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Currently, the use of phytochemical additives in animal diets is of interest as an alternative to replace subtherapeutic antibiotics in intensive production (Alagawany et al. 2020). This is mainly because these natural products reduce pathogen adherence, promote growth, mitigate the response to stress situations, increase bioavailability of nutrients for their absorption in the gastrointestinal tract (GIT), stimulate the secretion of digestive enzymes and modulate immunity and antioxidant activity (da Silveira et al. 2021).
Phytobiotics are natural products, rich in secondary metabolites, which are presented in solid form by a drying process or as essential oils (Kikusato et al. 2021). In the leaves of Jatropha curcas, which is a plant species of Euphorbiaceae family, native to American tropics, metabolites such as apigenin, vitexin, isovitexin, tannins, coumarins and flavonoids have been identified, responsible for several biological activities in animals and humans (Rahu et al. 2021). Generally, these natural products have been experimented on broilers and laying hens, but studies using quail as an animal model are scarce.
Quail production gradually increased, due to its excellent reproduction rate, its low feed conversion, the minimum requirements for its production, and its rapid growth and production cycle (Alagawany et al. 2020). However, meat and egg farming faces management, nutrition and health problems, which result in economic losses and make it impossible for birds to express their genetic potential (da Silveira et al. 2021). Jatropha curcas leaf powder has secondary metabolites with medicinal properties, which is evidence of its potential to improve the biological functions of poultry. The objective of this research was to perform the phytochemical screening of Jatropha curcas leaf powder and determine its effect on productivity, egg quality and blood biochemistry of laying quail.
Materials and Methods
J. curcas leaves and sample preparation. Thirty kilograms of leaves were randomly taken from 20 J. curcas trees, approximately four years old, out of a total of 150 trees, in November, 2019 in Santa Isabel-Bayamo-Granma area, Cuba. This region is characterized by a flat topography and brown carbonate soil. Diversity of size, structure and optimal classification of leaves, identified in the Department of Botany of the Faculty of Agricultural Sciences of the University of Granma, was considered for the collection (Más et al. 2017). After collecting, impurities were removed from leaves (mainly dust and dirt). They were dried under the sun for seven days on perforated cardboard sheets and moved twice a day. Afterwards, leaves were crushed in a parallel blade mill until obtaining a 1 mm powder (Martínez et al. 2020).
Successive extractions. To achieve the greatest depletion of sample, the successive extraction scheme was used with solvents of increasing polarity: ethanol and water. Dry powder, obtained from J. curcas 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 prepare the alcoholic extract and 50 mL of distilled water to obtain the aqueous extract (Más et al. 2017).
Phytochemical screening. Secondary metabolites were qualified in the ethanolic and aqueous extract, according to the protocol proposed by Fajardo et al. (2018). The phytochemical characterization was carried out at the Center for the Study of Applied Chemistry (CEQA, initials in Spanish) of the Faculty of Technical Sciences, belonging to the University of Granma, Cuba.
Experimental location. The in vivo experiment was carried out from February to March 2020 at the “Comandante Manuel Fajardo” poultry farm, located in Jiguaní, Granma, Cuba. Mean relative humidity of this region is 80%; mean minimum temperature of 26.5 °C and mean maximum of 31.1 °C.
Diets, animals and treatments. A total of 150-layer quail (Coturnix japonica), 25 weeks old, were randomly distributed into three treatments and five repetitions per treatment during 70 d. Treatments consisted of the inclusion of 0, 0.5 and 1.0 % of J. curcas leaf powder in quail diets. Results of Suwarta and Suryani (2019) were considered to select addition levels of J. curcas leaf powder. Diets were formulated from corn meal and soybean cake, as recommended by UECAN (2011). They were weekly prepared and J. curcas leaf powder was added in the feed factory. Its composition and contribution are shown in table 1.
Table 1 Composition and contribution of the laying quail diet (25 to 35 weeks)
| Ingredients | Inclusion level, % |
|---|---|
| Corn meal | 42.75 |
| Soybean meal | 42.87 |
| Plant oil | 3.25 |
| Salt | 0.30 |
| DL-methionine | 0.08 |
| Monocalcium phosphate | 1.79 |
| Calcium carbonate | 7.50 |
| Choline chloride | 0.33 |
| Mineral and vitamin premix1 | 1.13 |
| Calculated contribution, % | |
| Metabolizable energy, MJ/kg | 11.50 |
| Crude protein | 21.00 |
| Ca | 3.20 |
| Available P | 0.50 |
| Lysine | 1.21 |
| Methionine+ Cystine | 0.72 |
| Tryptophan | 0.26 |
| Threonine | 0.56 |
| Ether extract | 1.67 |
| Crude fiber | 2.40 |
1Each kg contains: vit. A, 10 x 106 I.U.; vit. D3, 1.5 x 106 I.U.; 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 10 laying quails were housed. Animals received 35 g of feed/quail/ d. Water was supplied ad libitum through a nipple drinker per cage and 16 h of daylight were offered each day. The experiment had an adaptation period of 15 d (Martínez et al. 2020). The experimental animals were not provided with drugs or therapeutic veterinary care during the experimental stage.
