Effects of supplementing hen diet with Lavandula angustifolia and/or Mentha spicata essential oils on production performance, egg quality and blood variables of laying hens

Abstract Background Organic products of animals are getting more accepted by consumers. Using herbal additives may lead to more health animal products. In this research it is hypothesized that Lavandula angustifolia and/or Mentha spicata essential oils would be helpful to enhance production performance in laying hens. Objectives This experiment was conducted to evaluate the effects of Lavandula angustifolia and Mentha spicata essential oils on performance, egg traits and blood variables in laying hens. Methods 144 Lohmann LSL‐Lite laying hens from 42 until 56 weeks of age were used in a completely randomized design in four treatments and six replicates (six birds per replicate). The treatments consisted of: (a) control group (basal diet), (b) basal diet supplemented with 250 mg/kg diet lavender essential oil (LEO), (c) basal diet supplemented with 250 mg/kg diet mint essential oil (MEO), and (d) basal diet supplemented with both LEO and MEO. Results Using LEO and/or MEO did not affect body weight changes, feed intake, egg weight, egg index, yolk index, Haugh unit, egg shell weight and egg shell thickness. Feeding LEO, individually or in combination with MEO, did not affect FCR compared with the control group (p < .05), however, feeding MEO individually increased feed conversation ratio (FCR) compared to LEO and the control group during 42–56 weeks (p < .05), as well as decreasing egg mass compared to LEO (p < .05). Feeding LEO increased egg production compared to MEO and combination of MEO and LEO (p < .05). Conclusions In conclusion, dietary supplemental MEO (250 mg/kg) may increase FCR, and LEO (250 mg/kg) is more effective than MEO (250 mg/kg) for egg production and egg mass purposes; besides MEO (250 mg/kg) negatively affected FCR compared with the control group. In addition, no specific beneficial effect of dietary supplemental MEO and/or LEO on the other measured variables was detected.


| INTRODUC TI ON
The use of most antibiotic growth promoters has been banned in many countries, especially in the European Union since 2006 (Butaye et al., 2000). Herbal additives are given to animals or birds to improve their physiological and productive performance. The antimicrobial effect of some medicinal plants is reported (Valero & Salmeron, 2003).
Medicinal plants, their extracts and essential oils have a wide range of activities, including inhibitory action on pathogens, effects on physio-pathologies and activity in different body systems, e.g. endocrine and immune system (Francois, 2006). In layers and broilers, herbs and spices are not just appetite and digestion stimulants, but they can influence other physiological functions, help to sustain good health and welfare and also improve their performance (Frankic, Voljc, Salobir, & Rezar, 2009). The ajwain essential oil (Trachyspermum ammi L.) supplementation had a positive effect on the FCR, intestinal morphology and microbiota counts of breeder quails (Hajiaghapour & Rezaeipour, 2018).
The exact mechanisms by which plant components may alter blood profile are not fully understood; for instance, it is reported that peppermint (Mentha piperita) may prevent lipid peroxidation; besides, phenolic compounds and antioxidant activities found in peppermint tea may have hypoglycemic effects. It is also reported that peppermint (Mentha piperita) may interact with chromium resulting in insulin-sensitive cell receptors or binding activity; in addition, some studies suggest that essential oils could increase insulin sensitivity-receptors. The above-mentioned mechanisms could lead to improve nutrient digestibility and modulate beneficial microbiota and also blood profile in laying hens' gut, which in turn may result in alter quantity and quality of their production. Nevertheless, exact mechanisms of action of phytogenic feed additives are still not fully understood.
The plants of this family are a rich source of polyphenols, flavonoids and carvone, thus possessing strong antioxidant properties (Bimakr et al., 2011;Gulluce et al., 2007). The broiler chicks fed diet supplemented with of 2% spearmint was lower significantly in the serum total cholesterol compared with the control group and other groups (Abu Isha, El-Hamid, Ziena, & Ahmed, 2018). It is stated that the improvement of FCR resulted from the increase in appetite due to the stimulation of salivary and gastric glands by spearmint oil, the decrease in pathogenic bacteria and better digestibility; besides, it is suggested that spearmint oil may stimulate salivary and gastric glands, and decrease bacteria which in turn improve digestibility and FCR (Abu Isha et al., 2018qouted from Amal, 2012. Abu Isha et al., (2018) reported that, the addition of spearmint essential oil, to the diet increased significantly the feed intake of broiler chicks (Abu Isha et al., 2018). In addition, it is reported that feeding spearmint increases feed intake and consequently improves growth in broiler chickens (Saleh, Ijiri, & Ohtsuka, 2014). Galib, Al-kassi, and Noor (2010) stated that the broiler chicks fed on peppermint (Mentha piperita) powder consumed significantly more feed consumption compared to the control group.
On this basis, we hypothesized that inclusion of Lavandula angustifolia and/or Mentha spicata into laying hens' diets would be helpful to enhance production performance, egg traits and blood biochemical variables; besides, assessing the probable synergistic interaction between dietary Lavandula angustifolia and Mentha spicata can be mentioned as novelty of the current study.

