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The use of antibiotics as additives in animal feeding, especially in birds and pigs, was a common practice from the 50s of the last century. However, the alarming increase in resistance to antibiotics is currently a matter of concern for the scientific community, due to the problem that this causes in the treatment of infectious diseases (Más et al. 2015). Besides, the growth promoter antibiotics (GPA) can increase the number of resistant strains, as well as transfer crossed resistance to other microorganisms (Ljubojević et al. 2016). Despite restrictions on the use of GPAs in the European Union, many countries still use them regularly in poultry production (FAO 2016).
The current alternatives to the use of antibiotics are natural products (Vinus et al. 2018). In the poultry industry, prebiotics, probiotics and phytobiotics have been researched with the objective of improving health status, reducing pathogenic microorganisms and modulating a better immune response (Markowiak and Śliżewska 2018). Plant additives are considered an alternative to replace antibiotics, from the technical, economic and biological point of view, due to the safety of their inclusion and their null residuality (Castillo-López et al. 2017). Many benefits of medicinal plant powders have been reported in birds, such as the increase of nutrient digestibility, immune stability, competitive exclusion of microorganisms in the gastrointestinal tract, better intestinal health, and positive changes in the final product (egg and meat.) (Vinus et al. 2018).
The guava tree (Psidium guajava) is native of Tropical America. The archaeological remains place it in Brazil or somewhere between Mexico and Peru (Singh, 2018). Its leaves and bark are used as phytopreparations in animals and humans, due to their antibacterial, antiemetic, anti-inflammatory, anthelmintic, antiseptic, antitoxic, astringent, carminative, spasmodic and tonic properties (Naseer et al. 2018). Salazar et al. (2017) found improvements in egg production and mass conversion, when they included P. guajava powder as part of a mixture of medicinal plants.
Also, the crude extract of P. guajava leaves reduced the serum concentration of malondialdehyde (MDA) as an indicator of oxidative stress in laying hens (Boonthium et al. 2010). As well, Zargar et al. (2020) reported that the use of P. guajava leaves reduced histopathological lesions and cholesterol of the broiler carcass. Geidam et al. (2015) showed that with the use of the leaves of this medicinal plant the diarrheal syndrome in broilers with E. coli decreased. Despite the chemical benefits of P. guajava, few scientific studies have been performed to show its benefits as a zootechnical additive in diets destined for laying hens. The objective of this study was to evaluate the phytobiotic effect of Psidium guajava leaf powder on the productivity and quality of the egg of laying hens.
Materials and Methods
Phytochemical screening of Psidium guajava leaves. To prepare the sample, leaves of 20 Psidium guajava trees were taken, from the Cuban red dwarf variety, with approximately five years old, in Peralejo area Bayamo-Granma, Cuba. This region is characterized by a flat topography and brown carbonate soil. For the collection, the diversity of size, structure and optimal classification of leaves was considered, identified in the Departamento de Botánica, de la Facultad de Ciencias Agropecuarias de la Universidad de Granma.
After collecting, the impurities (mainly dust and dirt) were removed from the leaves and they were dried in the sun for seven days. Then, the leaves were milled until obtaining a 1 mm powder (Martínez et al. 2012a).
Successive extractions. To achieve the highest depletion of the sample, the successive extraction scheme was used with solvents of increasing polarity: ethanol and water (Fajardo et al. 2018). The dry powder obtained from the guava leaves was taken. Then, 5 g were weighed on an analytical balance (BS 2202S SARTORIUS, China) and 50 mL of 70% ethanol were added to make the alcoholic extract and 50 mL of distilled water to obtain the aqueous extract. Next, the extraction was carried out in a similar way (Más et al. 2017).
Phytochemical screening. The phytochemical analyzes were performed on the hydroalcoholic extract, according to the protocol proposed by Mijares et al. (2014) and Rodríguez et al. (2018). The tests of resins, Liebermann-Burchard (triterpenes and/or steroids), foam (saponins), ninhydrin (free amino acids), Dragendorff and Mayer (alkaloids), Baljet (coumarins), Fehling (reducing carbohydrates), ferric chloride (phenols or tannins), Borntrager (quinones), Shinoda (flavonoids) and anthocyanidins were tested. The phytochemical characterization was carried out at Centro de Estudio de Química Aplicada (CEQA), from Facultad de Ciencias Técnicas, belonging to Universidad de Granma, Cuba
Experimental location. The in vivo experiment was carried out at the Antonio Maceo poultry farm, in Bayamo, Granma, Cuba. The average relative humidity was 78 %, the average minimum temperature was 26.3 °C and the average maximum was 30.6 °C.
