Introduction

India is considered as one of the 12 enormous biodiversity centres having over 45,000 plant species. Recent field trials conducted on certain herbal formulations demonstrated the use of herbal preparations as growth promoters and have reported to reveal positive results with respect to body weight gain, feed efficiency, lowered mortality and increased livability in poultry (Kumar, 1991). The World Health Organization has recognized antibiotic resistance as a major and a growing problem related to the public health in both human and animal medicine. The alternative measure to overcome the problem of antibiotic resistance might be the use of phytochemicals and natural products (Reverter et al., 2014). They have several advantages effects over commonly used antibiotics and are generally considered as safe and their uses in the food industry have also attained popularity (Varel, 2002; Brenes and Roura, 2010). As the use of phytogenic substances have gain huge momentum, the plant family Lamiaceae has receives the greatest interest in the feed of poultry, with peppermint, oregano and thyme being the most popular representatives (Burt, 2004). The essential oils of Mentha plants have been reported to have anti-inflammatory, antimicrobial, antioxidant, anticarcinogenic, insecticide and analgesic properties (Shaikh et al., 2014) and also have widely used in the perfumery, pharmaceutical and food industries (Thawkar et al., 2016).

Out of a huge number of essential oil bearing plants belonging to Mentha species, (Mentha arvensis) locally known as pudina (Khan and Khatoon, 2008) constitutes the most important source of therapeutic agents and is used in the alternative systems of medicine (Naeem et al., 2011).The mint plant has been reported to contain many active ingredients such as menthol (40-50 %) (Malik et al., 2012), carvone (67.3 %), limonene (13.5%), 1,8- cineole (5.4%), linalool (2.8 %), menthone (2.9 %), and isomethone (1.2 %) (Younis et al., 2004) which provide positive effects on health and productivity (Ghazaghi et al., 2014). Therefore, looking to the role of M. arvensis, a feeding trial was conducted to study the effect of feeding different levels of pudina leaf powder supplementation on biochemical parameters in laying hens.

Material and Methods

A feeding trial of 12 weeks duration was conducted to evaluate the effect of incremental doses of pudina (Mentha arvensis) leaf powder in laying hens. Pudina leaf powder was procured from R.K.Dehydration, Gujarat. A total of 120 white leghorn laying hens of 36 weeks of age were used in the experiment. The experiment was carried out in Californian cages in a completely randomized design (CRD). The laying hens were randomly divided into four treatment groups each with six replicates of five birds each. The group T1 served as control in which feed was provided with no supplementation of pudina while T2, T3 and T4 were the treated groups in which Mentha arvensis leaf powder was added at 5.0, 7.5 and 10.0 g/ kg respectively.

The laying hens were housed in individual cage. The different treatment groups were provided similar housing and managemental conditions. The birds were provided ad lib feed and water throughout the feeding trial period of 12 weeks. At the end of the experiment (47^th week), blood was collected randomly from two birds of each replicate aseptically through the wing vein in sterilized disposable syringe. So, a total number of 48 blood samples were collected to study serum biochemical parameters. The collected blood sample was transferred in a clean test tube for serum separation to study the biochemical parameters using Span Diagnostic kits.

Statistical Analysis

All statistical analysis was done with the help of SPSS procedure and the data obtained during the experiment was further evaluated using one-way analysis of variance (ANOVA).

Table 1: Proximate analysis and nutritive value of dried pudina (Mentha arvensis) leaf powder

Proximate Analysis

Results and Discussion

Following biochemical parameters were studied using the serum obtained from the blood samples. As there is limited literatures available related to Mentha arvensis feeding in laying hens, therefore, comparative studies are made with other plants belonging to the genus Mentha.

Triglycerides

Pudina leaf powder supplementation had a reducing effect on serum triglycerides concentration (Table 2). Triglycerides values of group T1 (130.36 mg/dl) was significantly (P<0.05) higher than T2, T3 and T4 while the least value was recorded in T2 (106.98 mg/dl) group. The reduction of triglycerides values might be due to the reduction in lipid which is believed to be contributed by tocopherol and menthol contents in peppermint (Mansoub, 2011). According to Escop (2003), the reduction of triglycerides values was believed due to the presence of menthol and menthone in pudina leaves as they have the potential to decrease blood lipids in broiler chickens.

