Effect of dietary supplementation of peppermint on performance, egg quality, and serum metabolic profile of Hy-Line Brown hens during the late laying period

Abstract

This study was conducted to determine the effects of supplementation of dried peppermint (Mentha piperita L.) leaves in laying hen diets on laying performance, egg quality, and serum metabolic profile. A total of one hundred and fifty Hy-Line Brown laying hens (64-week-old), were assigned to five treatment diets including dry peppermint leaves at 0,5, 10, 15, or 20 g/kg, respectively, for 12 weeks. Each treatment had six replicates with five hens each. Over the course of the trial, incremental dietary peppermint leaves significantly increased (linear, P < 0.001) egg weight, egg production, egg mass and feed intake from 64-68, 68-72, 72-76 and 64-76 weeks of age. Moreover, feed conversion ratio was linearly decreased (P < 0.001) with increasing levels of peppermint in laying hens diet. The inclusion of 20 g/kg peppermint resulted in overall best performance. Eggshell percentage, eggshell thickness and Haugh unit of hens fed diets supplemented with peppermint leaves were greater (P < 0.01) than that of hens fed the control diet. However, peppermint supplementation did not influence other egg quality characteristics like albumen and yolk percentages and albumen height. Notably, serum cholesterol significantly decreased (P < 0.001) but serum total proteins increased (P = 0.015) with the increasing peppermint leaves levels. It can be concluded that peppermint leaves can be used as an effective feed additive to improve performance of laying hens during the late laying period.

Full text

Accepted Manuscript
Title: Effect of dietary supplementation of peppermint on
performance, egg quality, and serum metabolic profile of
Hy-Line Brown hens during the late laying period
Author: A.A.A. Abdel-Wareth J.D. Lohakare
PII: S0377-8401(14)00231-4
DOI: http://dx.doi.org/doi:10.1016/j.anifeedsci.2014.07.007
Reference: ANIFEE 13120
To appear in: Animal Feed Science and Technology
Received date: 20-2-2014
Revised date: 8-7-2014
Accepted date: 20-7-2014
Please cite this article as: Abdel-Wareth, A.A.A., Lohakare, J.D.,Effect of dietary
supplementation of peppermint on performance, egg quality, and serum metabolic
profile of Hy-Line Brown hens during the late laying period, Animal Feed Science
and Technology (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.07.007
This is a PDF file of an unedited manuscript that has been accepted for publication.
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Effect of dietary supplementation of peppermint on performance, egg quality, and
1
serum metabolic profile of Hy-Line Brown hens during the late laying period
2
A.A.A. Abdel-Waretha and J.D. Lohakareb,*
a Department of Animal and Poultry Production, Faculty of Agriculture, South Valley
3
University, 83523 Qena, Egypt
4
b College of Animal Life Sciences, Kangwon National University, Chunchon-200-701,
5
Republic of Korea
6
*Corresponding author at: College of Animal Life Sciences, Kangwon National 7
University, Chuncheon, South Korea. Tel:+82-33-250-8633, Fax:+82-33-244-8906. 8 E-mail addresses: jayant@kangwon.ac.kr (J.D. Lohakare), a_bkr1@yahoo.com (A.A.A.
Abdel-Wareth).

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Abstract
9
This study was conducted to determine the effects of supplementation of dried 10
peppermint (Mentha piperita L.) leaves in laying hen diets on laying performance, egg 11
quality, and serum metabolic profile. A total of one hundred and fifty Hy-Line Brown 12
laying hens (64-week-old), were assigned to five treatment diets including dry 13
peppermint leaves at 0,5, 10, 15, or 20 g/kg, respectively, for 12 weeks. Each treatment 14
had six replicates with five hens each. Over the course of the trial, incremental dietary 15
peppermint leaves significantly increased (linear, P<0.001) egg weight, egg production, 16
egg mass and feed intake from 64-68, 68-72, 72-76 and 64-76 weeks of age. Moreover, 17
feed conversion ratio was linearly decreased (P<0.001) with increasing levels of 18
peppermint in laying hens diet. The inclusion of 20 g/kg peppermint resulted in overall 19
best performance. Eggshell percentage, eggshell thickness and Haugh unit of hens fed 20
diets supplemented with peppermint leaves were greater (P<0.01) than that of hens fed 21
the control diet. However, peppermint supplementation did not influence other egg 22
quality characteristics like albumen and yolk percentages and albumen height. Notably, 23
serum cholesterol significantly decreased (P<0.001) but serum total proteins increased 24
(P=0.015) with the increasing peppermint leaves levels. It can be concluded that 25
peppermint leaves can be used as an effective feed additive to improve performance of 26
laying hens during the late laying period.
27
Keywords: egg quality, laying hens, peppermint, production, serum profile 28
Abbreviations: ADFom, acid detergent fiber; FCR, feed to egg mass ratio; HU, Haugh 29
unit; H, albumen height; aNDFom, neutral detergent fibre assayed with a heat stable 30
amylase; W, weight 31

