protein pattern of FAO/WHO.

Results of mineral content for dried pomegranate peel that presented in (Table 3) were in harmony with those noted by (Rowayshed et al. 2013) who reported that pomegranate fruit by-products were characterized with their richness with the most determined minerals.

Data of Table 4 that illustrated the vitamin contents of DPP cleared that it contained vit. B1 (Thiamine), vit. B2 (Riboflavin), vit. C (L-Ascorbic acid), vit. E (α-Tochoferol), and vit. A (Retinol). The result values were in agreement with those established by (Rowayshed et al. 2013). The determination of vitamins naturally occurred in DPP is considered one of the most important phytochemicals having the antioxidant, antimicrobial, and chemo preventive cancer properties and good standpoint in human nutrition (Huxley and Neil 2003). So, it could be mentioned that DPP considered a good source of vitamins.

Data obtained of fatty acid profile that illustrated in Table 5 cleared that DPP can be used as a good quality of fatty acids in sheep ration formulation. In addition, the present results were in agreement with those found by (Kotsampasi et al. 2014).

Polyphenolic fraction contents of DPP that presented in Table 6 are nearly in accordance with those obtained by Metche et al. (1996); Wang et al. (2004a, 2004b); Abbasi et al. (2008a, 2008b); Elfalleh et al. (2011); Abdel-Rahim et al. (2013) and Rowayshed et al. (2013).

Data of Table 7 showed that different experiment complete feed mixture was formulated as iso-caloric and iso-nitrogenous and their chemical analysis was covered the nutrient requirement for sheep according to NRC (1985).

Results of growth performance that illustrated in Table 8 recorded that when DPP incorporated at different levels (0.5, 1, and 2%), it caused an improvement in their gain while decreasing their feed intake and improving their feed conversion. These results in agreement with those reported by Sadq et al. (2016) who showed that final body weight was significantly (P < 0.05) higher in Karadi lambs fed 1% or 2% pomegranate peel as compared with lambs fed 4%. Also, incorporation DPP at level of 1 or 2% significantly (P < 0.05) decreased dry matter intake as expressed as g/h/d or g/kgW0.75; however, CFM2 that contained 0.5% DPP insignificantly (P < 0.05) decreased compared with the control one. These results are in agreement with those found by Reed (1995) and Kushwaha et al. (2013) who noted that high concentration of tannin in pomegranate peel powder may reduce intake, through their negative effect on palatability. In contrast Sadq et al. (2016) found that total dry matter intake increased (P < 0.05) significantly in lambs fed 1% pomegranate peel. On the other hand, dietary treatments insignificantly (P > 0.05) improved feed conversion that expressed as (g intake/g gain). The best feed conversion was found with incorporation DPP at 1% of TMR content (4.98). The present results are in harmony with those reported by Sadq et al. (2016) who established that the best improvements in feed conversion in Karadi lambs fed 1% pomegranate peels.

Data of economic evaluation (Table 9) mentioned that incorporation DPP in sheep ration formulation realized an improvement in their relative economic efficiency and decreased the feed costing per kg gain. These results were in agreement with those found by Denek and Can (2006); Omer and Abdel-Magid Soha (2015) who noted that the use of agro-industrial by-products in sheep rations has been successfully adopted as a strategy to reduce feeding costs and also to cope with the need to recycle waste material. Also, Romero-Huelva et al. (2012) noted that instead of 35% of concentrate with feed blocks containing waste fruits in lactating goat rations reduced animal feeding cost. Also, El Shaer et al. (1997) concluded that organic waste feed mixture used as non-conventional feed could be efficiently used as nutritious, palatable, and low-cost feed resources for small ruminants in Egypt.

Conclusion

From the data illustrated in the present study, it could be mentioned that dried pomegranate peel can be used safely in sheep feeding at level of 1% because this level realized the best growth performance and depressed the price of ration cost and recorded the best relative economical efficiency.

Availability of data and materials

“Not applicable” for that section.

Abbreviations

%:

Percentage

°C:

Degree centigrade

ADF:

Acid detergent fiber

ADL:

Acid detergent lignin

AOAC:

Official Methods of Analysis

Ca:

Calcium

CF:

Crude fiber

CFM:

Complete feed mixture

CP:

Crude protein

Cu:

Cobber

DHA:

Docosahexaenoic acid

DM:

Dry matter

DMI:

Dry matter intake

DPP:

Dried pomegranate peels

EE:

Ether extract

EPA:

Eicosapentaenoic acid

FAOSTAT-FAO:

Statistical database. Food and Agriculture Organization of the United Nations

Fe:

Iron

FW:

Final weight

g/h/d:

Gram per head per day

g/kg:

Gram per kilo gram

g/kgW0.75 :

Gram per kilo gram metabolic body weight size

GE:

Gross energy

K:

Potassium (K)

Kg/100 kg LBW:

Kilogram per 100-kg live body weight

kg/ton:

Kilogram per ton

LBW:

Live body weight

LE:

Egyptian pound

LE/ kg gain:

Egyptian pound per kilogram gain

Mg:

Magnesium

mg/100 g:

Millegram per 100 g

ml/min:

Mille per minute

Mn:

Manganese

MUFA:

Monounsaturated fatty acids

Na:

Sodium

NDF:

Neutral detergent fiber

NFE:

Nitrogen free-extract

NRC:

National Research Council

P:

Phosphorus

PUFA:

Polyunsaturated fatty acids

Se:

Selenium

SEM:

Standard error of the mean

SFA:

Saturated fatty acids

SPSS:

Statistical package for Social Sciences

TBWG:

Total body weight gain

TUFA:

Total unsaturated fatty acids

Vit:

Vitamin

Vit. A:

Retinol

Vit. B1 :

Thiamine

Vit. E:

α-Tochoferol

Vit. B2 :

Riboflavin

Vit. C:

L-Ascorbic acid

Zn:

Zinc

α-Linolenic:

Alpha linolenic

μ:

Overall mean

ω-3:

Omega three

ω-6 :

Omega six

ω6/ω3:

Omega six/Omega three

ω7:

Omega seven

ω-9:

Omega nine

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Acknowledgements

Our deep thanks for the workers in the Sheep and Goats’ Units for their help in conducting follow-up on the experimental animals during the feeding trial and cooperating in many steps that make this work possible.

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HAAO cooperated in the plane of work, field work, chemical analysis, arrangement data, statistical data, and writing, corrected the MS, and helped in the publication. SSA cooperated in the plane of work, field work, and following publication with the Journal (corresponding author). IMA cooperated in the plane of work, field work, and revision of the MS. All authors read and approved the final manuscript.

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Correspondence to Soha S. Abdel-Magid.

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Omer, H.A.A., Abdel-Magid, S.S. & Awadalla, I.M. Nutritional and chemical evaluation of dried pomegranate (Punica granatum L.) peels and studying the impact of level of inclusion in ration formulation on productive performance of growing Ossimi lambs. Bull Natl Res Cent 43, 182 (2019). https://doi.org/10.1186/s42269-019-0245-0

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