Effect of rosemary (Rosmarinus officinalis) leaf meal supplementation on production performance and egg quality of laying hens

Abstract

This experiment was conducted to investigate the supplemental effect of rosemary leaf meal (RLM) on egg production and egg quality in Lohmann-Brown laying hens. For each treatment, six replicates were performed with four 24-week-old laying hens for a total of 96 chickens. Dried rosemary leaf meal (RLM) was present in the commercial laying hen ration at 0, 1.7, 3.5 and 5.2%, respectively. The interactions between dietary treatments, egg storage temperatures (4 °C, 21.5 °C), and egg storage durations (7, 14, 21, 28, and 35 days) were examined in factorial designs using a fully randomized design. The data were analyzed using the Statistical Analysis System (SAS, 2014, Ver. 9.3) and Tukey's multiple range tests to separate the means. The inclusion of dried RLM at a level of 5.2% (p < 0.05) significantly improved the hens' daily egg production (78.9%). Feed conversion ratio (2.25) and egg weight (56.7 g) compared to control treatment. Similarly, at 3.5% and 5.2% RLM supplementation, eggshell weight (5.876 g) and eggshell strength increased significantly (p < 0.05) (compared to control, a weight of 3.76 kg/cm2 and a shell thickness (0.37 mm) was observed. In addition, the groups fed 5.2% RLM had significantly higher albumin weight (34.9 g/L), albumin height (6.9 mm), and yolk weight (15.6 g/L). l), yolk height (15.9 mm), yolk color (5.3 points), and Hough unit (83.9 points) than the control groups. Regarding the external characteristics of stored eggs, a group of hens fed at rates of 3.5 and 5.2% RLM showed significantly greater (p < 0.05) egg weight with increases in storage temperature and duration as a control group. There was a significant interaction effect between stored eggs collected from the supplemented groups in terms of egg weight, weight loss, shell weight, shell strength, and shell thickness stored at specific temperatures and storage durations. There was a decrease in albumen level and Haugh unit (HU) with an increase in storage temperature and duration over treatment (P < 0.05). Therefore, better external quality was observed in eggs collected from a group of chickens fed 4.5 and 5.2% RLM after 35 days of storage compared to a control group. Mean albumen height, albumen weight, HU, yolk height, and yolk color of eggs stored in the refrigerator (4 °C) were an exception for yolk weight, which was higher compared to room temperature (21.5 °C). Significantly low (P < 0.05) albumen height (6.61 to 2.96 cm), Hough unit (82.49 to 47.64 points), and yolk height (14.66 to 12.35 mm) were observed at 35 days storage recorded in the control group. In conclusion, supplementation with RLM at 3.5% and 5.2% improved the performance and quality of both fresh and stored eggs.

1. Introduction

Globally, poultry makes a significant contribution to food security and nutrition, providing energy, protein and essential micronutrients for humans, with short production cycles and the ability to convert a wide range of agri-food by-products and waste into meat Human-edible eggs [1]. In developing countries, rural poultry contributes significantly to poverty alleviation and improved food security [2]. In Ethiopia, lack of availability of sufficient feed, feed quality, feed costs, and lack of vitamin and mineral premixes in commercial poultry feeds in rural smallholder and large-scale poultry production systems are the most critical constraints affecting chicken productivity [3,4]. Finding unconventional, readily available, inexpensive and nutritious feed sources as alternative chicken feed ingredients is crucial. This situation justifies the evaluation of locally available complementary feed ingredients as feed additives for inclusion in a modern small-scale poultry production system. Therefore, it has become imperative to look for unconventional, available, cheaper and nutritious feeds as alternative chicken feed ingredients and supplements.

Rosemary (Rosmarinus officinalis L.) is a natural aromatic plant with high antioxidant activity that improves the performance of birds. Rosemary polyphenols (carnosol, carnosic acid, methylcarnosic acid, rosmarinic acid, ursolic acid, and many others) are the most physiologically active chemicals and have antibacterial, antioxidant, and antiphrastic, antiprotozoal, antifungal, and anti-inflammatory effects that can alter avian physiology [5]. Unlike synthetic antibiotics, herbal products leave no residue and are generally accepted as safe ingredients in the food industry and as growth promoters in animal feed [6,7]. A variety of medicinal plants are found in Ethiopia, including Rosmarinus officinalis [8] Likewise, aromatic and medicinal plants play an important role in the socio-cultural, spiritual and health aspects of Ethiopia.

