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Although scientific evidence shows that the use of subtherapeutic antibiotics can increase microbial resistance and cross-resistance to other microorganisms, as well as bioaccumulate in muscles and organs (Evangelista et al. 2022), still these synthetic products are commonly used for birds (Burnett et al. 2021) in Latin America. Even though poultry industry focuses more on the use and study of growth-promoting antibiotics in broilers, their inclusion in laying hens corresponds to the most critical stages, such as the beginning of laying and laying peak (Sharma et al. 2022). Recent studies demonstrated that several natural alternatives to preventive antibiotics can be used efficiently in diets and drinking water, such as prebiotics, probiotics, symbiotics, organic acids and medicinal plants (Torres et al. 2005 and Vlaicu et al. 2021). These natural products improve intestinal health, antioxidant capacity, immune status and productivity of laying hens (Khan et al. 2020).
Specifically, medicinal plants have some beneficial secondary metabolites that, included in low concentrations in chicken diets, can modify productive indicators and egg quality (Kim et al. 2011 and Wasilewski et al. 2015). Martínez et al. (2012, 2020) demonstrated that dietary supplementation with Anacardium occidentale and Psidium guajava leaf powder promoted egg production and quality. Also, Más et al. (2015) reported that the inclusion of 1.0 % of Morinda citrifolia leaf powder increased egg weight, shell thickness and yolk color, without decreasing the productivity of laying hens. Likewise, Olmo et al. (2013), by including up to 1.5 % of Moringa oleifera leaf powder in diets, found improvements in the productive indicators of hens at laying peak.
Más et al. (2017) reported that the mixtures of several medicinal plants such as Anacardium occidentale, Psidium guajava, Morinda citrifolia and Moringa oleifera modified the secondary metabolites in the hydro-alcoholic extract and several biologically important phytochemical compounds were identified. Salazar et al. (2017) noted that a mixture of medicinal plants improved the productivity of laying hens, perhaps due to the synergy of secondary metabolites. Despite these studies, it is necessary to continue research with other proportions of the mixtures of medicinal plants and verify their possible beneficial action in poultry. The objective of this study was to evaluate the phytobiotic effect of the mixed powder of these medicinal plants on productivity and quality of laying hens.
Materials and Methods
Experimental location and climatic characteristics. The work was carried out at the UEB Antonio Maceo, which is located on the Manzanillo highway km 12, in the town of La William Soler, Bayamo municipality, Granma province, Cuba. Mean relative humidity was 78 %, mean minimum temperature was 26.3 °C and mean maximum temperature was 30.6 °C.
Sample taking and preparing. An amount of 20 kg of leaves of P. guajava, A. occidentale, M. oleifera and M. citrifolia were collected from 20 trees approximately five years old in the peri-urban areas of Bayamo municipality, Granma. This territory is characterized by a flat topography and brown carbonated soil. Size diversity, structure and optimal classification of leaves were features considered for the collection, and were identified in the Department of Botany of the Faculty of Agricultural Sciences of the University of Granma, Cuba.
The leaves were dehydrated for seven days in the shade, on perforated cardboard sheets and removed twice a day. Then, they were placed in an oven (WSU 400, Germany) with air recirculation for 1 h at 60 °C. They were ground in a hammer mill with parallel blades, at 1 mm of size. Samples were stored at room temperature (26 ± 2 °C) in amber bottles to avoid decomposition of the active substances due to light. To prepare the mixed powder, the dry product obtained from leaves was taken and 40 % of A. occidentale, 20 % of P. guajava, 20 % of M. oleifera and 20 % of M. citrifolia were mixed, according to the results of the phytochemical screening performed by Más et al. (2017).
Diets, animals and treatments. A total of 120 White Leghorn laying hens (Hybrid L-33), 27 weeks of age, were placed, for 10 weeks, according to a completely randomized design with three treatments, 10 repetitions per treatment and four animals per repetition. The experimental treatments consisted of a basal diet (T1) and the addition of 0.5 (T2) and 1.0 % (T3) of mixed powder of leaves of medicinal plants (40 % A. occidentale, 20 % P. guajava, 20 % of M. oleifera and 20 % of M. citrifolia). The results of Rosabal et al. (2017) and Aroche et al. (2018) were considered to select the addition levels of mixed medicinal plant powder. In addition, the nutritional requirements recommended by UECAN (2007) were used for formulating the basal diet (table 1).
