BIHAREAN BIOLOGIST 10 (2): 118-122 ©Biharean Biologist, Oradea, Romania, 2016 Article No.: e151408 http://biozoojournals.ro/bihbiol/index.html

Effect of thyme (Thymus vulgaris) or peppermint (Mentha piperita) on performance, digestibility and blood metabolites of fattening Sanjabi lambs

Hassan KHAMISABADI1, Farokh KAFILZADEH1 and Bronamd CHARAIEN2,*

1. Department of Animal Science, Faculty of Agriculture, Razi University, Kermanshah, Iran. 2. Research Centre of Agriculture and Natural Resources of Kermanshah Province, Iran. *Corresponding author, B. Charaien, Tel: +989183334028, Work: +98-83-38358444, Fax: +98-08338370085, E-mail: [email protected]

Received: 02. March 2015 / Accepted: 10. April 2015 / Available online: 02. November 2016 / Printed: December 2016

Abstract. This study was carried out to investigate the effect of inclusion of dietary supplementation of peppermint (Mentha piperita) and Thymus vulgaris on growth performance, blood parameters of finishing lambs. Eighteen Sanjabi male lambs with average body weight of 21.6±1.5 (kg) (mean ± SE) in a completely randomized design were allocated to three treatments and six replications. During the finishing periods (90 days) the lambs were to weighted individually every 15 days and average daily gain and average daily feed consumption, feed conversion ratio, blood biochemical parameters, enzyme activity of alanine transaminase (ALT) and aspartate transaminase (AST) were determined. Statistical significant differences were found in daily weight gain, dry matter intake between three groups . Concentrations of cholesterol, nonesterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), blood creatinine levels were not influenced by addition of either thyme or peppermint to the diet. While triglyceride, glucose and urea were significantly lower in lambs received these plants in their diets. The results showed that addition of thyme or peppermint at a concentration of 3% on DM basis to diet or improve on nutrient digestibility and increased feed intake and average daily gain.

Key word: fattening lambs, Performance, Blood metabolites, peppermint, Thyme.