Productive indicators. Initial and final weights of the laying quails were individually determined at 25 and 35 weeks of age on a SARTORIUS digital scale (China), BL 1,500 model, with ± 0.10 g precision. The egg was weighed weekly, at a rate of 30 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%. Feed and nutrient intake was determined by offer and rejection method. To calculate mass conversion, consumed food, egg weight per repetition and number of laid eggs were considered. Viability was also determined at the end of the experiment. Percentage of unfit eggs (cracked, shell membrane and broken) was determined by the formula:
Egg external and internal quality. In weeks 30 and 35, 30 eggs/treatment were sampled to determine external and internal quality indicators. Egg weight was calculated with an OHAUS® digital scale (China), with a precision of 0.01 g. For the shape index (SI), the following expression was used:
The height of dense white and yolk was measured with a caliper, with ± 0.01 mm accuracy. Records of Haugh units (HU) were calculated by the relationship between egg weight (W) and height of the dense white (H):
Blood biochemistry. At 35 weeks of age, a fasting hematological test was performed on six laying quails per treatment. Blood was punctured from the left wing. One milliliter insulin syringes were used and the blood was placed in 2 mL vials with sodium heparin. Glucose, uric acid, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total cholesterol were determined by means of an ultraviolet spectrophotometer, Humalyzer brand, with enzymatic kits, commercialized by SPINREACT. SA. (Spain).
Statistical analysis. Data was processed by analysis of variance (Anova). In the necessary cases, Duncan (1955) test was applied to determine the multiple differences among means, according to the statistical program SPSS 23.0.1.2014 (SPSS Inc., Chicago, IL, USA).
Results and Discussion
Results of the phytochemical screening of Jatropha curcas leaf extracts are shown in table 2. The presence of alkaloids, coumarins, resins and anthocyanidins, saponins, free amino acids, tannins, quinones, flavonoids and mucilages is highlighted, metabolites responsible for different biological activities, when used in small concentrations in diets (Más et al. 2017).
Table 2 Phytochemical screening of Jatropha curcas leaf powder
| Trials | Ethanolic extract | Aqueous extract |
|---|---|---|
| Alkaloids | ++ | + |
| Coumarins | ++ | + |
| Resins and anthocyanidins | + | - |
| Triterpenes and steroids | - | - |
| Saponins | + | - |
| Free amino acids | ++ | + |
| Cardiotonic glycosides | - | |
| Reducing sugars | - | - |
| Phenols and tannins | +++ | ++ |
| Quinones | ++ | + |
| Flavonoids | ++ | ++ |
| Mucilages | + | + |
(-): Absence, (+): Presence, (++): Moderate, (+++): Abundant
The excess of tannins is considered an antinutritional factor because it limits the absorption of some nutrients, such as iron and proteins (Savón et al. 2007). However, small doses of these metabolites in diets can be efficient bactericides, fungicides, antioxidants and vasoconstrictors (Martínez et al. 2020). The flavonoids found in alcoholic and aqueous extracts may also be beneficial due to their phytoestrogen effect and antioxidant capacity. In addition, they influence on the synthesis of eicosanoids, platelet aggregation and oxidation of low-density lipoproteins (Tungmunnithum et al. 2018). Méndez et al. (2020) found that the high concentration of flavonoids and total phenols in J. curcas leaves induces an in vitro antioxidant effect. According to Kamboh et al. (2019), these secondary metabolites (flavonoids) have been used in poultry diets to improve the quality of the final product (meat and eggs).
The anthocyanidins found in J. curcas leaf powder have been used in poultry diets because they exert positive effects on inflammatory conditions related to their antioxidant capacity. Furthermore, they stimulate the immune system and increase lymphocyte proliferation and cytokine secretion (Changxing et al. 2018). Coumarins (++), identified by phytochemical screening in J. curcas leaves (table 2), have beneficial effects in small proportions, and are powerful anticoagulants and bactericides against strains of Staphylococcus aureos and Escherichia coli (Rahu et al. 2021).
According to Martínez et al. (2020), alkaloids are mainly found in the alcoholic extract and, to a lesser extent, in the aqueous extract, as occurred in the present study (table 2). Kikusato et al. (2021) demonstrated that small concentrations of alkaloids (mainly isoquinolines) reduce oxidative stress and postprandial intestinal inflammation in intensive productions and with heat stress. Despite the fact that other secondary metabolites (saponins, free amino acids, quinones and mucilages), responsible for the biological activity, were identified by phytochemical screening, results were not conclusive to relate them to a possible effect on production and quality of the egg of laying quails.
Table 3 shows the productive indicators of laying quails, fed with the dietary supplementation of 0.5 and 1.0 % of J. curcas leaf powder. Viability, live weight, feed intake, egg weight, mass conversion, and unfit eggs did not indicate significant differences among treatments. However, the addition of 0.5% of this medicinal plant (J. curcas) increased laying intensity by 1.89 % with respect to the remaining treatments (P <0.05).