Total Cholesterol

The results of the present experiment revealed that the addition of pudina powder in the diet of laying hens significantly (P<0.05) decreased serum cholesterol concentration (Table 2) in laying hens. The minimum cholesterol value was recorded in T4 group (138.30 mg/dl) which was significantly (P<0.05) lower than T1, T2 groups; meanwhile the maximum value was recorded in control group (168.33 mg/dl). The hypocholesteramic effect of pudina might be due to the menthol and menthone content which has the property to lower the activity of a liver enzyme HMG CoA reductase (a prime enzyme in cholesterol synthesis) and simultaneously lower cholesterol level. Cholesterol synthesis mainly takes place in liver (Poltowicz and Wesyk, 2005) with the maximum deposition of cholesterol in egg yolk. Mentha arvensis leaves exhibit highest content of phenol and flavonoids hence it is reported to have the greatest antioxidant property (Vishwakarma et al., 2014) and reduce cholesterol level.

The findings of present investigation corroborate with the previous study conducted by Abdel and Lohakare (2014) in which serum biochemical analyses in laying hens fed with various levels of peppermint leaves revealed that serum cholesterol linearly decreased with increasing experimental diet. Al-Fartosi and Al- Rekabi (2014) demonstrated that the phenolic compounds of leaves extract from Mentha longifolia and Mentha spicata in induced diabetic rats significantly reduced serum cholesterol level compared to untreated groups. In contrast, the results of the experiment conducted by Akbari and Torki (2014) failed to show the hypocholesterolaemic effects of dietary supplementation of peppermint together with chromium picolinate in broiler chicks.

HDL- Cholesterol

The supplementation of pudina leaf powder had an increasing effect on serum HDL cholesterol (Table 2). The mean values of group T4 (78.14 mg/dl) was significantly (P<0.05) higher than those of T1, T2 and T3. The results of the present study are in line with the findings of Mansoub (2011) who reported that elevation in HDL- cholesterol in serum of broiler might be due to tocopherol and menthol contents in peppermint. Similarly, Fallah et al. (2013) demonstrated that extract of peppermint increased HDL- cholesterol in broilers and is supposed to be due to the presence of active ingredients such as menthol and menthone.

LDL- cholesterol

Pudina leaf powder supplementation significantly had a decreasing effect on serum LDL-cholesterol values in white leghorn laying hens (Table 2). The mean values of T3 and T4 were significantly (P<0.05) lower than T1 and T2 groups. The minimum value recorded in T4 (38.63 mg/dl) group. The possible reason behind the reduction in LDL- cholesterol might be due to the reduction in blood lipid which is believed to be contributed by tocopherol and menthol (Mansoub, 2011), menthol and menthone (Escop, 2003; Falah et al., 2013) contents. The study conducted by Akbari and Torki (2014) reported that significant decrease in serum LDL- cholesterol might be due to the protective role of peppermint extract against heat stress condition.

VLDL-Cholesterol

The mean values of VLDL-Cholesterol ratio of T1, T2, T3 and T4 groups are presented (Table 2). The mean values of VLDL-Cholesterol of group T1 (26.07 mg/dl) of laying hens was significantly (P<0.05) higher than T2, T3 and T4 groups of laying hens. There is scarcity of literature regards to pudina powder supplementation on serum VLDL-cholesterol levels.

Glucose

The results of the present study indicated that there was significant (P<0.05) decreased in the concentration of glucose due to the addition of pudina leaf powder (Table 3). Serum glucose value of T1 (190.82 mg/dl) group devoid of pudina leaf powder was significantly (P<0.05) higher than T2, T3 and T4 groups while the least glucose level was reported in T4 group (170.60 mg/dl) of laying hens.