Introduction compounds of Thyme (Thymus vulgaris) are thymol, carvacrol, terpineol, trans-thujanol-terpineol, tepinene, In recent years, trends and search for ‘natural’ feed additives cymene, borneol and linalool, with thymol being in the in the livestock. Industry has stimulated interest in plant highest concentrations (∼ 360 mg/g; Giordani et al. 2004, secondary compounds or plant extracts such as essential oils Sebeşan & Cărăban 2008). 500–1000 mg/l of thymol has as potential alternatives to growth promoters for animal resulted in accumulation of amino acids, and reduction of production (Thakare 2004, Westendarp 2005). They have ammonia N concentrations in vitro incubations of ruminal been shown to exhibit selective antibacterial activity, and fluid (Brochers 1965, Castillejos et al. 2006 a,b). Newboldet may inhibit degradation of protein in the rumen, thereby al. (2004) investigated the effect of specific blend of essential potentially increasing the intestinal supply of amino acids oil compounds (the major components were thymol, (AA) to the animal host (Wallace 2004).Using plants and guajacol and limonene) on rumen fermentation in sheep. plant extracts in livestock production has expanded after the Deamination of amino acids measured in vitro in rumen ban of use of antibiotics and growth promoters, including fluid removed from the sheep decreased by 25% although ionophores, in livestock nutrition. Bioactive plants and plant the ammonia concentration was unaffected. compounds, when included as feed components have a Hosoda et al. (2005) reported that digestibility of dry range of effects on digestibility and blood parameters in the matter, organic matter, crude protein, neutral detergent ruminant. They may affect blood parameters in sheep by fiber, acid detergent fiber, and gross energy in cows fed the maintaining of plasma glucose level (Raghuvansi et al. 2007), diet additionally supplemented with peppermint were while others may increase (Mohammed et al. 2004) plasma lowered in comparison with that of cows fed no glucose concentration in steers, or alter serum insulin peppermint. Ando et al. (2003) reported that peppermint concentration in young holstein bulls consuming high levels feeding to steers changed the concentration of ruminal of concentrate (Devant et al. 2007). Alterations in serum ammonia-nitrogen but did not affect nutrient digestibility. aspartate transaminase, lactate dehydrogenase, Alanine Therefore the objective of the present study was to aminotransferase, iron, bilirubin, urea, total protein, albumin investigate the influence of addition of peppermint or thyme and calcium concentrations, elevated serum creatinine and to the diet of finishing lamb on growth performance, phosphorus have also been reported in sheep (Adam1998, nutrient digestibility, blood metabolites and rumen Adam et al. 2000, Aslaniet al. 2004, Mahgoubet al. 2008). parameters. Sheep consuming feeds containing phenols and condensed tannins had reduced haematocrit (Mahgoub et al. 2008), While those consuming castor bean had increased Material and methods haematocrit (Aslani et al. 2007).Thyme (Thymus vulgaris L) and of peppermint (Mentha piperita L) are a medicinal herbs The experiment was carried out in Mehregan Research Station of Agriculture and Natural Resources Research Centre of Kermanshah, in the Lamiaceae family, cultivated worldwide for culinary, Iran. Eighteen male lambs from Sanjabi (S) weighing 21± 1.50 Kg cosmetic perennial, and medical proposes these species have (mean initial body weight ± standard error) were used All lambs special functions such as antiseptic, antimicrobial and were born from synchronized ewes maintained under identical antioxidant (Sengul et al. 2008).The main secondary condition with their dams and had access to concentrate, alfalfa hay metabolites in peppermint menthol and menthone together and water ad libitum until weaning at 90 days age. After weaning with several other minor constituents, including pulegone, lambs were drenched against parasites and enterotoxaemia and menthofuran and limonene (Nair 2001). While the active housed in individually iron pens (2m × 1 m), and assigned to one of Effect of thyme or peppermint on performance, digestibility and blood metabolites of fattening Sanjabi lambs 119 the three dietary treatments for 90 days (including 3 weeks of stored at −20 °C for later determination of the NH3-N concentration adaptation period) prior to slaughter. The three diets used were the as described by Chaney et al.(1950). control CON (70% concentrate and 30% alfalfa hay, where the At monthly intervals before the daily feed delivery, blood was concentrate contained corn grain, barley, soybean meal, beet pulp withdrawn from the jugular vein of all lambs from each treatment molasses, salt, vitamin, limestone, sodium bicarbonate and a mineral group. Samples were collected using 6-mL vacutainer tubes (16×100 premix), Peppermint (3% Peppermint, 27% alfalfa and 70% mm; BD vacutainer SST Plus blood collection tubes—Becton concentrate) and the Thyme diet (3% Thymus vulgaris, 27% alfalfa, Dickinson, Franklin Lakes, NJ, USA). The tubes were immediately 70% concentrate). Fresh peppermint and Thymus vulgaris samples transferred to the laboratory, centrifuged (3000×g, 10 min, +4 °C) were collected from the experimental farm of agricultural research and serum was harvested for later analyses of urea nitrogen, and development Institute in the middle of spring. Herbs samples nonesterified fatty acids (NEFA), cholesterol, triglycerides, keratin were dried in shade by shad. The active compounds of the methanol and ß-hydroxyl butyric acid. In serum samples, the concentration of extract from thyme and peppermint were determined by cholesterol, serum urea nitrogen (BUN), total protein, albumin and chromatography. The major compounds of peppermint Shade-dried glucose was determined by an automated biochemical analyzer were (L)-Menthol (37.69%), (L)-Menthone (21.79%), Isomenthone (Biotechnical, Targa 3000, Rome, Italy) using commercial kits [Pars (17.42%) and Menthyl acetate (6.21%). In the case of shad-dried Azmoon Company, Catalogue Numbers: glucose (1-500-017), thyme, the most important compounds of the extract were thymol albumin (1-500-001), total protein (1-500-028), BUN (1-400-029), (49.72%), carvacrol (50.0%). The proximate chemical analysis of the cholesterol (1-500-010), triglyceride (1-500-032), Tehran, Iran] both plant samples was carried out following standard methods of Nonesterified fatty acids (NEFA) and ß-hydroxyl butyric acid[ AOAC (2000). Additionally, samples were analyzed for neutral England Company, Catalogue Numbers: NEFA (), ß-hydroxyl detergent fibre (NDF), and acid detergent fibre (ADF) according to butyric acid() according to the manufacturer’s instructions. Van Soest et al. (1991). Values for NDF and ADF are expressed Statistical Analysis: Statistical analysis of experimental data inclusive of residual Ash. was performed using the GLM procedure of SAS Institute Inc., Cary, NC, USA). All data were analyzed by the Duncan’s new multiple