Diets, animals and treatments. A total of 160 White Leghorn L-33 hens, 27 weeks old, were randomly distributed in four treatments and 10 repetitions per treatment during 70 d. The treatments consisted of a control diet (T0) and the addition of 0.5 (T1), 1.0 (T2) and 1.5 % (T3) of Psidium guajava leaf powder. The results of Más et al. (2016) and Aroche et al. (2018) to select the addition levels of P. guajava leaf powder were considered. In addition, the same control diet for laying hens was used, applied in a previous experiment (Rosabal et al. 2017), formulated from corn and soybean cake, as recommended by UECAN (2007).
Experimental conditions. The experimental unit consisted of a 40 x 40 cm metal cage, where four hens were housed. The birds received 110 g of food/hen/d. The water was supplied ad libitum through a nipple/cage and 16 hours of lighting were offered each day. The experiment had an adaptation period of 15 days (Martínez et al. 2012b). The experimental birds were not given medicine or therapeutic veterinary attention during the experimental period.
Productive indicators. The initial and final weights of the laying hens were performed individually at 27 and 37 weeks of age on a SARTORIUS digital scale, model BL 1500, with precision ± 0.10 g. The egg weight was weekly carried out: 20 eggs/treatment, between 8:30 and 9:30 a.m. and the average weight was calculated.
For determining laying intensity, total egg production/week/treatment was considered and one egg/d/ housed bird was assumed as 100%. Mass conversion was calculated when considering food intake, egg weight per repetition and number of eggs laid. At the end of the experiment, the viability was also calculated. The percentage of unfit eggs (cracked, without an eggshell and broken) was calculated by the formula: % unfit eggs (UE), %UE =
External and internal quality of the egg. In week 37, a total of 20 eggs/treatment were sampled to determine the external and internal quality indicators of the egg. The egg weight was determined with an OHAUS® (China) digital scale with a precision of 0.01 g. To calculate the shape index (SI) the formula used was:
A Russian vernier with ± 0.01 mm precision was used for measuring the eggshell thickness at the egg’s equator and at the upper and lower poles. The shell surface was determined according to Carter (1975) formula, where Area=
The height of the dense white and the yolk was measured with a height gauge, with ± 0.01 mm accuracy. The yolk color was determined by Roche’s range of 15 colors. The records of Haugh units (HU) were calculated by the relation between the egg weight (W) and the height (H) of the dense white using the formula:
Statistical analysis. The data were processed by analysis of variance (Anova), one-way, in a totally random design. When necessary, Duncan (1955) Test was used to determine the multiple differences between means, according to the SPSS version 23.0 statistical program. The percentage of unfit eggs was analyzed by comparison of proportions using the COMPARPRO 1.0 program (Font et al. 2007).
Results and Discussion
The results of the phytochemical screening of Psidium guajava leaf extracts (table 1) show the diversity of secondary metabolites in the hydroalcoholic extract (mainly flavonoids), metabolites responsible for different biological activities when they are used in small concentrations in diets (Salazar et al. 2019 and Martínez et al. 2020). Also, there was not alkaloids, coumarins and resins in the hydroalcoholic extract of P. guajava leaves.