Kothai (2005) reported that the phytochemical compound, flavonoid in pudina, is responsible for reducing the blood glucose level and simultaneously increase the number of pancreatic beta cells. Mansoub (2011) reported that reduction in glucose level in serum of broiler might be due to the decline in lipid in serum of broilers which is believed to be contributed by tocopherol and menthol contents in peppermint. Akbari and Torki (2014) reported that decline in serum glucose level might be due to the protective action of peppermint extract against heat stress condition. Khamisabadi et al. (2015) demonstrated that extract of peppermint had decreasing effect on serum glucose in broiler was believed owing to the presence of major components like menthol and menthone, which have the potential to decrease blood lipids in broiler (Escop, 2003).

The results of present study correlate with the findings of Barbalho et al. (2011) who reported that the offspring from diabetic dam treated with peppermint showed significant decrease in glucose level as compared to control group. On the contrary, Abdel and Lohakare (2014) concluded that there was no significant effect of peppermint leaves feeding on blood glucose level in laying birds.

Total Protein

Administration of pudina leaf powder in the feed of laying hens significantly (P<0.05) improved serum total protein levels (Table 3). Serum total protein value of T4 (5.58 mg/dl) group was significantly (P<0.05) higher than T1 and T3 but non- significant difference was noted between the groups T2 and T4. Mansoub (2011) reported that elevation in total protein levels in serum of broilers might be due to the reduction in lipid in serum of broilers which is believed to be contributed by tocopherol and menthol contents in peppermint. According to the experiment conducted by Fallah et al. (2013) extract of peppermint had increased total protein concentration in broiler.

Albumin

Supplementation of pudina leaf powder supplementation had an increasing consequence on serum total albumin concentration (Table 3). Albumin value in T4 (2.15 mg/dl) group was significantly (P<0.05) higher than the T1 (1.66 mg/dl) group with non- significant differences among T2, T3 and T4 groups. The results of the present investigation are in accordance with previous research conducted by Nayak et al. (2014) who demonstrated that broilers fed with combination of 4 g Mentha piperita and 2 ppm ochratoxin showed an increase serum albumin concentration at all stages of growth. In contrast to the present findings, no difference in mean values of albumin between the treated and untreated groups fed with Mentha piperita leaves in laying hens (Abdel and Lohakare, 2014) and in Japanese quail fed with Mentha spicata (Ghazaghi et al., 2014) were noted. Increase in albumin concentration revealed that there is no possible reason related to certain factors associated with protein synthesis and leakage of albumin as a consequence of kidney damage (Huff et al., 1988). Thus, supplementation of pudina leaf powder can be recommended in laying hens.

Globulin

The concentration of globulin had increased significantly due to the inclusion of pudina leaf powder (Table 3). Globulin values in T2 and T4 groups were significantly (P<0.05) higher than the T1 group with no significance difference among T2, T3 and T4 groups of laying hens. The findings of present experiment on serum globulin level are supported by Zerehdaran et al. (2016) who observed significant increase in serum globulin levels in broiler fed with peppermint plant powder. On the contrary, Mesbahzadeh et al. (2015) found non-significant increase in the serum globulin level in male wistar rats supplemented with peppermint extract. Higher value of serum globulin reflexes higher amount of immunoglobulin. As the gamma fractions is the largest portion of globulin, it can be concluded that the dietary supplementation of pudina might increases immune response.

Albumin/ Globulin Ratio

Albumin/ Globulin Ratio was linearly increased in pudina supplemented groups, however, the increase was no significant among different treatment groups (Table 3). Akbari and Torki (2014) reported that increase in albumin/ globulin ratio might be due to the protective role of peppermint extract against heat stress condition.

Uric Acid

The supplementation of pudina leaf powder in serum uric acid of laying hens had a remarkable reducing effect (Table 3). The mean values of T1 (3.22 mg/dl) was the maximum and significantly (P<0.05) higher than T3 and T4, while T3 (2.78 mg/dl) had the lowest value of uric acid. In the present study, the possible reason for reduction of serum uric acid concentration in groups supplemented with pudina may be the lower intake and more efficient use of proteins in the diet. The results of the present experiment are in line with the earlier observations noted by Sharifi et al. (2010) where they observed that a significant reduction in serum uric acid concentration was observed with the inclusion of peppermint essential oil in broiler chicks. The serum uric acid levels of different groups in the present experiment clearly indicate that there is no adverse effect of pudina leaf powder supplementation on renal function of laying hens.