range tests using the GLM procedures of SAS. The following model Table 1. Ingredient (% DM) of experimental diets fattening sangabi was used: Yij = μ+Ti +eij where Yij represents an observation; μ is lambs. the overall mean; Ti is the effect of the treatments and eij is the Ingredient diet Peppermint Thyme Control residual error. Significantly different means were then further Peppermint 3 - - differentiated using the least significant difference comparison Thymus - 3 - procedures. All statistical tests were conducted at 95% confidence level. Performance data and blood biochemistry were analyzed by Corn grain 25 25 25 mixed model base on repeated measurement. Alfalfa 27 27 30

Barley 22.5 22.5 22.5

Soybean meal 15 15 15 Result Molasses 5 5 5

Salt 0.5 0.5 0.5 Growth performance Mineral supplement* 0.5 0.5 0.5 The growth performance, are shown in Table 2. The average Vitamin supplement* 0.5 0.5 0.5 Sodium bicarbonate 0.5 0.5 0.5 daily feed consumption (g) was not significantly (P>0.05) Limestone 0.5 0.5 0.5 different between the peppermint and T. vulgaris treatment. Metabolizable 2.47 2.44 2.45 The average daily feed consumption (g) in both groups energy(Mcal/kgDM) received medicinal plants was greater than the control Crude Protein (%) 16.10 16.00 16.23 (P<0.05). Calcium (%) 0.750 0.657 0.641 In the present study, using peppermint and T. vulgaris in Phosphorous (%) 0.435 0.448 0.427 the diets increased dry matter intake, which might be related to the high carvacrol present in both of Herbs added to the *Contained (IU/kg): ß-carotene (600,000), cholecalciferol (200000), and tocopherol (200mg), antioxidant (2500mg), calcium (195g), phosphorous (80g), diets. The average daily gain in peppermint treatment was magnesium (210000mg), manganese (2200mg), ferrous (3000mg), copper significantly higher than the other treatment. There were no (300mg), zinc (300mg), cobalt (100mg), iodine (120mg) and selenium (1.1mg). significant differences in feed conversion ratio among

dietary treatments (P>0.05). All lambs had access to water adlibitum during the fattening period. The ration formulated according to the NRC, 2007 In vivo digestibility recommendations. Feces samples were taken from rectal for five The result of in vivo digestibility mthod Acid Insulable Ash days in order to estimate the digestibility of dietary constituents, (AIA) of diet contains peppermint and T. vulgaris are shown based on acid insoluble ash procedure according to the Van Keulen in Table 3. The results suggest that the addition of &Yang (1977). At the end of collection days, feces were weighed and homogenized and a sub-sample was taken. peppermint and T. vulgaris in fattening lambs diets had no Lambs were weighed at twice a week intervals, after 16h fast effect on dry matter, crude protein, ether extract, NDF and (with access to water), and feed consumed daily were measured. ADF digestibility but increased calcium and phosphorus Samples at the end of the finishing period, were collected just before digestibility. (0 h) and at 2, 4 h after feeding. Approximately 50 mL of liquid samples were collected via the mouth using oesophageal sampling Blood metabolites filtered through a triple cheesecloth layer, and the pH was The result of blood serum parameters in during a fattening immediately measured using a digital pH meter (Digmed Model period are shown in Table 4, 5 and 6. Serum metabolites DM21; Digicrom, São Paulo, Brazil). After the pH reading, 1 ml of were not affected by treatments in the end of fattening H2SO40.1 N was added to each sample. At the end of each experimental period, the sample was made for each animal and period. Serum concentrations of Glucose at the middle of

120 H. Khamisabadi et al.

Table 2. Effect of addition of peppermint and Thymuse vulgaris on performance in fattening sangabi lambs.

Parameters Peppermint Thyme Control SEM p-value Growth performance Average daily feed consumption (g) 1494.37a 1488.53a 1185.55b 40.570 0.001> Average daily gain(g) 298.15a 265.83b 228.70b 11.870 0.046 FCR 5.70 6.34 6.01 0.250 0.612

FCR, feed consumed (kg) for 1 kg body weight gain; CON: Control; Peppermint: 3% Pep+ control; Thyme: 3%Thyme+ control. Means within rows with different superscript are significantly different at p<0.05.