Table 1 Phytochemical screening of P. guajava leaves
| Secondary metabolites | Hydroalcoholic extract |
|---|---|
| Mayer and Wagner (alkaloids) | - |
| Baljet (coumarines) | - |
| Foam (saponins) | + |
| Shinoda (flavonoids) | ++ |
| Fehling (reducing sugars) | + |
| Bortrager (quinones) | + |
| Libermamn-Burchard (triterpenes and steroids) | + |
| Ninhidrina (free amino acids) | + |
| Anthocyanidines | + |
| Ferric chloride (Phenols and tannins) | + |
| Resins | - |
Legend:(-): Absence, (+): Presence, (++): Abundant
The tannins present in the Psidium guajava leaves have been described by Más et al. (2015) and Mapatac (2017) as anti-nutritional factors, when they are in excess in diets or drugs, because they limit the absorption of some nutrients such as iron and amino acids (Sobral-Souza et al. 2019). However, it has been reported that these polyphenolic compounds, in small concentrations, can be efficient anti-inflammatory, astringent, fungicidal, vasoconstrictive, and antioxidants (Xu et al. 2017). In this sense, Huang et al. (2018) reported that tannins can inhibit the growth of enterobacteriaceae and decrease the circulating cholesterol, when reducing the absorption of this lipid and expelling it through the feces.
In turn, the presence of flavonoids in the hydroalcoholic extract of the plant under study corroborates what was stated by Vargas et al. (2006), who detected that guava leaves are important sources of flavonoids. The extracts and leaves of guava (Psidium guajava) have a group of new components, only present in these plants, as is the case of gammapyrones, which belong to the of glycosylated flavonoids subclass. Afzal et al. (2019) found that extracts from P. guajava leaves regulate the functioning of the enteric nervous system in the digestive system, showing its antidiarrheal function.
Jiménez-Escrig et al. (2001) reported that the leaves and the juice of the P. guajava fruit reduced the activity of the free radicals DPPH (2.2-Diphenyl-1-Picrylhydrazyl) in in vitro tests and the oxidation of low-density lipoproteins induced by the copper.
Other authors suggest that these compounds (flavonoids) have phytostogenic activity, since they are structurally similar to natural estrogens (17 beta estradiol) as well as synthetic ones (Yang et al. 2000). Ismail et al. (2012) had reported a decrease in the growth of Staphylococcus aureus and Escherichia coli, when using guava leaves with high flavonoid content in their diets.
The presence of anthocyanidins in the Psidium guajava powder is related to the decrease in oxidative stress and the stimulation of the immune system, due to the proliferation of lymphocytes and the secretion of cytokinins (interleukin II) by the activated lymphocytes (Wu et al. 2018). Shipp and Abdel-Aal (2010) reported that anthocyanidins are natural water-soluble pigments, which can increase the color of the egg yolk as an added value to the product. According to these results, the beneficial secondary metabolites in Psidium guajava leaves could benefit the biological response in birds.
Although other secondary metabolites (saponins, reducing sugars, quinones, triterpenes and steroids), responsible for the biological activity, were identified by phytochemical screening, the results are not conclusive to relate them to a possible effect on the production and quality of the egg of lying hens. Table 2 shows that different additions of P. guajava leaf powder did not statistically change (P> 0.05) the viability, food intake, unfit eggs and the initial and final weight of laying hens. However, the addition of 0.5 % of this medicinal plant increased the laying intensity by 3.28% and the egg weight by 1.67 g; in addition to reducing the mass conversion by 0.14 kg / kg in relation to the control treatment and the other treatments with P. guajava leaf powder (P <0.05).
Table 2 Effect of P. guajava leaf powder on the productive performance of laying hens
| Indicators | Control | 0.50 | 1.00 | 1.50 | SE± | P value |
|---|---|---|---|---|---|---|
| Viability, % | 100 | 100 | 100 | 100 | ||
| Initial live weight, g | 1633 | 1625 | 1626 | 1625 | 5.849 | 0.411 |
| Final live weight , g | 1667 | 1660 | 1670 | 1686 | 9.519 | 0.143 |
| Laying intensity, % | 80.18b | 83.46a | 79.89b | 79.65b | 0.890 | <0.001 |
| Food intake , g/bird/d | 110 | 110 | 110 | 110 | ||
| Mass conversion, kg/kg | 2.61a | 2.41b | 2.62a | 2.58a | 0.033 | <0.001 |
| Egg weight, g | 53.08c | 54.75a | 53.89b | 53.43bc | 0.260 | 0.023 |
| Cracked eggs , % | 0.22 | 0.21 | 0.22 | 0.22 | 0.098 | 0.060 |
| Eggs without an eggshell, % | 0.04 | 0.08 | 0.04 | 0.04 | 0.039 | 0.160 |
| Broken eggs, % | 0.00 | 0.00 | 0.00 | 0.00 | ||
a,b,cMeans with different letters in the same row differ at P<0.05 (Duncan 1955)
The viability (100%) showed the safety of the Psidum guajava leaf powder used up to 1.5 % in the diets of laying hens during 10 experimental weeks, without causing morbidity and mortality (table 2). Apparently, P. guajava leaf powder, added in small concentrations (up to 1.5 %) in the diet of laying hens, did not cause adverse effects, since the metabolites did not modify the viability, live weight and food intake.