Calcium

The statistical analysis of serum calcium concentration in the present study showed non- significant difference among different groups (Table 3). In agreement with these results, Abdel and Lohakare (2014) suggested that there were no significant effects of peppermint leaves supplementation on serum calcium concentration in laying hens.

Creatinine

The laying hens of treatment groups had numerically higher value of serum creatinine compared to the control group but no significant difference were noted (Table 3). The results of the present study showed contrasting results to those of Thangapandiyan et al. (2013). They reported significant decrease in the level of serum creatinine with combined supplementation of peppermint leaf extract and cadmium in comparison with cadmium intoxicated rats. Serum concentration of creatinine is widely used as an indicator of kidney function test.

Serum creatinine as well as uric acid concentrations of different groups were within the normal range which indicate that there is no adverse effect of pudina supplementation on renal function of laying hens.

Serum Aspartate Aminotransferase (AST)

The AST values revealed a significant (P<0.05) decreasing effect due to pudina leaf powder supplementation in laying hens (Table 3). The highest and lowest value of AST were recorded in T1 (175.45 mg/dl) and T2 (166.32 mg/dl) respectively. The lower values of AST might be probably due to the hepato-protective property of the pudina. Liver contains high levels of ALT and AST enzymes. ALT is found in cytoplasm and AST in mitochondria of hepatic cells (Adzet et al., 1987). Destruction of liver leads to release of these enzymes in blood stream and subsequently elevate the enzyme levels (Parmer et al., 2012). Thus increased levels of these enzymes signifies the liver damage condition (Hetrog and Hollmann, 1998). Owing to the presence of active ingredient such as alpha- tocopherol, caffeic acid and menthol, peppermint extract have strong protective role against arsenic induced liver toxicity in mice.

Study conducted by Patil and Mall (2012) also showed that Mentha arvensis has significant hepatoprotective activity. Contrary to the present findings, Alamgeer et al. (2013) reported that aqueous methanolic extract of Mentha longifolia unaffected the elevated level of AST in normotensive and hypertensive rats.

Serum Alanine Aminotransferase (ALT)

There was a significant reduction in ALT levels after inclusion of different levels of pudina leaf powder in the basal diet of laying hens (Table 3). The ALT concentration in group T1 (27.22 mg/dl) was significantly (P<0.05) higher than T2, T3 and T4 groups while the minimum value was noted in T4 (26.61 mg/dl). In the present study, it could be stated that values of ALT in the treatment groups were lower as compared to control group which might be probably due to the hepato-protective property of the Mentha arvensis. In accordance to the findings of present study Rajesh et al. (2013) reported that the elevated levels of serum biochemical markers ALT in induced hepatic damage rats were found decreased with addition of ethanol extract of Mentha arvensis leaves at 400 mg/kg. The present study is on the contrary of the findings suggested by Alamgeer et al. (2013) that the aqueous extract of Mentha longifolia L. unaffected the biochemical parameters related to hepatic function such as ALT compared with the control group.

Table 2: Effect of pudina leaf powder on lipid profiles (mean ± S.E.) in laying WLH

Values with different superscripts row wise differ significantly (P<0.05)

Table 3: Effect of pudina leaf powder on serum biochemical profile (mean ± S.E.) in laying WLH

Values with different superscripts row wise differ significantly (P<0.05)

Conclusion

The effect of administration of dietary levels of pudina leaf powder at different levels on certain biochemical parameters resulted beneficial effect on serum biochemical parameters in White Leghorn hens with less or no adverse effects on hepatic and renal functions test. The dietary level 10.0 g/kg was considered to result the overall best performance. It is possible to produce lean eggs to a certain extent by manipulating diets. However, further studies are needed to determine the effect of pudina leaf powder at higher levels in laying hens.

Acknowledgement

Authors are thankful to the Dean and Head of Department, LPM, College of Veterinary and Animal Sciences, G. B. Pant University of Agriculture and Technology, Pantnagar, India for providing necessary facilities to conduct the research experiment. Assistance provided by Departments of Animal Nutrition, Animal Genetics and Breeding, College of Veterinary and Animal Sciences, G. B. Pant University of Agricultural and Technology for this research work is also thankfully acknowledged.

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