| Laboratory animals, experimental design and treatments
All experimental protocols were adhered and were approved by

| Productive performance and egg quality
The productive performance of the laying hens including hen-day egg production, feed intake and egg weight were recorded daily, and feed conversation ratio (FCR) and egg mass were calculated. To evaluate egg quality characteristics including egg quality traits yolk Haugh units were calculated from the records of albumen height and egg weight using the HU formula (Eisen, Bohren, & McKean, 1962). The shell thickness was a mean value of measurement at three locations on the egg (air cell, equator and sharp end) by using a dial and pipe gage. The yolk colour was scored with the aid of Roche Yolk Colour Fan. Yolk height and yolk diameter were measured by a tripod micrometer (Mitutoyo, 0.01 mm) and compass (Swordfish, 0.02 mm), respectively, then the yolk index was calculated. Egg length and width were individually recorded and used to calculate egg shape index.

| Blood biochemical variables
At the end of the experiment, eight hens were randomly selected from each treatment and blood samples were collected from the brachial vein into a 5-ml syringe. The collected blood samples were centrifuged at 805 g for 10 min, and the sera was frozen at −20°C until the analysis. Serum samples were thawed at room temperature and were analysed for glucose, triglyceride, albumin, uric acid and cholesterol according to the manufacture recommendations (Pars Azmun); besides, the enzyme activity of plasma glutathione peroxidase was measured according to the manufacture recommendations (Randox Laboratories kit).

| Statistical analysis
All data obtained from the trials were subjected to the analysis of variance procedure of statistical analysis system (SAS, 2001, Version 8.02) according to completely randomized design. Means were separated by Duncan's new multiple range test. The level of significance was determined at <.05. The following model was considered for is the main effect of MEO, RC ij is interaction between the effect of LEO and MEO, and (e ij ) is the residual error. The effects of the main factors were not considered, whenever the interaction was significant (Figures 1-3).

| Production performance
The productivity data of laying hens are summarized in

| Egg quality traits
Supplementation of the layer diet with LEO and/or MEO had no significant effects on egg weight, egg index, yolk index, HU, yolk colour, egg shell weight and egg shell thickness (p > .05) ( Table 4).