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1. Introduction 32
Phytogenic substances have several advantages over commonly used antibiotics 33
since they are generally recognized as safe and are commonly used items in the food 34
industry (Varel, 2002; Brenes and Roura, 2010).The plant family Lamiaceae has received 35
the greatest interest in poultry feed, with peppermint, thyme and oregano as the most 36
popular representatives (Burt, 2004). These medicinal plants active components which 37
are often called phytobiotics or botanicals are secondary metabolites in medicinal plants 38
with positives effects on animal health and productivity (Windisch et al., 2008; Ghazaghi 39
et al., 2014). 40
Peppermint (Mentha piperita L.) is widely used in herbal medicine and believed to 41
be particularly beneficial in building the immune system and antimicrobial properties, as 42
well as strong antioxidant properties and enhance appetite, mainly due to its active 43
components (Dorman et al., 2003: Yalçin et al., 2012).The peppermint plant is an 44
aromatic perennial herb cultivated in Egypt and in most part of the world, has 45
traditionally been used in medicine. Peppermint leaves contains about 0.5-4% essential 46
oils that is composed of 25-78% Menthol, 14-36% Menthone, 1.5-10% Isomenthone, 2.8-47
10% Menthyl acetate, 3.5-14% Cineol (Grigoleit and Grigoleit, 2005; Bupesh et al., 48
2007; Aziz et al., 2011). Most of the farmers in Egypt region prefer to keep their hens to 49
late production period for higher egg weight and economic aspects rather than 50
substituting them with new birds. However, egg production and egg quality (e.g shell and 51
albumin characteristics) decreased as age advances (Nobakht et al., 2006). 52
There are few research reports available on its practical usage as feed additives in 53
poultry nutrition especially on broilers and the results showed that peppermint leaves had 54

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a growth promoting efficacy at an early stage of broilers life (Ocak et al., 55
2008;Toghyaniet al., 2010) but there is a scarcity of reports on its usage in layer diets. 56
For this reason the present study was conducted to evaluate the potential of different 57
levels of peppermint leaves as feed additives in laying hens during late period in order to 58
observe their influence on feed intake, performance, egg quality and serum metabolic 59
profile.
60
61
2. Materials and methods
62
2.1. Experimental birds, design and feed preparation
63
The present study was conducted in the breeding farm of agricultural research centre 64
of the Agriculture faculty, South Valley University, Qena, Egypt. A total of one hundred 65
and fifty Hy-Line Brown laying hens (average weight =1.720 g, 64 weeks age)were 66
assigned to five treatment diets including dry peppermint leaves at 0,5, 10, 15, or 20 g/kg, 67
respectively, for 12 weeks. Each treatment had six replicate cages with five hens each. 68
Replicates were equally distributed into upper and lower cage levels to minimize the cage 69
level effect. Five hens were housed in a 60×60×40 cm3 cage. 70
All hens were housed in an environmentally controlled house with temperature 71
maintained at approximately 24°C. The house had controlled ventilation and lighting 72
(16L:8D). All hens were supplied with feed and water for ad libitum consumption. 73
Animal housing and handling procedures during experimentation were in accordance 74
with guidelines of the Institutional Animal Care and Ethics. 75
The hens were fed diets in mash form during the experiment (64-76 weeks of age). 76
The basal diet was formulated (Table 1) according to recommendations of NRC (1994). 77