Moreover, Rosmarinus officinalis also known as “Yetibs kitel” in Ethiopia is used to flavor a wide range of dishes and spices. Rosemary officinalis, like other Lamiaceae species, contain flavonoids and phenolic acids [9], also reported, in biological systems, phenolic chemicals can slow down oxidative processes. Research to date has focused on studying the oil extracted from rosemary as a feed additive in broilers and laying hens. For example, Chicken supplemented with 0.5% rosemary powder achieved improvement in weight gain, and FCR [10]. Similarly [11], reported that 0.6% supplementation of rosemary powder improved egg count and egg mass in laying hens of all ages. Also [12], found that supplementation with 200 mg/kg REO increased egg weight, decreased feed intake, and improved feed conversion ratio.

In animal feeds, rosemary is used as an alternative to synthetic antioxidants and antimicrobials as it promotes nutrient digestion, reduces microbial growth, and delays lipid oxidation [[13], [14], [15]]. According to Refs. [16,17], rosemary as feed additives enhances production performance and antioxidant enzyme activity under temperature-stress conditions and improves eggshell quality. In another report [18], found that supplementation of 5000 or 10000 mg/kg rosemary herb to diets of laying hens had no effect on feed intake, egg weight, egg mass, feed conversion ratio, or average body weight. There are limited studies on the potential effects of rosemary leaf meal (RLM) on production performance and quality of fresh and stored eggs in Ethiopia considering that the rosemary plant is readily available in the country. This being the case, the objective of this study is to evaluate the effect of rosemary leaf meal on egg production and egg quality in Lohmann Brown laying hens.

2. Materials and methods

2.1. Description of the study area

The study was conducted in Jimma Town, located 346 km from Addis Ababa at latitude 7o41′N and Longitude 36o50′E, 1750 m above sea level.

2.2. Rosemary collection and preparation

Fresh rosemary leaf was purchased at the local market of Mana District, Jimma Zone. It was chopped into pieces with a knife, spread out in the shade and dried for an average of 3–5 days on average, and ground using a mechanical grinder to pass through a sieve of 2 mm.

2.3. Chemical analysis

The nutritional composition and nutrient analysis (% dry matter) of experimental diets was presented in Table 1. The proximate compositions of RLM powder and commercial concentrate were analyzed at Jimma University College of Agriculture and Veterinary Medicine in the Animal Nutrition Laboratory following standard procedure [19]. The ME value was calculated as 37*CP+ 81.8*EE+35.5*NFE according to the formula of [20].

Table 1.

Nutritional composition and nutrient analysis (% dry matter) of experimental diets.

Supplementation of rosemary leaf meal	0%	1.7%	3.5%	5.2	Rosemary leaf meal
Ingredients
Toasted soybean meal	20	20	20	20
Maize grain	49	49	49	49
Meat and bone meal	2	2	2	2
Nouga seed cake	14	14	14	14
Wheat bran	8	8	8	8
Vitamin and mineral premix	2	2	2	2
Limestone flour	4.5	4.5	4.5	4.5
Salt	0.5	0.5	0.5	0.5
Rosemary leaf meal	0	1.7	3.5	5.2
Nutrient composition
Dry matter (%)	89.5	92.8	94	94.4	89.4
Crude protein (%)	17.37	18	18.5	18.8	15.9
Ether extract (%)	4.59	5.56	6.9	7.6	5.4
Crude fibre (%)	6.16	6.9	7.2	7.4	9.64
Ash (%)	8.4	8.5	8.67	8.74	7.54
Nitrogen free extract (%)	52.98	61.8	61.6	62.4	50.92
Metabolizable energy (kcal/kg)	14.6	14.68	17.8	16.24	14.1

^aGuizotiaabyssinica; Vitamin premix per kg of diet: vitamin A: 2.7 mg; vitamin D3: 0.05 mg; vitamin E: 18 mg; vitamin K3: 2 mg; thiamine: 1.8 mg; riboflavin: 6.6 mg; pantothenic acid: 10 mg; pyridoxine: 3 mg; cyanocobalamin: 0.015 mg; niacin: 30 mg; biotin: 0.1 mg; folic acid: 1 mg; choline chloride: 250 mg; antioxidant: 100 mg; Fe: 50 mg; Mn: 100 mg; Zn: 100 mg; Cu: 10 mg; I: 1 mg and Se: 0.2 mg.

2.4. Management of the experimental layers

Lohmann-Brown layers were used for the experiment. Ninety-six (96) Lohmann Brown layers of 24 weeks of age were used for the experiment. The experimental house was properly cleaned, disinfected, and well-ventilated before the arrival of the layer's chicks, and the birds were vaccinated against Newcastle disease, fowl typhoid, fowl pox infectious bursal disease, and Marek's disease. The birds were fed on the experimental ration for two weeks of acclimatization period before the start of the feeding trial. Feed and water were given ad libitum. The experimental period lasted 12 weeks from July 23 - November 15, 2021.