Table 1 Composition and contribution of the diet of laying hens (27-37 weeks)
| Ingredients | Inclusion level, % |
|---|---|
| Corn meal | 67.70 |
| Soy cake meal | 21.00 |
| Salt | 0.33 |
| DL-methionine | 0.10 |
| Monocalcium phosphate | 1.34 |
| Calcium carbonate | 9.16 |
| Choline chloride | 0.07 |
| Premix1 | 0.30 |
| Calculated contribution (%) | |
| ME (MJ/kg) | 11.64 |
| CP | 15.50 |
| Ca | 3.80 |
| Total P | 0.66 |
| Available P | 0.40 |
| Lysine | 0.73 |
| Methionine | 0.33 |
| Methionine+cystine | 0.61 |
| Tryptophan | 0.20 |
| Threonine | 0.56 |
| Ether extract | 1.67 |
| Linoleic acid | 0.80 |
| Crude fiber | 2.40 |
1Each kg contains: vit. A 10 x 106 IU; vit. D3 1.5x 106 IU; vit. K3 2,100 mg; vit. E 10,000 mg; thiamine 800 mg; riboflavin 2,500 mg; pantothenic acid 10,000 mg; pyridoxine 2,500 mg; folic acid 250 mg; biotin 100 mg; vit. B12 15 mg; manganese 60,000 mg; copper 8,000 mg; iron 60,000 mg; zinc 50,000 mg; selenium 200 mg; iodine 800 mg; cobalt 500 mg; antioxidant 125,000 mg.
Experimental conditions. The experimental unit consisted of a 40 x 40 cm metal cage, where four hens were housed. The animals received 110 g of feed/hen/day. Water was supplied ad libitum through a nipple per cage and 16 hours of lighting was provided each day. The experiment had an adaptation of 14 d (Martínez et al. 2012). During the experimental stage, the birds were not given any medication or therapeutic veterinary care.
Productive indicators. Laying hens were weighed individually at 27 and 37 weeks of age on a SARTORIUS digital scale, BL 1500 model, with precision ± 0.10 g. Egg weight was recorded weekly at 25 eggs/treatment, between 8:30 to 9:30 a.m. and mean weight was calculated. To determine laying intensity, total egg production/week/treatment was considered and one egg/day/housed bird was assumed as 100 %. Mass conversion was calculated considering the feed consumed, egg weight per repetition and the number of laid eggs. Viability of hens was computed at the end of the experiment. The percentage of unsuitable eggs (cracked, shell-less, broken and dirty eggs) was calculated by the formula:
External and internal quality of eggs. In week 37, 30 eggs/treatment were sampled to determine their external and internal quality indicators. Egg weight was determined with an OHAUS® (China) digital scale, with ± 0.01 g of precision.
The following formula was used for calculating shape index (SI):
To measure shell thickness at the equator and at upper and lower poles of the egg, a Russian-made caliper was used, with an accuracy of ± 0.01mm. Shell surface was determined by Carter's (1975) formula:
Albumin and yolk height was measured with a height caliper with an accuracy of ± 0.01 mm. Yolk color was determined by Roche color fan of 15 colors (Odunsi 2003). The records of Haugh units (HU) were calculated by the relationship between egg weight (W) and albumin height (H) using the formula: (Rosabal et al. 2017)
Statistical analysis. Data was processed using simple classification analysis of variance (ANOVA), in a completely randomized design. In necessary cases, Duncan (1955) test was applied to determine multiple differences between means, according to the SPSS version 23.0 statistical program. The percentage of unsuitable eggs (cracked, dirty, broken and egg-shell) was analyzed by comparison of proportions using the COMPARPRO 1.0 program (Font et al. 2007).
Results and Discussion
Table 2 shows the effect of mixed leaf powder of A. occidentale (40 %), P. guajava (20 %), M. citrifolia (20 %) and M. oleifera (20 %) on the growth performance of laying hens. (27 to 37 weeks). Viability, body weight (initial and final), egg weight and dirty and shelled eggs did not show differences (P>0.05) among experimental treatments. However, the addition of 1.0 % of the mixed powder of medicinal plant decreased the percentage of cracked eggs and improved laying intensity and mass conversion (P<0.05) compared to the other treatments. The birds, in all treatments, consumed 100 % of the feed provided and no broken eggs were found.