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The basal diet contained 0, 5, 10, 15 or 20 g/kg of dried peppermint leaves, respectively, 78
for five treatment diets. First, the peppermint leaves of different levels were mixed 79
separately in 3 kg of the basal diet each and then they were added to the required feed 80
amount for the prescribed experimental period. The chemical analysis of peppermint 81
leaves used in this study is shown in Table 2. 82
2.2. Performance parameters
83
Hen-day egg production, feed consumption, egg weight, and hen mortality were 84
recorded daily. Feed conversion ratio was calculated as grams of feed intake per gram of 85
egg mass produced. Egg mass was calculated by multiplying egg weight by egg 86
production rate. All production variables were determined for each replicate. 87
2.3. Egg quality parameters
88
The parameters relative to egg quality were evaluated at 76 weeks of age. Twelve 89
eggs were randomly collected per treatment (2 egg per replicate) to determine these 90
parameters. The collected eggs were weighed. The eggs were broken out individually 91
onto a glass plate surface and allowed to sit for 5 min. The heights of yolk and albumen, 92
and the diameter of yolk were measured using the callipers. Yolks were separated from 93
albumen manually, and both were weighed. 94
The weight of shell, albumen and yolk were divided by whole egg weight and then 95
multiplied by 100 to determine percentage weight. Eggshell thickness (without inner and 96
outer shell membranes) was measured at the middle part of the eggshell using (QCT) 97
shell thickness micrometer (Technical Services and Supplies Ltd, England). Haugh units 98

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(HU) were calculated from the records of albumen height (H) and egg weight (W) using 99
the following formula: HU = 100 log10 (H−1.7 W0.37 + 7.56), according to Haugh (1937). 100
2.4. Blood sampling and laboratory analyses
101
At the end of experimental period, 12 birds from each treatment were randomly 102
selected (2 birds per replicate). The blood was drawn from wing vein using sterilized 103
needles and syringes in vacutainer tubes for serum collection. Feed was not withdrawn 104
from the feeder before blood was collected. After the serum was separated naturally, it 105
was centrifuged for 10 min (3,000 × g) at room temperature. Serum was collected in 106
tubes and stored at -20oC until further analysis. Serum glucose, total cholesterol, total 107
proteins, calcium, and phosphorus were measured spectrophotometrically by using 108
commercial kits (Spectrum chemical company, Obour City - Cairo, Egypt). 109
2.5. Chemical analysis
110
The diet and peppermint leaves were analyzed for moisture by oven drying (930.15), 111
ash by incineration (942.05), protein by Kjeldahl (984.13), and ether extract by Soxhlet 112
fat analysis (920.39), as described by the AOAC International (2000). Calcium was 113
measured using atomic absorption spectrometry and phosphorus was analysed 114
colorimetrically (method 10.6.1; VDLUFA, 2007). Starch and sugar contents of the diets 115
were quantified using official European Union methods (Anonymous, 2009). The 116
contents of neutral detergent fibre (assayed with a heat stable amylase, aNDFom) and 117
acid detergent fibre (ADFom), both expressed exclusive residual ash, were determined 118
sequentially without sodium sulphite (method 6.5.1 for aNDFom and 6.5.2 for ADFom; 119
VDLUFA, 2007). Lysine and methionine (after oxidation) were analysed using an amino 120

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acid analyser after hydrolysis (6 M HCl) of the diets (method 4.11.1; VDLUFA, 2007). 121
The gross energy content of the diets was measured using an adiabatic bomb calorimeter 122
(model C 200; IKA, Heitersheim, Germany). 123
2.6. Statistical analysis
124
The statistical analysis was performed using a completely randomized design and the 125
general linear model (GLM) procedure of SAS 9.2 (SAS Institute, 2009). The model only 126
included the level of supplementation. Pens were the experimental units for all analysis. 127
Orthogonal polynomial contrasts were used to determine the linear and quadratic effects 128
of the increasing levels of inclusion and Duncan multiple range test was used to compare 129
means. Significance was declared at P<0.05; P-values less than 0.001 are expressed as 130
“<0.001” rather than the actual value. 131
3. Results 132
3.1. Egg production 133
Supplementation with peppermint leaves showed effect on hen-day egg production, 134
egg weight, egg mass and feed to egg mass ratio (FCR; Table 3). There was no mortality 135
and the general health status of laying hens was good throughout the experimental period. 136
The higher inclusion levels of peppermint leaves significantly increased the egg weight, 137
egg production, egg mass and feed intake as well as improved FCR (P<0.001) from 64-138
68, 68-72, and 72-76 weeks of age. For overall experiment period (64-76 weeks of age), 139
the peppermint leaves supplementation resulted in a linear increase (P<0.001) in egg 140
weight and egg mass with the increasing levels in the diets. Likewise, dietary peppermint 141