2.5. Experimental design and treatment

A total of 96 twenty-two-weeks-old Lohmann Brown layers were used for the experiment. The layers were randomly assigned to four treatment groups in a Completely Randomized Design (CRD). Each treatment group was replicated 6 times to obtain a total of 6 groups of 4 layers each under each treatment. The experimental diet contained 1.7, 3.5, and 5.2% RLM respectively. The commercial ration contained 2898.9 Kcal metabolizable energy and 17.37 crude proteins adequate to meet the energy and protein requirements of Lohmann Brown layers as recommended by Ref. [21]. Experimental diets were balanced and composed to be isocaloric and isonitrogenous varying only in the levels of RLM in the diets (Table 1). The layers were randomly assigned to the experimental diets within individual cages measuring 50 × 50 × 45 cm. A nipple drinker and trough feeders were installed in the cages. Programmable lighting and adequate ventilation were installed in the birdhouse. All experimental procedures were performed in compliance with the general guidelines of the animal ethical committee.

2.6. Laying performance and egg quality

2.6.1. Production data

The birds were weighed at the beginning and the end of the trial using an electronic weighing balance with an accuracy of 0.05g. Weight gain was calculated as the difference between the final and the initial weight of the hens. Eggs were collected daily (morning and afternoon) and evaluated weekly. The collected eggs from each group were weighed to determine total egg production (%) and average egg weight (g). Egg mass was calculated as the number of eggs multiplied by egg weight per treatment. An electronic balance was used to weigh the eggs (0.01g minimum weight). Daily feed intake per group was determined by weighing the feed offered and the leftover (refusal) the following morning. The average egg weight was multiplied by the total number of eggs produced to get the total egg mass.

Daily egg mass per hen was computed by dividing the total egg mass by the number of hens that were initially housed and the total number of days in which the hens were inlaying. The feed conversion ratio (feed consumed/egg mass) was then calculated as grams of feed: grams of egg mass produced [22].

Eggs were examined for interior and exterior quality. Three eggs were randomly collected from each treatment replication to measure egg quality every week.

Eggs were weighed, and then their length and width were determined before breaking. To test both internal and external egg quality parameters, the egg was gently shattered on a glass plate (35 × 25 cm). Yolks were separated from albumen. Any albumen that had adhered to the egg shells was removed. The weight of the albumen was estimated by subtracting the weight of the yolk and shell from the total weight of the egg. The egg shape index was computed as the ratio of egg width to length, and the yolk index as the average yolk height divided by yolk diameter (mm) following the removal of the yolk from the albumen. A tripod micrometer was used to measure the yolk height to the nearest 0.01 mm, and a vernier caliper was used to measure the yolk diameter to the nearest 0.05 mm. The Roche Color Fan (Printed in Switzerland), a common colorimetric technique with a 1–15 scale, was used to determine the yolk's color. The Haugh unit was calculated according to Ref. [23].

The eggs were examined for shell quality based on shell thickness (with shell membrane) which was measured using a micrometer screw gauge. The breaking strength of the eggshells (kg/cm2) was measured by an egg force reader). The shell weight was determined by digital balance.

2.6.2. Egg quality data

Sixty fresh eggs from each treatment and a total of 240 eggs were used for both storage temperatures. Eggs were stored at 4 °C and 21.5 °C for 7, 14, 21, 28, and 35 days. The stored eggs were identified and analyzed at each corresponding storage time and temperature. Of the 240 eggs, 120 eggs were stored at room temperature (21oc), and 120 eggs were stored in the refrigerator (4oc). Six eggs from each treatment were randomly collected from both storage methods after the following storage times (7, 14, 21, 28, and 35 days) to determine egg quality traits. Eggs were kept on egg trays assigned to the refrigerator's cooling compartment in the fridge compartment of the refrigerator. For room temperature storage, the egg was placed in a well-ventilated room, eggs were weighed at each sampling time, and loss measurements of egg weight during storage were calculated as follows:

$$\text{Total}\text{egg}\text{weight}\text{loss}(\mathrm{g})=\text{Initial}\text{weight}(\mathrm{g})-\text{final}\text{weight}(\mathrm{g}).\text{Weight}\text{loss}\text{percentage}\text{was}\text{calculated}\text{using}\text{day}0\text{egg}\text{weight}(\mathrm{g})$$

$$\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}(\%)=\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}(\mathrm{g})$$

$$\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}(\mathrm{g})\times 100$$

The external egg quality parameters such as egg length and egg width were measured with a Vernier caliper in centimeters and the egg shape index was obtained as a ratio of egg width to length. The shell thickness of the dry eggshell was measured with a micrometer screw gauge. The mean of three points (the narrow, wide, and middle) was taken as the shell thickness. Weight loss of whole eggs during storage was adapted from (Suresh et al., 2015) according to the following formula, Weight loss = Initial egg weight – final egg weight after storage. Percentage weight loss was estimated from day 0 egg weight (g). Weight loss (%) = total Weight loss (g)/initial weight (g) × 100.