Table 3. Effect of Peppermint and Thymus vulgaris on in vivo digestibility in fattening Sanjabi lambs.

Composition (%) Peppermint Thyme Control SEM p-value Dry matter 84.86 86.49 85.05 0.061 0.515 Crude protein 77.65 75.17 76.96 0.667 1.14 Ether extract 77.93 75.43 76.58 0.074 1.28 NDF 58.04 59.50 65.19 0.424 2.20 ADF 49.76 45.96 50.13 0.183 2.56 Calcium 53.98a 44.21a 37.30b 2.99 0.05 Phosphorus 66.65a 53.61a 50.66b 2.99 0.03

Means within rows with different superscript are significantly different at p<0.05.Con: Control; Peppermint: 3% Pep+ control; Thyme: 3%Thyme+ control. Means within rows with different superscript are significantly different at p<0.05; NDF: Neutral detergent fiber; ADF: Acid detergent fiber.

Table 4. Effect of peppermint and Thymus vulgaris on blood metabolites at the first of fattening Sangabi lambs.

Blood metabolites Peppermint Thyme Control SEM p-value Albumin (g/dL) 3.42 3.43 3.52 0.043 0.625 Globulin(mg/dL) 2.40 2.06 2.48 0.081 0.112 Glucose (mg/dL) 80.25 74.00 70.00 2.670 0.277 Protein (g/dL) 5.82 5.50 6.00 0.098 0.127 Cholesterol (mg/dL) 56.25 50.33 55.20 2.870 0.754 Triglycerides (mg/dL) 12.00 12.50 11.00 0.850 0.797 keratin (mg/dL) 1.03b 1.30a 1.23a 0.039 0.002 Urea N, mg/dL 27.26 38.61 29.55 2.410 0.184 NEFA (mmol/L) 0.31b 0.43a 0.42a 0.023 0.050 BHBA 0.54 0.36 0.43 0.043 0.308 ASAT (U/L) 106.75 75.33 79.20 8.490 0.184 ALT (U/L) 18.00 10.33 10.60 1.880 0.177

Means within rows with different superscript are significantly different at p<0.05. NEFA: Non-esterified fatty acids; BHBA: beta-Hydroxybutyric acid; ASAT: Aspartate transaminase; ALT: Alanine transaminase.

Table 5. Effect of peppermint and Thymus vulgaris on blood metabolites at the middle of period in fattening Sangabi lambs.

Blood metabolites Peppermint Thyme Control SEM p-value Albumin (g/dL) 3.62 3.68 3.64 0.029 0.723 Globulin(mg/dL) 2.52 2.70 2.64 0.061 0.507 Glucose (mg/dL) 87.00a 72.40b 73.40b 2.090 0.001> Protein (g/dL) 6.14 6.38 6.28 0.064 0.326 Cholesterol (mg/dL) 45.00 53.40 48.80 2.180 0.313 Triglycerides (mg/dL) 11.20a 8.40ab 6.90b 0.766 0.053 keratin (mg/dL) 0.988 1.05 1.01 0.019 0.423 Urea N, mg/dL 38.60b 54.06a 50.78a 2.330 0.005 NEFA (mmol/L) 0.32 0.31 0.35 0.020 0.755 BHBA 0.37 0.32 0.35 0.025 0.798 ASAT (U/L) 71.20 90.60 83.00 4.620 0.238 ALT (U/L) 9.60 11.00 10.20 0.520 0.581

Means within rows with different superscript are significantly different at p<0.05. NEFA: Non-esterified fatty acids; BHBA: beta-Hydroxybutyric acid; ASAT: Aspartate transaminase; ALT: Alanine transaminase. Effect of thyme or peppermint on performance, digestibility and blood metabolites of fattening Sanjabi lambs 121

Table 6. Effect of peppermint and Thymus vulgaris on blood metabolites at the end of f period in fattening Sangabi lambs