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leaves (5, 10, 15 or 20 g/kg) increased hen-day egg production linearly (P<0.001) by 142
6.63%, 6.68%, 8.99% and 9.89% than control, respectively (Table 3). Moreover, 143
peppermint leaves significantly (linear, P<0.001) increased feed intake during the 144
experimental period. The feed conversion ratio decreased linearly (P<0.001) with the 145
increasing peppermint levels. 146
3.2. Egg quality
147
In relation to egg quality (Table 4), dietary addition of peppermint leaves (5, 10, 15 148
or 20 g/kg) to laying hens for 12 weeks resulted in a linear increase (P=0.007) in egg 149
shell percentage, and increased both linearly and quadratically (P<0.001) eggshell 150
thickness in a dose-dependent manner. Furthermore, with the increasing levels of dietary 151
peppermint leaves (5, 10, 15 or 20 g/kg), Haugh unit linearly increased by 3.02%, 4.89%, 152
5.04% and 6.56% than control, respectively (Table 4). However, dietary peppermint 153
leaves had no effects on albumen and yolk percentages as well as albumen height 154
(P=0.057) in the late laying period of hens. 155
3.3. Serum biochemical parameters
156
The serum biochemical analyses revealed that serum cholesterol linearly decreased 157
(P<0.001) with increasing peppermint leaves levels in the diets (Table 5). However, 158
serum total protein of laying hens was increased (P=0.015) with increasing levels of 159
peppermint leaves. On the other hand, there were no significant effects of peppermint 160
leaves on serum calcium, phosphorus and glucose (Table 5). 161
4. Discussion
162

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Due to scarcity of available reports on effect of peppermint leaves on laying hens, 163
comparison was done with other studies that used herbs of the similar family Lamiaceae. 164
The beneficial effects of dietary inclusion of herbs on gut health, digestion of nutrients 165
and intestinal integrity have been reported earlier (Cross et al., 2007; Brenes and Roura, 166
2010). These beneficial effects might be directly associated with improvements in laying 167
performance. In the present study, the general health status of laying hens was good 168
during the experimental period (64-68, 68-72, 72-76 and 64-76 weeks of age) which 169
could be related to the house environmental system. Generally, the birds given diets 170
containing peppermint leaves had the greatest overall egg production, egg mass and egg 171
weight compared with the birds given control diet proving that peppermint had beneficial 172
action in the oviposition process, imperative on the conversion of digested feed into eggs. 173
The peppermint leaves significantly improved feed conversion ratio and this could be 174
related to increased efficiency of feed utilization. Moreover, the increase in productive 175
performance of hens due to this medicinal plant could be attributed to its content of 176
essential oils. These active components (cineole, citral, geraniol, linalool and menthol) 177
have shown to possess antimicrobial, antifungal and antioxidant activities as well as 178
improved digestion and absorption of dietary nutrients (Pattnaik et al., 1997; Grigoleit 179
and Grigoleit, 2005; Bupesh et al., 2007: Radwan et al., 2008) that might have improved 180
the performance and production efficiency of hens in this study. 181
Moreover, peppermint leaves has the ability to improve feed intake which was 182
increased with the increasing levels of supplementation. Increased feed intake was earlier 183
reported (Al-Ankari et al., 2004) in broilers fed habek mint (Mentha longifolia) at 200 184
g/kg, hence palatability was not a concern as levels in our study was quite low. In the 185

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present study, addition of peppermint improved egg weight, egg production, egg mass 186
and FCR of laying hens. 187
The statistical analysis of the present study showed that the dietary peppermint 188
leaves at 0.5% up to 2.0% enhanced the bird production status during the late laying 189
period. However, most of the previous studies have been investigated in early stages. 190
Such as the results of Ocak et al. (2008) and Toghyani et al. (2010) demonstrated that 191
birds given diets containing peppermint leaves are efficacious in supporting the growth 192
performance of broiler chicks at early stages of life. It was reported earlier that using 2% 193
blends of ziziphora, menta, peppermint and nettle improved egg mass and feed 194
conversion in laying hens (Sayedpiran et al., 2011). 195
In this study, egg weight, egg mass and egg production significantly increased with 196
increasing dietary peppermint leaves at all stages. In agreement with Abdel-Wareth et al. 197
(2013) where addition of thyme or oregano leaves at 10, 20, 30 g/kg in hen diets had 198
significantly increased (P<0.01) egg weight, egg mass and hen-day-egg production 199
during the periods of 68 to 72 weeks of age, are consistent with our results. Dietary 200
supplementation with thyme leaves at the levels of 0, 0.1, 0.5 and 1% to basal diet 201
improved the egg production rate by 6.12%, 5.16% and -2.06% compared to control, 202
respectively (Bölükbaşi and Erhan, 2007). Also, addition of 0.5% thyme, oregano or 203
rosemary leaves to laying hens diet numerically increased egg weight, egg mass, egg 204
production and feed intake (Radwan et al., 2008). In the present study, it could be stated 205
that dietary peppermint leaves have positive response on production performance of 206
laying hens attributed to the essential oils present in it. 207