For internal egg quality traits, the egg was broken open on a flat surface and the yolk was separated from the albumen. A vernier caliper was used to measure the widest horizontal circumference of the yolk. Yolk height was measured as the height of the yolk at the midpoint with a tripod micrometer. The yolk index was calculated by dividing the yolk height by its diameter. Albumen width was measured at the widest horizontal circumference with a Vernier caliper. Albumin height was measured as the height of the chalazae at a point midway between the thinner and outer circumference of the white with a tripod micrometer and the index was calculated by dividing the height of the albumen by its diameter.

2.7. Statistical analysis

All collected data were subjected to analysis of variance (ANOVA) for completely randomized designs using the General Linear Models (GLM) procedure of the Statistical Analysis System [24]. Treatment means were compared with Tukey multiple range test at (p < 0.05).

3. Results

3.1. Laying performance

Feed intake, hen daily egg production percentage, egg weight, and egg mass indicated an increase as the level of rosemary inclusion increased from 0% (control) to 5.2% (T4), and the feed conversion ratio decreased from 0% to 1.7%, 4.5 and 5.2% inclusions (Table 2). The FCR obtained from all supplemented groups was significantly lower (p < 0.05) as compared to the control groups. The daily feed intake of experimental layers fed on 5.2% rosemary powder was significantly higher (p < 0.05) than that of the control group (Table 2). Supplementing RLM to layers feed significantly affected the production performance of layers as compared to the control group (Table 2). Egg weight and egg mass produced by birds who received treatment four (T4) were significantly (p < 0.05) higher compared to birds receiving control and T1, T2, and T3 (Table 2). Regarding the current result inclusion of RLM at the level of 5.2% improved egg weight (by 1.49g) as compared to the control (Table 2).

Table 2.

Feed intake, %HDEP, egg weight, egg mass, and FCR as affected by RLM.

Parameter	Control	T2	T3	T4	SEM	P-Value
HDEP (%)	73.79b	73.89b	78.70a	78.89a	0.047	<.0001
Feed intake (g/h/d)	104.72d	105.63c	106.76b	107.66a	0.021	<.0001
Egg weight (g)	55.22d	55.49c	56.19b	56.71a	0.015	<.0001
Egg mass (g)	42.55d	43.77c	45.44b	47.82a	0.002	<.0001
FCR (g feed/g egg mass)	2.46a	2.41b	2.37c	2.25d	0.001	<.0001

^{a b c d}: Means with different superscripts within a row are significantly different (P < 0.05). HDEP= Hen Day Egg Production, FCR= Feed Conversion Ratio, P- Value = Probability Value, SEM= Standard Error of Mean.

3.2. Effects of rosemary leaf meal on egg quality parameters

3.2.1. External quality characteristics of fresh egg

The effect of external egg quality parameters of RLM is shown in Table 3. There is no significant (p < 0.05) difference in egg width and egg length obtained from birds fed either any level of experimental feed or control group. On the other hand, shell weight, shell thickness, and shell strength were affected by the level of rosemary leaf powder supplementation. The result of mean shell weight, shell thickness and shell strength obtained from a group of birds fed 3.5% and 5.2% of rosemary leaf powder was significantly (p < 0.05) higher as compared to control groups.

Table 3.

External egg quality characteristics as affected by RLM supplementation.

Parameter	Control	T2	T3	T4	SEM	P-Value
Egg width (cm)	4.43	4.44	4.46	4.45	0.006	0.9681
Egg length (cm)	5.29	5.29	5.32	5.30	0.011	0.9880
Shell weight (g)	5.31b	5.31b	5.82a	5.87a	0.098	<.0001
Shell thickness (mm)	0.32b	0.32b	0.35a	0.37a	0.004	<.0001
Shell strength (kg/cm2)	3.10b	3.33b	3.58a	3.76a	0.098	<.0001

^{a b c d}: Mean with different superscripts within a row are significantly different (P < 0.05). P- Value = Probability Value, SEM= Standard Error of Mean.

3.2.2. Effects of rosemary leaf meal on internal quality of fresh eggs

The internal quality of fresh egg quality characteristics is shown in Table 4. Mean Albumen height, Albumen weight, Yolk height, Yolk weight, Yolk color, and HU were significantly (p < 0.05) affected by rosemary supplementation. Supplementation with rosemary leaf powder resulted in improvements in internal egg quality as the level of supplementation increased from 0 to 5.2%.

Table 4.

The internal egg quality characteristics as affected by RLM supplementation.