It has been recognized that a high supplementation with phytochemicals can decrease the assimilation of nutrients, cause diarrhea and weight loss (Huang et al. 2018). Ni et al. (2016) reported that excessive intake of certain secondary metabolites can cause symptoms related to anti-nutritional factors in laying hens (lower food intake, low egg production and mortality).
The increase in the laying intensity, with the addition of 0.5 % of P. guajava powder, showed the effectiveness of this medicinal plant as a promoter of egg production in laying hens. Ghasemi et al. (2010) reported similar responses when adding garlic (Allium sativum) and thyme (Thymus vulgaris) powders in the diets of laying hens, confirming the beneficial effect of natural products, such as P. guajava powder. The possible phytoestrogenic effects of the polyphenolic compounds detected in P. guajava powder (table 2) could also influence on the improvement of egg production and mass conversion (Purdom et al. 1994 and Ghasemi et al. 2010). Çiftci et al. (2012) found that a higher circulation of estrogens in blood improves egg production in laying hens. Regarding the secondary metabolites and their beneficial functions in P. guajava leaf powder, their direct effect on the productivity of laying hens can be affirmed, since they cannot synthesize them.
The higher egg weight (T1) with this medicinal plant could be associated with the ideal intestinal conditions of birds, which favor the increase and development of the beneficial flora, since the secondary metabolites present in the P. guajava leaves can decrease the pathogenic bacteria (Más et al. 2015) and contribute to the competitive exclusion of the gastrointestinal tract (GIT). This effect in the intestine favors the absorption of Ca in the diets and its incorporation in the shell formation (Sahin et al. 2018). Apparently, the tannins in the medicinal powder did not affect the absorption of sulfur amino acids, since these amino acids are the main promoters of egg weight. It should highlighted that the higher addition (T2 and T3) of this natural product (P. guajava) decreased the productive response of laying hens compared to T0, perhaps due to the fact that the secondary metabolites exerted some antinutritional effects, without apparent affections in animals.
The eggs fit for intake were not affected by the supplementation of the natural product. Rosabal et al. (2017) and Salazar et al. (2017) reported similar results, when using small proportions of medicinal plants in the diets of laying hens. These data are important, if it is takes into account that larger eggs are more prone to shell break (Taylor et al. 2016), which shows that the additive increases the egg weight, without affecting its commercialization, since according to Jones et al. (2018) this indicator influences on the profitability of poultry activity.
Table 3 shows the effect of P. guajava powder on the external and internal quality of the eggs of laying hen. It was showed that the addition of P. guajava leaves did not modify the shape index, yolk height, shell surface, egg weight and Haugh unit, at 37 weeks of age. However, the shell thickness and the yolk color increased (P <0.05) with the addition of 0.5 % with respect to the other experimental treatments. In addition, in T3 the height of the dense white decreased (P <0.05) in relation to the control, without differences with the T1 and T2.