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This study showed no significant differences in yolk and albumen percentages as 208
well as albumen height among treatments. However, there were significant effects on 209
eggshell percentage, eggshell thickness and Haugh unit of laying hens at 76 weeks of age. 210
Increased shell weight and shell thickness by addition of peppermint leaves may be 211
attributed to its main active components that possess antibacterial and antioxidant 212
activities (Dorman et al., 2003: Yalçin et al., 2012). It could be postulated that 213
peppermint may provide a healthy environment in uterus (e.g. site of calcium deposition) 214
and consequently increase shell weight and shell thickness more than the control 215
treatment. These improvements in eggshell and Haugh unit are important for the egg-216
food industry, because peppermint may enhance the safety and stability of eggs. The 217
Haugh unit score is known as an indicator of egg freshness and is related to shelf life 218
(Williams, 1992; Özek et al., 2011). Our findings are in agreement with earlier study that 219
showed supplementation of thyme improved shell weight and shell thickness compared to 220
hens fed control diet (Ali et al., 2007). It is also reported that 2% blends of ziziphora, 221
menta, peppermint and nettle improved egg Haugh unit in laying hens (Sayedpiran et al., 222
2011). Dietary supplement with 36 mg/kg essential oils had increased Haugh unit score in 223
laying hens egg at 68 weeks of age (Özeket al., 2011). In contrary, Haugh unit results of 224
our study did not agree with the results of Botsoglou et al. (2005) and Florou-Paneri et al. 225
(2005) that reported Haugh unit score was not affected by oregano oil supplementation in 226
the diet. However, more research is needed to further explore the influence of peppermint 227
leaves and its essential oils on egg quality parameters, particularly in regard to their mode 228
of action on egg composition in laying hens of different ages. Addition of thyme or 229
oregano leaves at 10, 20, 30 g/kg in hen diets had no significant effect on egg quality in 230

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feeding trials during the periods of 68 to 72 weeks of age is also reported recently 231
(Abdel-Wareth et al., 2013). 232
To our knowledge, no publication exists on peppermint leaves and its effects on late 233
laying periods in hens. This study showed no significant differences in serum glucose, 234
calcium and phosphorus in laying hens among the treatments and the values remained 235
within the normal range (Azzam et al., 2011; Weber et al., 2013). However, it is of 236
interest to note that peppermint leaves decreased the serum cholesterol levels and 237
increased total proteins in laying hens during the late laying period, but the values are 238
within the normal physiological range (Azzam et al., 2011;Özek et al., 2011; Bozkurt et 239
al., 2012; Weber et al., 2013). These results agree with those of Abdel-Wareth et al. 240
(2013) that reported addition of thyme or oregano leaves at 10, 20, 30 g/kg in hen diets 241
decreased(P<0.01) serum cholesterol and increased total proteins, but serum calcium, 242
phosphorus and glucose were not influenced at 68 to 72 weeks of age similar to our 243
results. Moreover, total lipids and cholesterol concentrations tended to decrease in the 244
blood of growing Japanese quails fed medicinal plant-diets (containing thyme flowers or 245
Chamomile flower heads) as compared to the control group is reported earlier (Genedy 246
and Zeweil, 2003). Likewise, cholesterol concentrations of birds fed 1.0 g thyme 247
flowers/kg diet had significantly lower values than the control group (Zeweil et al., 248
2006). Many other reports indicated that essential oils decreased cholesterol 249
concentration and increased total proteins in serum (Lee et al., 2003; Bölükbaşi et al., 250
2008; 2009; 2010), similar to our results. However, in contrary, addition of 1.0% (thyme 251
or rosemary) or 0.5% Curcuma longa did not decrease total cholesterol and LDL- 252
cholesterol in laying hen blood significantly (Radwan et al., 2008). Similarly, other 253