Blood metabolites Peppermint Thyme Control SEM p-value Albumin (g/dL) 3.64 3.76 3.64 0.040 0.406 Globulin(mg/dL) 2.40 2.62 2.74 0.071 0.144 Glucose (mg/dL) 88.80 90.00 87.00 1.300 0.673 Protein (g/dL) 6.04 6.38 6.38 0.088 0.203 Cholesterol (mg/dL) 42.60 50.60 60.40 3.180 0.061 Triglycerides (mg/dL) 9.20 11.30 12.20 0.871 0.381 keratin (mg/dL) 1.15 1.15 1.13 0.019 0.882 Urea N, mg/dL 37.13 42.50 44.72 1.510 0.120 NEFA (mmol/L) 0.32 0.37 0.39 0.016 0.256 BHBA 0.31 0.41 0.47 0.033 0.143 Bilirobin (mg/dL) 0.058 0.030 0.028 0.006 0.105 ASAT (U/L) 84.80 115.60 116.00 9.030 0.289 ALT (U/L) 12.60 13.20 15.60 0.952 0.427

Table 7. Effect of peppermint and Thymus vulgaris on rumen parameters at the middle of period in fattening Sangabi lambs.

Rumen parameters Peppermint Thyme Control SEM p-value pH 0h 7.24 6.80 7.06 0.091 0.144 2h 6.05 5.98 6.33 0.133 0.598 4h 5.81 6.00 6.36 0.164 0.437 NH3-N(100mol/mg) 0h 2.51 3.74 11.61 2.030 0.121 2h 6.53 6.58 12.32 1.230 0.064 4h 4.37 6.66 6.31 0.889 0.592

fattening period, was affected by peppermint treatment Discussion which was significantly higher than the T. vulgaris and control group. Such decreasing in the Glucose in peppermint Our results reveal that the average daily gain of peppermint treatment could be due to major active compounds such as treatment was highest among treatments. Yang et al. (2007) Menthol and Menthone content of peppermint. Serum urea reported that addition of sinnam-aldehyde or carvacrol (200 nitrogen concentration in the middle of fattening period was mg/kg diet) into diet based on corn grain and barley had no lower in lambs fed peppermint compared to those fed the effects on dry matter intake, average daily gain, feed thyme and control diet. This result is somewhat surprising efficiency, carcass characteristic, meat quality in 180 days of because it is consistent with the tendency for lower ruminal finishing lambs. ammonia concentration observed in lambs fed the The increase in feed intake and consequently average peppermint diet compared to lambs fed the diet containing daily gain in peppermint treatment were due to increasing in thyme and no supplement. Blood keratin (mg/dL) and appetite (Russell et al. 2003). NEFA (mmol/L) at the first of fattening period were Bampidis et al. (2005) reported similar results on significantly lower in peppermint than that other treatment. fattening lambs; he showed that using of peppermint Serum concentrations of aspartate (AST) and alanine powder (4 and 8 kg/1000kg) had no effect on growth transaminase (ALT) were not affected by peppermint and T. performance. Urea is synthesized in the liver from ammonia vulgaris which suggests no change in fat mobilization. absorbed from the rumen or gut, and so a urea concentration in blood is positively correlated with the ruminal Ruminal pH and ammonia-N concentration of ammonia (Davidson et al. 2003). Chavez et The mean of ruminal fermentation parameters is presented al. (2011) also reported addition of 200mg/ kg DM of the in Table 7. The different treatments had no effects on carvacrol and sinnam-aldehyde had no effect on average ruminal pH.Compared with the control, in the peppermint daily gain. Hosoda et al. (2006) has shown that and T. vulgaris containing diets ammonia-N concentrations concentrations of glucose, triglyceride, NEFA, total protein, were reduced (P < 0.05) by 70% and 50% in 0h and 2h after in plasma were unaffected by peppermint treatments( 5% feeding, respectively, By contrast, T.vulgaris no decreased in of the diet on a dry matter basis) in holestin steers. 4h after feeding. In this study, reduction in ruminal Wanapat et al. (2013) found lemongrass meal ammonia-N concentrations with the use of peppermint and supplementation at 100 g/d plus peppermint powder at 10 T. vulgaris in the diet of lamb could probably resulted from a g/d (LP), decreased ruminal NH 3 –N concentration in beef. greater concentration of to carvacrol, sinnama-ldehyde and However, Patra & Zhongtang (2012) observed that essential thymol which is effective in proteolysis bacterial and oil of peppermint and thyme (1g/liter) did not affect decreased proteolysis of feed protein. ammonia N concentration in batch cultures. These differences among the experiments may be due to soil composition, climateand harvest stage of herbs. The results 122 H. Khamisabadi et al. in this study are consistent with those of Castillejos et al. Hosoda, K., Kuramoto, K., Eruden, B., Nishida, T., Shioya, S. 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