| Production performance
Several medicinal plants (e.g., garlic, onion, thyme, spearmint, pepper, yucca, black cumin (black seed), ginger) have been used in animal and poultry (layers and broilers) production due to their health benefits, e.g., spearmint leaves is used as carminative, antispasmodic and choleretic in human reported by Al-Kassie (2010). It is hypothesized that herb mixture (garlic, onion, thyme, spearmint, pepper, yucca, black cumin seed, and ginger) might have several vital benefits of such as antioxidant, antimicrobial and anti-inflammatory, which raise its use as herbal feed additives as alternative to antibiotics in broilers diets (Saleh, Ebeid, & Abudabos, 2018). The ajwain essential oil (Trachyspermum ammi L.) supplementation did not affect egg weight, egg mass, egg production, and feed intake in quail breeders (Hajiaghapour & Rezaeipour, 2018). Spearmint is used as an antispasmodic, choleretic and carminative (Al-Kassie, 2010). It is reported that, addition of spearmint essential oils, to the diet increased significantly the feed intake of broiler chicks (Abu Isha et al., 2018), Feeding spearmint increases feed intake and consequently improves growth in broiler chicks (Saleh et al., 2014).
Some aromatic herbal essences like lavender contain phytoestrogens. According to Saleh, Ahmed, and Ebeid (2019) inclusion of mixed sources of phytoestrogens (combination of 1 g/kg flaxseeds and 1 g/kg fenugreek) in diets improve laying performance, egg quality, the antioxidative status, hormonal profile and steroidogenesis in aged laying hens. Generally it is assumed that essential oils of herbs and spices might improve the palatability of feed due to their flavourful characteristics, hence could promote feed intake when added to layers and broilers diets (Hertrampf, 2001;Williams & Losa, 2001).
In this study, the addition of LEO and MEO essential oils did not influence feed intake and body weight changes during the trial period. In agreement, Nasiri-Moghaddam, Hassanabadi, and Bidar (2012) reported that there was no difference in feed intake values between broilers fed control and those fed lavender essence (350 mg/kg) at the period of 22-42 days age.
The feed conversion ratio (FCR) describes the hens' overall efficiency in converting ingested feed mass into egg mass over a specific period of time. An important claim often made for phytogenic feed additives is improvement of the FCR and thereby enhancing the intestinal availability of essential nutrients for absorption (Langhout, 2000;Williams & Losa, 2001 In an experiment curcumin (0.5 g), capsaicin (0.015 g), piperine (0.02 g), ginger (0.05 g), cumin (1.25 g), asafoetida (0.25 g), ajwan (0.2 g), fennel (0.5 g), coriander (2.0 g), mint (1.0 g), garlic (0.5 g) and onion (2.0 g) could shorten the time of feed passage through digestive tract in rats (Platel & Srinivasan, 2001). Herbs with growth promoting activity increase the stability of feed and beneficially influence the gastrointestinal ecosystem mostly through growth inhibition of pathogenic microorganisms' growth (Windisch, Schedle, Plitzner, & Kroismayr, 2008). Therefore, it might be possible that the increase in digestion and absorption of essential nutrients due to increasing the enzyme activity and/ or inhibition of pathogenic microorganism's growth could be the main reason of medicinal plants to accelerate the performance.  (Ocak et al., 2008). The controversy among these studies might be due to the differences in mint resources, which vary according to species, active ingredient, harvest time, etc (Brenes & Roura, 2010).
The phytogenic feed additives as performance enhancers for laying hens have primarily been supplemented to increase the utilization of the limit-fed diet and, in turn, improve egg production (Bozkurt et al., 2009). In the current study, supplementing MEO individually or combined with LEO significantly decreased egg production and also egg mass values during 42-46 weeks; in addition, egg weights in MEO and LEO groups were not influenced compared to the control group. Egg production was significantly higher in LEO group compared to MEO and LEO + MEO groups during 42-56 weeks. LEO feeding also significantly increased egg mass compared to MEO group during 42-56 weeks. According to some authors, diets with higher inclusion levels of essential oils compounds had toxic effects (Krishan, & Narang, 2014). In this case, decline in egg production in treatment 4 (LEO + MEO), may be due to the accumulation of the essential oils and its probable toxicity; however, more adequate toxicological study must be carried out to clarify this assumption.
The results of this experiment are in accordance with the results of Nasiri-Moghaddam, Hassanabadi & Bidar who did not observe any increases in egg weight, feed intake and egg production percentage by using lavender essence in the diets of layers (Nasiri-Moghaddam et al., 2012). In addition, Bozkurt et al. showed that supplemented diets with mixture of essential oil (i.e., oregano oil, laurel leaf oil, sage leaf oil, myrtle leaf oil, fennel seed oil and citrus peel oil) (24 and 48 mg/kg of diet) did not affect egg production and egg weight of broiler breeders (Bozkurt et al., 2009). In another study (Botsoglou et al., 2005), the effects of dietary aromatic plant extracts (rosemary at 5 g/kg diet, oregano at 5 g/kg diet and saffron at 20 mg/kg diet) on the laying performance of hens from 32 to 40 weeks of age were investigated and the results showed no significant differences in egg production and egg weight among the treatment groups.
These observations partially support the hypothesis that herbs and their associated essential oils may favourably affect hen performance but the number of experimental studies conducted under standardized field conditions with larger numbers of hens and for longer durations, preferably over a whole laying cycle, is still limited (Bozkurt et al., 2009). In the current research, the essential oils did not increase egg production and egg mass, but as numerical, the highest egg production percentage and egg mass were found in LEO group.
Most of findings showed that improving FCR was related to increase in the amount of egg mass; consequently, increase in egg mass might lead to improved FCR Sayedpiran, Nobakht, & Khodaei, 2011). In a study, the impact of feeding different amounts of eucalyptus (eucalyptus is similar to lavender due to containing cisteol) on productive performance, egg quality, blood variables and immune response in Japanese quails was investigated and it indicated enhanced daily weight gain, number of egg, egg weight, egg mass, improved FCR and quality of egg in the diets with eucalyptus compared to the control diet (Hassan et al., 2011). The results of our experiment are not in accordance with the results of Hassan, et al. that observed increase in egg weight, feed intake and production percentage by using eucalyptus powder in the diets of quails (Hassan et al., 2011). In another study, applying 3 g of eucalyptus per kilogram of diet improved the laying hens' productive performance and immunocompetence; moreover, FCR was improved in the hens given 3 g of eucalyp-