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studies indicated that a diet supplemented with herbal plant (chicory powder; Mansoub, 254
2011) and 36 mg/kg essential oils (Özek et al., 2011) had no effects on serum glucose of 255
laying hens in line with our results. 256
There exists conflicting reports concerning the influence of supplemental herbs or 257
essential oils on blood biochemical profile. It was reported that using 2% blends of 258
ziziphora, menta, peppermint and nettle did not affect blood biochemicals in laying hens 259
(Sayedpiran et al., 2011), and diet supplemented with 36 mg/kg essential oils had no 260
effect on serum total cholesterol of laying hens (Özek et al., 2011). And serum calcium 261
concentration was significantly decreased by dietary supplementation of bergamot oil 262
(1.5 ml/kg) to laying hens during the late laying period (75 week old) (Bölükbasi et al., 263
2010). For clear conclusion, further systematic studies might be considered to elucidate 264
the effect of peppermint leaves at different stages of production in laying hens. 265
Conclusion
266
The effects of different dietary levels of peppermint leaves on productive 267
performance, egg quality and serum biochemistry showed that 12 weeks administration 268
of these leaves have beneficial effects on egg production, egg weight, egg mass, feed 269
intake, FCR, HU, eggshell and serum cholesterol during the late laying period in Hy-Line 270
brown hens. More detailed studies are needed in future to determine the optimal dietary 271
inclusion level and its economic impact. 272
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396

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396 Table 1
397
Ingredient composition and chemical analysis (g/kg) of the basal diet 398
Ingredients g/kg
Corn 679
Soybean meal 178
Soybean oil 8
Corn gluten meal 25
Di-calcium phosphate 19
Limestone 82
Vitamin and mineral mixa 2
DL-Methionine 2.5
Sodium chloride
Choline
4
0.5
Determined analysis, g/kg
Dry matter 889
Ash 114
Crude protein 182
Ether extract 42.4
ADFom 41.2
aNDFom 102
Calcium 37.1
Phosphorus 5.43
Starch 376
Sugar 42.1
Lysine 0.96
Methionine 0.39
Gross energy (MJ/kg) 16.0
aProvided the following per kg of diet: vitamin A, 12,000 IU; vitamin D3, 7200 IU; 399
vitamin E, 20 IU; vitamin B1, 2.5 mg; vitamin B2, 5 mg; vitamin K, 3 mg; vitamin B12, 400

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1.5 µg; pyridoxine, 0.225 µg; pantothenic acid, 10 mg; niacin, 35 mg; folic acid, 1.5 mg; 401
biotin 125 mg; Mn, 90 mg; Cu, 7.5 mg; Zn, 65 mg; Fe, 50 mg; Se, 0.1 mg. 402

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Table 2
403
Chemical composition and nutritive value of peppermint leaves on drymatter basis 404
Chemical compositiona
Dry matter (g/kg) 942
Ash, % 14.9
Gross energy (MJ/Kg) 12.8
Crude protein, % 16.2
Neutral detergent fiber, %
Calcium,%
Phosphorus,%
28.2
1.97
0.31
aAll analyses were carried out in triplicate405