Parameter	Control	T2	T3	T4	SEM	P-value
Albumen weight (g)	33.56d	33.95c	34.64b	34.94a	0.012	<.0001
Albumen height (cm)	6.11d	6.31c	6.59b	6.91a	0.004	<.0001
Yolk height (mm)	14.35d	14.59c	15.41b	15.90a	0.017	<.0001
Yolk weight (g)	14.34d	14.62c	15.12b	15.61a	0.013	<.0001
Yolk color	4.18d	4.55c	4.86b	5.33a	0.018	<.0001
Haugh Unit (HU)	79.12d	80.36c	82.05b	83.94a	0.021	<.0001

^{a b c d}: Mean with different superscripts within a row are significantly different (P < 0.05). P- Value = Probability Value, SEM= Standard Error of Mean.

4. Interaction effect of egg quality parameters versus storage duration and temperature

There was a significant interaction effect (p < 0.05) on egg weight, weight loss, shell weight, shell strength, and shell thickness of eggs stored at specific temperatures and egg storage times (Table 5). Internal egg quality parameters (albumen height, albumen weight, yolk height, yolk weight, and HU and yolk color) were significantly affected by different storage temperatures and storage duration (Table 6).

Table 5.

External quality of stored eggs as affected by Rosemary Leaf Meal supplementation under different storage temperatures and storage duration.

Treatments	IEW	FEW	EwL	Ewd	EL	Shw	ShT	EshSt
1	54.21c	50.81d	3.40a	4.05d	5.12d	5.07c	0.28d	4.97c
2	54.22c	51.20c	3.07b	4.06c	5.15c	5.14b	0.29c	5.04b
3	55.32b	53.04b	2.28c	4.26b	5.40b	5.16b	0.32b	5.06b
4	55.45a	53.45a	1.99d	4.28a	5.41a	5.41a	0.33a	5.31a
P-Value	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
Storage
Refrigerator (4oc)	54.96a	52.63a	2.33b	4.21	5.31	5.22a	0.31a	5.12a
R.T 21.50c)	54.63b	51.61b	3.04a	4.19	5.31	5.20b	0.30b	5.10b
P-Value	<.0001	<.0001	<.0001	–	–	0.0289	<.0001	0.0289
Duration
7	55.66a	54.87a	0.78e	4.22a	5.27b	5.25a	0.32a	5.15a
14	55.14b	52.94b	2.20d	4.20b	5.27b	5.25b	0.31b	5.15a
21	54.53c	51.51c	3.02c	4.20b	5.31a	5.21b	0.30c	5.11b
28	54.38d	50.97d	3.41b	4.20b	5.27b	5.20b	0.29d	5.10b
35	54.28d	50.28e	4.00a	4.10c	5.27b	5.20b	0.28e	5.11b
P-Value	<.0001	<.0001	<.0001	<.0001	<.0001	0.0026	<.0001	0.0026
P-Value
storage*Duration	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	0.0063	<.0001
storage*Treat	0.0034	<.0001	<.0001	<.0001	0.0001	0.0002	0.1049	0.0002
Duration*Treat	<.0001	<.0001	<.0001	<.0001		0.0610	0.0837	0.0610
Stor*Dur*Treat	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	0.2513	<.0001
S.E.M	0.022	0.004	0.023	0.002	0.013	0.027	0.003	0.027
Cv	0.315063	0.123194	6.158872	0.2777517	0.247916	3.172083	1.761709	3.234336

^{a b c d} = Means with different superscripts within a row are significantly different (P < 0.05). IEW=Initial Egg Weight, FEW=Final Egg Weight, Ewt L = Egg Weight Loss, Ewd = Egg width, EL = Egg Length, EShSt = Shell Strength, ShW = Egg Shell Weight, ShT = Egg Shell Thickness, SEM = Standard Error of Mean, CV= Coefficient of Variance, P- Value = Probability Value.

Table 6.

Internal egg quality of stored eggs affected by RLM supplementation under different storage temperatures and storage duration.

Treatment	AW	AH	YH	YW	YC	HU
1	30.59d	4.00d	12.86d	13.40d	2.98d	53.04d
2	30.94c	4.04c	13.21c	13.43c	3.32c	60.55c
3	32.59b	4.78b	14.01b	13.53b	6.23b	67.45b
4	32.76a	4.91a	14.18a	13.67a	6.47a	68.34a
P-Value	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
Storage
Refr(40c)	32.40a	5.16a	13.93a	13.45b	5.26a	72.21a
R.T (21.50c)	31.04b	3.50b	13.20b	13.56a	4.24b	52.48b
P-Value	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
Duration (Days)
7	33.83a	6.61a	14.66a	13.31e	6.75a	82.49a
14	32.61b	5.02b	14.05b	13.38d	5.83b	71.15b
21	31.44c	3.87c	13.58c	13.44c	4.83c	59.39c
28	30.68d	3.20d	13.00d	13.63b	3.48d	51.06d
35	30.04e	2.96e	12.55e	13.79a	2.85e	47.64e
P-Value	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
P-Value
storage*Duration	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
storage*Treat	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
Duration*Treat	<.0001	<.0001	<.0001	<.0001	0.0575	<.0001
Storage*Dur*Treat	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
S.E.M	0.012	0.003	0.006	0.014	0.115	0.006
CV	0.101996	0.365519	0.180041	0.088237	7.139295	0.400543