Table 2 Phytobiotic effect of mixed powder from leaves of medicinal plants on the productive performance of laying hens (27 to 37 weeks)
| Experimental treatments | |||||
|---|---|---|---|---|---|
| Indicators | Control | 0.5 % | 1.0 % | SE ± | P value |
| Initial body weight, g | 1638 | 1593 | 1650 | 23.2 | 0.0603 |
| Laying intensity, % | 80.18b | 81.18b | 85.53a | 0.93 | 0.0011 |
| Egg weight, g | 53.80 | 53.21 | 53.74 | 0.27 | 0.1903 |
| Feed intake, g/bird/d | 115.00 | 115.00 | 115.00 | ||
| Mixed powder intake | 0.00 | 0.55 | 1.10 | ||
|
|
0.00 | 0.22 | 0.44 | ||
|
|
0.00 | 0.11 | 0.22 | ||
|
|
0.00 | 0.11 | 0.22 | ||
|
|
0.00 | 0.11 | 0.22 | ||
| Mass conversion, kg/kg | 2.73a | 2.71a | 2.52b | 0.03 | 0.0015 |
| Cracked eggs, % | 0.22a | 0.18a | 0.08b | 0.01 | 0.0014 |
| Shell-less eggs, % | 0.09 | 0.00 | 0.04 | 0.03 | 0.1009 |
| Dirty eggs, % | 0.45 | 0.48 | 0.38 | 0.03 | 0.7004 |
| Final body weight, g | 1667 | 1648 | 1656 | 25.31 | 0.2605 |
a,b Means with different letters in the same line, differ at P<0.05, Duncan (1955)
Poultry viability (100 %) demonstrated the safety of mixed powder used during the 70 experimental days. Similar results were reported by Martínez et al. (2020), Rabelo-Ruiz et al. (2021) and Ruesga-Gutiérrez et al. (2022), who demonstrated that phytobiotic additives used in small concentrations do not cause morbidity and mortality in birds. Likewise, increasing levels of the medicinal mixed powder did not modify feed intake, previously verified by Más et al. (2015) and Rosabal et al. (2017) when they used up to 1.0 % of the leaf powder of A. occidentale and M. citrifolia, respectively in the diets for laying hens.
Likewise, the results of hen weight, egg weight and the percentage of shell-less and dirty eggs could be due to the fact that the secondary metabolites of the plants for the mixed powder did not cause symptoms related to anti-nutritional factors (table 2). As reported by Savón et al. (2007) and Ni et al. (2016) an excess of anti-nutritional factors can decrease body weight, productive behavior, absorption of amino acids (mainly sulfur) and shell thickness. However, the mixed powder of medicinal plants (1 %) reduced cracked eggs by 0.14 % compared to the control (table 2). Mosayyeb et al. (2023) found a lower percentage of unsuitable eggs when using phytobiotics in the diets of laying hens. These authors inferred that this effect was a result of the improvement of intestinal health and nutrient absorption.
Laying intensity increased (P<0.05) with the supplementation of 1.0 % of the medicinal mixed powder, demonstrating that small concentrations of these medicinal powders, rich in secondary metabolites, promote egg production (5.35 %) in hens at full peak. of posture. In this sense, Más et al. (2017) identified metabolites responsible for biological activity such as triterpenes, steroids, saponins, coumarins, quinones and anthocyanidins in the mixture of A. occidentale (40 %), P. guajava (20 %), M. oleifera (20 %) and M. citrifolia (20 %).
Salazar et al. (2017) reported that inclusion of 1.0 % of the mixture of P. guajava (40 %), M. oleifera (20 %), A. occidentale (20 %) and M. citrifolia (20 %) increased egg production at 3.75%, although without modifying the other productive indicators. Kong et al. (2006) found that the dietary use of a mixture of several Chinese medicinal plants improved productivity and immune status of broilers, the authors demonstrated the synergy of the chemical compounds in this phytobiotic product researched. Likewise, Chen et al. (2003) reported that the mixture of medicinal plants (synergistic effect) improved cellular immunity and productivity in fast-growing birds. It should be considered that the secondary metabolites identified in the leaves of the medicinal mixed powder have antioxidant, anti-inflammatory and antimicrobial properties (Rafeeq et al. 2023), which had a positive effect on animal response.