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406
Table 3
407
Effect of peppermint leaves on performance of laying hens 408
Peppermint leaves (g/kg) P-value
Items 0 5 10 15 20 SEM1 Lin2 Quad3
64-68 weeks of age
Egg weight, (g/hen/day) 63.76e 64.61d 65.51c 66.28b 66.94a 0.152 0.001 0.410
Hen-day production, (%) 74.75d 77.82c 78.09 c 79.67b 80.74a 0.228 0.001 0.004
Egg mass, (g/hen/day) 47.67e 50.28d 51.15c 52.79 b 54.05a 0.203 0.001 0.016
Feed intake, (g/hen/day) 109.0d 111.1c 113.3b 113.3 b 115.9a 0.430 0.001 0.053
Feed conversion ratio 2.28a 2.21b 2.21b 2.14 c 2.12 c 0.013 0.001 0.441
68-72 weeks of age
Egg weight, (g/hen/day) 64.25d 65.05c 66.46b 66.77b 67.44a 0.176 0.001 0.053
Hen-day production, (%) 75.42e 79.81d 80.93c 82.30b 83.28a 0.262 0.001 0.001
Egg mass, (g/hen/day) 48.47 e 51.92d 53.79 c 54.95 b 56.17a 0.267 0.001 0.001
Feed intake, (g/hen/day) 109.3d 112.5c 114.0b 114.3ab 115.4a 0.378 0.001 0.001
Feed conversion ratio 2.26 a 2.17 b 2.12 c 2.08d 2.06 d 0.012 0.001 0.006
72-76 weeks of age
Egg weight, (g/hen/day) 65.32d 66.99c 66.99b 67.92a 68.65a 0.145 0.001 0.001
Hen-day production, (%) 76.23 c 81.29 b 81.56 b 83.56 a 84.21a 0.443 0.001 0.001
Egg mass, (g/hen/day) 49.79d 54.45c 55.39 b 57.55a 57.80a 0.300 0.001 0.001
Feed intake, (g/hen/day) 111.2d 114.5c 115.9ab 116.7ab 117.2a 0.378 0.001 0.001
Feed conversion ratio 2.23 a 2.10 b 2.09 b 2.03 c 2.02 c 0.013 0.001 0.001
64-72 weeks of age
Egg weight, (g/hen/day) 64.27e 65.21d 66.21c 67.08b 67.74a 0.301 0.001 0.562
Hen-day production, (%) 75.36c 80.01b 80.48b 82.14a 82.81a 0.871 0.001 0.065
Egg mass, (g/hen/day) 48.43d 52.16c 53.28b 55.10a 56.09a 0.585 0.001 0.054
Feed intake, (g/hen/day) 109.7c 112.7b 114.7a 115.0a 116.0a 0.894 0.001 0.121
Feed conversion ratio 2.27a 2.16b 2.15b 2.09c 2.07c 0.022 0.001 0.193
a-d Means not sharing a common superscript in a row are significantly different (P<0.05) 409

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1Standard error of the means 410 2,3Linear and quadratic responses, respectively, to dietary inclusion levels411

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412 Table 4
413
Effect of peppermint leaves on egg quality parameters of laying hens 414
Peppermint leaves (g/kg) P-value
Items
0 5 10 15 20
SEM1
Lin2 Quad3
Albumen, % 60.98 60.60 60.96 61.47 61.57 0.597 0.289 0.619
Yolk, % 30.75 30.70 30.09 29.49 29.64 0.671 0.116 0.876
Shell, % 8.26b 8.69ab 8.94ab 9.04a 9.12a 0.226 0.007 0.322
Shell thickness, mm 0.323c 0.334b 0.335b 0.336ab 0.338a 0.081 0.001 0.001
Haugh unit, score 85.54b 88.12ab 89.72ab 89.85ab 91.15a 1.503 0.010 0.479
Albumen height, mm
7.97 8.10 8.38 8.47 8.67 0.281 0.057 0.952
a-d Means not sharing a common superscript in a row are significantly different (P<0.05) 415
1Standard error of the means 416
2,3Linear and quadratic responses, respectively, to dietary inclusion levels417

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418
Table 5
419
Effect of peppermint leaves on serum metabolites of laying hens 420
Peppermint leaves (g/kg) P-value
Items
0 5 10 15 20
SEM1
Lin2 Quad3
Cholesterol (mmol/L) 4.48a 4.43ab 4.30bc 4.18cd 4.16d 0.407 0.001 0.738
Total protein (g/L) 62.00b 62.33ab 62.67ab 63.67a64.00a0.577 0.015 0.764
Glucose (mmol/L) 12.82 12.87 12.95 13.02 12.95 0.096 0.210 0.487
Calcium (mmol/L) 3.28 3.28 3.29 3.29 3.29 0.004 0.510 0.839
Phosphorus (mmol/L) 2.31 2.32 2.32 2.37 2.35 0.026 0.142 0.984
a-d Means not sharing a common superscript in a row are significantly different (P<0.05) 421
1Standard error of the means
422
2,3Linear and quadratic responses, respectively, to dietary inclusion levels 423
424

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Statement of Conflict of Interest 424
The authors hereby declare that no competing and conflict of interests exist. 425
426

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Highlights 426
1. Peppermints leaves can be used as a feed additive in layers diet at late laying 427
stages. 428
2. Peppermint leaves at 5-20 g/kg improved egg production performance and egg 429
quality. 430
3. It reduces serum cholesterol and increased total proteins in serum. 431
432