^{a b c d e} Mean with different superscripts within a column are significantly different (P < 0.05). AH= Albumen Height, AW= Albumen Weight, HU= Haugh Unit, YW= Yolk Weight, YH= Yolk Height, YC= Yolk Color, T = Treatment, SEM = Standard Error of Mean, CV= Coefficient of Variance, P- Value = Probability Value.

There was a significant interaction effect between the treatments on albumen height, albumen weight, yolk height, yolk weight, and HU and yolk color. Internal quality parameters were significantly decreased as storage time increased from 0 to 35 days. Again, a significant interaction effect was observed as storage temperature increased from 4oc to 21oc. (Table 6). Accordingly, the low nutrient loss was observed in the group of birds fed 5.2% of rosemary leaf stored at 4oc storage temperature and a storage period of 35 days compared to the control group.

5. Discussion

Rosemary Leaf Meal (RLM) supplementation in Lohman Brown laying hens can improve overall production performance and egg quality. Numerous studies have concluded that the supplementation of rosemary leaf meal in the diet of laying hens has a positive impact on the performance and egg production of the hens and improves the immunity and antioxidant status of the hen [16].

Supplementing a laying hen diet with rosemary leaf meal powder resulted in a significant improvement in laying performance. Supplementation of laying hen feed with approximately 5.2% RLM powder increased daily weight gain and final body weight of the birds. Feed intake, percentage of daily egg production by the hens, egg weight and egg mass improved as the level of RLM supplementation increased from 0% to 5.2%. The daily feed intake of the experimental laying hens fed with 5.2% rosemary leaf powder was significantly higher than in other groups. The improvement in body weight gain, egg weight and feed intake could be attributed to the presence of active bio-ingredients in rosemary that could have a beneficial effect on digestion and feed intake by improving the microflora of the gastrointestinal tract. Bioactive components in RLM such as flavonoids, phenolic acids and terpenoids [25]directly affect some digestive enzymes such as amylases and proteases [26] and improve poultry growth by improving the absorbing cells in the gut [27]. Consistent with this [28,29], indicate that rosemary and its essential oil supplementation have a significant impact on feed intake. In addition [5], RLM has been reported to increase nutrient absorption and appetite, digestion of bile acids, as well as lipid absorption, synthesis, and salivary secretion. On the contrary [30] reported decreased feed intake and improved feed efficiency in groups supplemented with RMO (100–300 mg/kg). The discrepancy between the current study and the previous report could be due to the differences in chicken breed and overall management system during the trial period.

The supplementation of RLM in a layer diet significantly affected mean egg production. Chickens that received 3.5 and 5.2% RLM powder had a significantly higher percentage of egg production compared to the control group. Consistent with the current study [31], reported that supplementation with dried rosemary improved the egg-laying capacity of chickens by 1.79%. The current study also agreed with [11] who showed that productive performance traits and egg quality were improved by supplementing laying hen diets with 3000 mg/kg rosemary powder. Contrary to current evidence [32], reported that there was no difference in egg production in the group supplemented with 200 mg/kg rosemary essential oil compared to the control group. The difference, contrary to our findings, could be due to the synergistic effect of two different essential oils (cinnamon and rosemary) used in the previous report. The improvement in egg production characteristics from RLM supplementation could be due to the nutrients contained in the RLM, which could result in improved digestion, nutrient uptake, and a direct or indirect anabolic effect on host tissues. It could also be due to the effects of the biological activity of phenolic compounds such as carnosol and carbolic acids found in rosemary, which can result in higher feed efficiency and conversion resulting from improved production performance [33].

The highest egg mass was recorded in a group of birds fed 5.2% RLM compared to the control group. This could be due to the positive effect of biochemical components in rosemary, which increase feed conversion. The current study agreed with [10,11] who reported that egg production and egg mass were increased by 0.5% and 0.6% RLM supplementation in the layer feed. On the contrary [34] reported that dietary supplementation with rosemary oil (at a level of 200 mg/kg) had no effect on egg mass in all RLM-supplemented treatment groups.

The FCR obtained from all supplemented groups was significantly lower compared to the control groups. This indicates that laying hens fed 3.5 and 5.2% RML had higher feed efficiency compared to the control. This means that the lower the FCR, the higher the performance achieved by the chickens [35]. The result was consistent with (12)who reported that a dietary supplement of 200 mg/kg rosemary essential oil improved feed conversion rates in laying hens. The result was also consistent with [36] who reported that the use of 0.5% rosemary leaves in the diet improved feed efficiency in birds.