Table 3 Effect of the P. guajava leaf powder on the external and internal quality of the egg of laying hens (37 weeks)
| Indicators | ||||||
|---|---|---|---|---|---|---|
| Control | 0.50 | 1.00 | 1.50 | SE± | P value | |
| Egg weight, g | 55.20 | 55.40 | 55.20 | 55.40 | 1.792 | 0.758 |
| Shape index, % | 73.75 | 75.07 | 77.89 | 75.74 | 0.005 | 0.201 |
| Shell thickness, mm | 0.20c | 0.26a | 0.22bc | 0.23b | 1.131 | <0.001 |
| Height of the dense white, mm | 6.31a | 6.17ab | 6.03ab | 5.97b | 0.122 | 0.030 |
| Yolk height, mm | 6.93 | 6.99 | 7.00 | 6.96 | 0.235 | 0.052 |
| Haugh unit | 78.21 | 79.84 | 78.23 | 78.06 | 0.110 | 0.545 |
| Shell surface, cm2 | 44.90 | 44.44 | 45.75 | 45.01 | 0.874 | 0.456 |
| Yolk color | 6b | 7a | 6b | 6b | 0.567 | 0.003 |
a,b,cMeans with different letters in the same row differ at P<0.05 (Duncan 1955)
The highest thickness of the shell with T1 could be due that better intestinal health increased Ca absorption in the intestinal lumen, since according to Savón et al. (2007) ideal pH conditions are needed by the insolubility or instability of this mineral. In addition, the possible phytoestrogenic action of the secondary metabolites could increase the incorporation of calcium into the shell. According to Elaroussi et al. (1994), estrogens are the main transporters of calcium carbonate from the intestine to the bones and, in turn, enter to the homeostatic cycle of Ca. This can avoid the problems in the imbalance of this mineral in the bones, the incidence of problems in the legs and the shell thickness. However, further studies are necessary to validate this hypothesis. The T2 and T3 had results equivalent to the control, with worse results with respect to T1. According to Martínez et al. (2013), the beneficial effect of medicinal plants will depend on the chemical structure of the secondary metabolites, their concentration and level of addition in the bird diets.
Despite the beneficial effect of secondary metabolites of P. guajava leaves on the diets of laying hens, they did not improve the height of the dense white. On the contrary, this portion of the egg gradually decreased with the addition of P. guajava (table 3). Apparently, the intake of 1.65 g/bird/d of P. guajava (1.5 %) caused lower amino acid efficiency for the formation of this structure, since according to Keener et al. (2006) the amount of white depends on the balance in amino acids which provided by the diet protein. A deficiency in lysine or methionine reduces the weight of the albumen and decreases the concentration of all free amino acids. In addition, Rodríguez et al. (2011) reported that albumin is characterized by its homogeneity, given by the liquid and thick white. The proportion of this structure varies depending on many factors, particularly the age of the egg.
Although the height of the dense white showed statistical differences, this did not influence on the Haugh units, which did not vary with the addition of the medicinal plant. Similar results found Rosabal et al. (2017) and Salazar et al. (2017). However, they did not agree with Martínez et al. (2012b), who found an increase in this indicator when using up to 1.5 % of Anacardium occidentale leaf powder. Nematinia et al. (2018) showed that the Haugh unit is at its maximum value, when the egg has just been laid or when it has cooled, which denotes a good degree of freshness.
The P. guajava leaf powder did not modify the yolk height, which corresponds to that reported by Martínez et al. (2012b), Más et al. (2015) and Salazar et al. (2017), when they used various phytobiotics in the diets of laying hens. This structure of the egg is determined by the lipid content. Apparently, the secondary metabolites in the medicinal plant did not modify the metabolism of this biomolecule. In addition, other factors can determine this portion of the egg, such as egg weight, productive stage, age, and breed (Grobas et al. 2001).
The addition of phytobiotic additives in the diets of laying hens can determine the intensity of the yolk color, without affecting the performance of the birds, which is acceptable for the table egg industry, bakeries and other industries (Odunsi 2003). However, it will depend on the concentration of the natural colorant in the plant material. In this study there was an increase in the yolk color with the addition of 0.5 % of P. guajava leaf powder, which could be due to the presence of anthocyanidins determined in the phytochemical screening (table 1). The natural colorant influences on the pigmentation of the yolk (Carrillo et al. 2005). However, the results show that the faster formation of the egg, with the addition of 0.5 % of P. guajava powder, could determine the highest deposition of pigments in the yolk, since in the treatments with 1 and 1.5 % there was a decrease of the yolk color (table 3).
The P. guajava leaf powder has a wide variety of secondary metabolites responsible for biological activity. The 0.5 % of P. guajava leaf powder is recommended in the diets of laying hens in their full laying peak to improve productivity and some indicators of egg quality.