Also, the inclusion of 1.0 % of the mixed powder of medicinal plants promoted productive efficiency (mass conversion), due to the increase in egg production, with equal feed intake among treatments (table 2). According to Hidayat et al. (2021), extracts or bioactive compounds from plants used in animal feed have an anti-inflammatory and antimicrobial effect, which modifies intestinal permeability and villi structure and, consequently, increases productive efficiency of birds.
Saleh et al. (2019) recommended the use of phytochemical products to improve productivity, immune activity and ovarian development in laying hens. However, Ghasemi et al. (2010) found no changes in mass conversion when they used a mixture of garlic and thymol.
Table 3 shows that the experimental diets did not modify (P>0.05) the external and internal quality of eggs of laying hens (37 weeks). According to Martínez et al. (2020), different effects can be elucidated when medicinal plants are used orally, because it will depend on the chemical structure of secondary metabolites, their concentration and addition level in poultry diets. Thus, eggshell thickness and surface area did not change due to the effect of experimental diets (P>0.05). This shows that the phytobiotic used up to 1.0 % does not affect the absorption of calcium, phosphorus or other minerals. However, studies by Salazar et al. (2017) showed that the inclusion of 1.0 % of a medicinal mixed powder increased eggshell thickness in laying hens.
Table 3 Effect of dietary supplementation of mixed powder of leaves of medicinal plants on the external and internal quality of the egg of laying hens (37 weeks)
| Experimental treatments | |||||
|---|---|---|---|---|---|
| Indicators | 0 % | 0.5 % | 1.0 % | SE± | P value |
| Egg weight, g | 59.40 | 62.20 | 58.40 | 1.26 | 0.1291 |
| Shape index, % | 74.48 | 76.41 | 73.18 | 1.50 | 0.3475 |
| Eggshell weight, g | 7.80 | 7.92 | 8.06 | 0.34 | 0.8661 |
| Eggshell thickness, mm | 0.27 | 0.26 | 0.27 | 0.01 | 0.8046 |
| Eggshell surface area, cm2 | 47.28 | 48.83 | 46.72 | 0.70 | 0.1275 |
| Dense white height, mm | 6.19 | 6.28 | 6.32 | 0.10 | 0.6748 |
| Yolk height, mm | 7.35 | 7.29 | 7.39 | 0.14 | 0.8829 |
| Haugh units | 78.21 | 77.90 | 79.43 | 0.83 | 0.4172 |
| Yolk color | 6 | 6 | 6 | ||
The secondary metabolites present in the medicinal mixed powder did not modify (P>0.05) the albumin height and the Haugh units (table 3). This structure of the egg depends on the balance of amino acids to form albumin proteins, such as albumin, conalbumin, ovomucin and lysozyme.
The deficiency of essential amino acids reduces albumin height and Haugh units (Ullah et al. 2022). Salazar et al. (2017) and Martínez et al. (2022) found no changes in albumen height and Haugh units when they used up to 1.0 % of a mixed powder of medicinal plants and P. guajava leaf powder in laying hens, respectively. The groups with the phytobiotic did not modify yolk height, apparently, the secondary metabolites to make up the medicinal powder did not alter the metabolism of lipids, rich in this structure of the egg, although other studies are necessary to verify this hypothesis. Other experiments with phytobiotics in hens indicated a similar effect (Más et al. 2015 and Salazar et al. 2017).
This phytobiotic derived from a mixture of medicinal plants did not modify yolk color. All experimental groups indicated the same feed intake and, therefore, of yellow corn, rich in zeaxanthin (tables 1 and 2), as the main pigment in the diet (Calvo-Brenes and O'Hare 2020). However, Más et al. (2015) reported that the supplementation of A. occidentale and M. citrifolia leaf powder pigmented the egg yolk, compared to the control treatment, due to the presence of anthocyanidins. Apparently, the low level of inclusion of these medicinal plants in the mixture, or perhaps a possible antagonism of several secondary metabolites in the mixed powder, inhibited the action of this natural dye. Other studies are necessary to elucidate this possible antagonism.