The supplementation of RLM powder in a laying hen feed resulted in a significant improvement in mean shell weight, shell thickness and shell strength as the supplementation level in birds receiving 3.5 and 5.2% compared to the other birds of 0% increased to 5.2% and higher in this experiment. The improvement in eggshell properties could be due to adequate levels of calcium (317 mg/kg) and phosphorus (66 mg/kg) as well as various bioactive ingredients. In addition [37], concluded that essential oil supplementation decreased mineral excretion and improved Ca bioavailability.

The current study agrees with [11,16] who reported that peak eggshell weight and shell thickness were significantly increased by dietary RML. This can be explained in part by the fact that essential oils may have a beneficial effect on the rate of mineral absorption, particularly calcium and magnesium ions [38]. In addition [12], reported that eggshell thickness increased significantly in a group of chickens supplemented with 250 mg/kg rosemary essential oil compared to control groups. In contrast [34], reported that eggshell weight and eggshell thickness were not significantly affected by supplementation with 200 mg/kg or 250 mg/kg rosemary essential oil compared to the control group. This variability in results could be related to the dose, combination effect, and constituents of essential oils, as well as their metabolism and use in birds. An improvement in shell thickness when supplementing with rosemary could be attributed to carnosic acid and rosmarinic acid, which are the main antioxidant and antimicrobial compounds in rosemary [39]. In addition [17], reported that crushing strength was increased by the addition of rosemary to 250 mg/kg rosemary essential oil in the diet and had a positive effect on eggshell quality in laying hens. According to Ref. [40], essential oil (EO) can modulate the absorption of cations such as Ca2+ through direct interaction with epithelial transport proteins such as those of the transient receptor potential. The same author concluded that plasma calcium levels were significantly increased by essential oil supplementation.

Internal egg quality parameters such as mean albumen height, albumen weight, yolk height, yolk color and Haugh unit were increased in the group of birds fed 5.2% rosemary leaf powder compared to the control group. The increase in protein levels and Haugh unit score found in the current study indicates better storage potential for eggs from RLM-fed hens. Good antioxidant activity is responsible for good protein quality. According to Ref. [41], the improvement in protein quality through natural products is related to their high bioavailability, antioxidant function and ability to maintain the intestinal microflora. The naturally occurring antioxidant flavonoids may support this assumption [42].

Consistent with the current results [10,34] a significant effect of the inclusion of rosemary essential oil (200 mg/kg) in the diet of laying hens on increasing yolk weight, egg albumin and yolk index was reported. Consistent with current evidence [43], it has been reported that the beneficial effects of rosemary oil on digestibility may have led to greater absorption of minerals and other dietary nutrients, and thus larger egg yolks.

The highest Haugh unit score was achieved by a group of birds fed 5.2% rosemary powder compared to 0, 1.7 and 3.5%. The improvement in HU compared to the current study could be due to the biochemical composition of RLM enhancing the natural antioxidant capacity of the inner egg quality. It could also be because RLM had a positive impact on the protein levels of the supplemented groups, since it was a key component in calculating the Haugh unit. The current result is consistent with [44] who reported that the Haugh unit score was higher in rosemary groups than in control groups. Contrary to current evidence [12], it was reported that the yolk fraction and Haugh unit were reduced by the addition of 200 mg/kg rosemary essential oil (REO) to laying hen feed.

The deepest yellow yolk color was measured for a group of chickens fed at 5.2%, significantly compared to a control group. The improvement in yolk color of the supplemented group may be due to the chemical composition of rosemary leaves, as this has a positive effect on yolk color. Consistent with the current finding, dietary intake of 200 mg/kg rosemary oil (RO) promoted higher iron absorption, as this mineral is responsible for the intense yellow color of egg yolk [45].

There was a significant interaction effect between stored eggs collected from the supplemented groups in terms of egg weight, weight loss, shell weight, shell strength, and shell thickness stored at a given temperature and storage duration. However, there is no published literature on the effects of RLM on the quality of externally stored eggs. In stored eggs from birds fed 5.2%, egg weight loss was significantly less, followed by 3.5%. The weight loss of the eggs from the control group (0%) and 1.7% increased with increasing storage temperature and storage time. This means that as the storage time increases, the egg weight decreases, leading to weight loss. Egg weight loss corresponds to protein water loss through eggshell pores during the gas exchange process (escape of moisture and carbon dioxide) that occurs continuously after laying and can be accelerated by inadequate storage conditions, mostly at high temperatures. The decrease in egg weight and increase in egg weight loss found in the current study was similar to the finding of [46] who reported that the storage temperature increased from 4 to 21.5 °C. There was a reduction in egg weight, which in turn led to increased egg weight loss in the stored eggs. Weight reduction has been associated with an increase in egg moisture loss with increasing storage temperature [47]. The rate at which gases and moisture escape from the tray pores during storage depends on the storage environment (temperature 4 °C or >27 °C, relative humidity and airflow). Consistent with the current results [48,49], egg weight loss was shown to be affected by storage time and temperature.