| Egg quality traits
Supplementation of the layer diet with Lavandula angustifolia and Mentha spicata essential oils had no significant effects on egg weight, egg index, yolk index, HU, yolk colour and egg quality

| Blood metabolites
No significant influence of experimental diets on albumin, uric acid, glucose, triglyceride and cholesterol was observed. There were some changes by addition of MEO, but the changes did not reach statistical significance. The results of this study revealed that LEO and MEO consumption could not change blood variables significantly. Most of spices and herbs enhance the synthesis and excretion of bile acids in the liver.
As bile acids had beneficially effects on lipids' digestion and absorption, it could improve the lipids' digestion and absorption, which led to increase in the level of blood triglyceride (Srinivasan, 2005). Khursheed et al. mentioned that a variety of essential oil compounds, such as menthone, menthol and geraniol have been shown to suppress the hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (Khursheed et al., 2017). However, in the present study, the effect of the essential oils on blood triglyceride was not significant.
Changes in blood biochemical variables in the present experiment are not supported by other researchers (Nobakht & Mehmannavaz, 2010;Sayedpiran et al., 2011), who reported increase in feed intake and decrease in blood triglyceride level by adding 2% of Lamiaceace menthapiperita. In a research, mixture of three medicinal plants (0.5% Malva silvestris, 1% Alhaji maurorum, 0.5% Mentha spicata) decreased blood glucose of broilers (Nobakht & Shahryar, 2010). In addition, using 0.5% of Mentha pulegium powder significantly improved the performance and reduced the blood glucose of broilers .
Akbari and Torki reported that peppermint extract has increased total protein and HDL and decreased blood serum concentrations of total cholesterol, triglycerides, LDL and glucose of broilers (Akbari & Torki, 2014). Fallah, Kiani, & Azarfar also reported that peppermint extract (200 meg/kg) has increased albumin, HDL and significantly reduced total cholesterol, triglycerides, LDL and glucose in broilers (Fallah, Kiani, & Azarfar, 2013). It seems as some components of peppermint including menthol and menthone have a potential to decrease blood lipids in broilers (Escop (European Scientific Cooperative on Phytotherapy), 2003). Mansoub also reported that peppermint extract (0.75%, 1%, 1.5%, and 2%) increases albumin, total protein and HDL and also significantly decreases glucose, triglycerides, total cholesterol and LDL in serum of broilers; he also suggested that the main reason for the reduction of serum lipids in broilers is due to the active ingredients of peppermint leaves such as tocopherol and menthol (Mansoub, 2011). Our observation in this experiment on blood biochemical variables is not in agreement with Hardari, Nobakht, and Safamehr (2010) report, who stated that using 1.5% of M. pulegium L.
had positive effects on blood cholesterol level.
Definitely, several variables such as supplementation methods (in diet or drinking water), the supplemental level, its level in the basal diet, bioavailability, stress condition, degree of stress, as well as duration of usage could be, at least in part, probable reasons for contradictory results in miscellaneous experiments.

| CON CLUS ION
Based on the results of the present study, it can be concluded that dietary supplemental MEO (250 mg/kg) may increase FCR, and LEO (250 mg/kg) is more effective than LEO (250 mg/kg) for egg production and egg mass purposes; besides MEO (250 mg/kg) negatively affected FCR compared with the control group.

CO N FLI C T O F I NTE R E S T
The authors of this manuscript have no conflicts of interest to declare. Validation; Writing-original draft.

E TH I C A L S TATEM ENT
All the experimental proceedings in this experiment were approved by the Animal Ethics Committee of Razi University, Kermanshah, Iran.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.343.