Eggs stored for 35 days and collected from 5.2% to 3.5% RLM, respectively, were found to have significantly higher shell weight and shell thickness compared to the control groups. The result of the recorded shell weight for a group of birds fed 1.7, 3.5 and 5.2% was higher compared to the control groups. However, there is no published literature to support or refute our results. In addition, shell weight decreased with increasing storage temperature and duration. The current results are similar to the report of [49] who found that egg weight is significantly affected by storage time and temperature. Consistent with the current study [50], reported that the weight of the shell is part of the total weight of the egg, so a decrease in egg weight would reflect a decrease in shell weight as moisture is lost from the shell. The egg weight was significantly influenced by the storage temperature.

Eggshell thickness was negatively affected by storage time, which may be due to RLM's antioxidant activity. The current results show that shell thickness decreased with storage time, which is in contrast to the result of [51] who reported that shell thickness was not significantly affected by storage time. On the other hand, in the group of birds to which 5.2% RLM was added, a significantly higher shell strength was observed. A lower shell breaking strength was observed in the control groups. The current study found that storage temperature had no effect on egg width and egg length of the stored eggs. The result was similar to the results of [52] who reported that storage temperature had no significant effect on egg width, egg length, and egg shape index of the stored eggs.

There is a significant interaction effect between treatment, storage duration and temperatures on the weight of the stored egg. This interaction showed that stored eggs from 0%-fed laying hens showed a loss in egg weight compared to other groups due to longer storage time. Eggs stored in the refrigerator (4 °C) had a significantly higher egg weight than eggs stored at (21.5 °C). Significantly higher egg weight losses were observed in eggs stored at room temperature (21.5 °C). Storage duration had a significant impact on egg weight and egg weight loss. With increasing duration of egg storage from 7 to 35 days, the weight of stored eggs decreased and weight loss increased. The current study agreed with [53] who pointed out that a significant association between storage time and temperature is known in egg weight loss, with higher weight loss observed in eggs without reduced film stored at room temperature for longer periods became. In a study examining eggs stored for up to 35 days [54], an average weight loss of 9.20% was found in eggs stored in excessive temperature and humidity conditions. Similar results were reported by Ref. [55] who confirmed a significant egg weight loss of 0.65 g to 1.03 g in eggs stored consecutively for 5 and 10 days at 21 °C.

The current study showed that tray weight is affected by storage temperature. Shell thickness decreased with increasing storage time. Lower shell thickness losses were recorded after 7 days of storage, while high shell thickness losses were recorded after 35 days of storage. However, storage temperature had no effect on egg width and egg length. Storage temperature significantly affected shell thickness. However, studies on the effects of RLM on the oxidative stability of shell eggs are lacking. There was a significant interaction between RLM and storage temperature in egg weight loss. The interaction showed that the weight loss of stored eggs was clearly evident when the degree of entrapment increased from 3.5 to 5.2% with increasing storage temperature from 4 °C to 21.5 °C. The current study showed that rosemary leaf powder had a positive interaction effect on protein height, protein weight, yolk height, yolk weight, and HU and yolk color.

The mean protein height, protein weight, HU, yolk height, and yolk color of eggs stored in the refrigerator (4 °C) were higher than those stored at room temperature (21.5 °C), with the exception of the yolk weight. In eggs stored at room temperature (21.5 °C), a significantly higher yolk weight was achieved than at refrigerator temperature (4 °C) and also increased with storage time. The results were also consistent with [56] who reported that yolk weight increases with increasing storage temperature.

6. Conclusion

Supplementing commercial layer feed with RML powder at levels of 3.5% and 5.2% improved live weight gain, egg production performance and egg quality in Lohmann-Brown layers. Improved feed intake and feed conversion rates were achieved in the groups of layers fed with 3.5% and 5.2% RLM powder. The supplementation of RML in commercial laying hens improved the shelf life of eggs and minimized the severe effects of temperature and storage time on stored eggs.

Funding statement

This research received no funding.

Author contribution statement

Seman Kedir: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.

Metekia Tamiru Tasew; Ashraf Alkhtib: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Dawit Adisu Tadese; Melaku Mulugeta; Ashenafi Miresa; Jane Wamatu; Emily Burton: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Lishan Takele: Contributed reagents, materials, analysis tools or data; Wrote the paper.

Data availability statement

Data will be made available on request.

Additional information

No additional information is available for this paper.

Declaration of competing interest

The authors declare that there is no competing interest.