Table 3 Effect of J. curcas leaf powder on productive indicators of laying quails (25-35 weeks)
| Productive indicators | J. curcas leaf powder (%) | SE± |
|
||
|---|---|---|---|---|---|
| Control | 0.5 | 1.0 | |||
| Initial weight, g | 187.00 | 188.00 | 188.00 | 0.49 | 0.179 |
| Final weight, g | 192.00 | 193.00 | 193.00 | 7.45 | 0.069 |
| Viability, % | 100.00 | 100.00 | 100.00 | ||
| Laying intensity, % | 78.35b | 80.24a | 78.09b | 0.65 | 0.042 |
| Feed intake, g/quail/d | 31.13 | 31.64 | 31.55 | 0.16 | 0.058 |
| Egg weight, g | 11.07 | 11.15 | 11.10 | 0.05 | 0.074 |
| Mass conversion, kg/kg | 3.59 | 3.54 | 3.64 | 0.08 | 0.059 |
| Unfit eggs, % | 0.25 | 0.27 | 0.25 | 0.03 | 0.090 |
a,b Means with different superscripts in the same line differ at P<0.05
Viability demonstrates the innocuity of the used phytobiotic (J. curcas) up to 1 % in diets of laying quail during 70 experimental days (table 3). These results coincide with those reported by Khalifa and Noseer (2019), Suwarta and Suryani (2019) and Zeweil et al. (2019), who found no morbidity and mortality in laying quail when they used ginger (Zingiber officinale), cinnamon (Cinnamomum verum), turmeric (Curcuma longa) and licorice (Glycyrrhiz glabra) as phytochemical additives, respectively.
The use of these medicinal compounds in quail diets has been little studied and applied mainly due to the rusticity of this poultry species, which shows great resistance to environmental changes and diseases (Nain et al. 2011). However, Radwan et al. (2008) and Suwarta and Suryani (2019) demonstrated that the use of 0.5% of cinnamon and turmeric powder improved the production of quail eggs, because of the antioxidant and anti-inflammatory effect of these natural compounds. Similar results were found in this experiment, where the use of 0.5 % of J. curcas leaf powder increased laying intensity by 1.89 % (table 3), which shows that this medicinal plant, rich in anti-inflammatory (alkaloids), antioxidant (flavonoids) and bactericide (tannins and coumarins) compounds have a beneficial effect on this poultry species. Also, the possible phytoestrogen effects of polyphenolic compounds detected in J. curcas powder (table 2) could promote egg production (Ghasemi et al. 2010). In this regard, reports of Çiftci (2012) refer that a greater circulation of estrogens in blood improved egg production of laying hens.
Feed intake, egg weight and mass conversion did not change (table 3) (P> 0.05), despite the inclusion of J. curcas leaf powder rich in secondary metabolites (tables 2 and 3). Similar results were found by Zeweil et al. (2019) and Santos et al. (2019), when they used small concentrations of phytochemical compounds in diets of laying quail. However, Suwarta and Suryani (2019) reported that increasing levels of secondary metabolites gradually reduced feed intake in laying quails. These authors base this result on the idea that the chemical compounds decreased food palatability. This result shows that the addition of up to 1 % of J. curcas leaf powder does not cause symptomatic intestinal disorders, which could trigger an increase of mass conversion and a decrease of feed intake and productivity (egg weight and production) of laying quails.
Savón et al. (2007) reported that a high concentration of secondary metabolites (mainly tannins) reduces productive indicators of these animals. Specifically, the tannins identified by phytochemical screening (table 2; +++) in the medicinal powder did not affect the absorption of sulfur amino acids, since these amino acids directly influence on egg weight. Likewise, eggs fit for consumption were not affected by the supplementation of the natural product. Salazar et al. (2017) and Martínez et al. (2020) reported similar results, when using small proportions of medicinal plants in the diets of laying birds.
Table 4 shows the external and internal quality of the egg of laying quails, when 0.5 and 1 % of J. curcas leaf powder were added to the diet. In both sampled weeks (30 and 35), the inclusion of 0.5 % of J. curcas leaf powder increased (P <0.05) the height of the dense white and Haugh unit with respect to the remaining treatments, although in the week 30, Haugh unit was not statistically different in the groups with medicinal powder. There was no difference of egg weight, shape index and yolk height (P> 0.05) due to the effect of the experimental treatments.
Table 4 Effect of J. curcas leaf powder on the external and internal quality of the egg of laying quails
| Egg quality | J. curcas leaf powder (%) | SE± | Value of P | ||
|---|---|---|---|---|---|
| Control | 0.5 | 1.0 | |||
| 30 weeks | |||||
| Egg weight, g | 10.87 | 11.41 | 10.87 | 0.20 | 0.106 |
| Shape index, % | 88.36 | 88.67 | 89.80 | 0.63 | 0.185 |
| Dense white height, mm | 4.23b | 4.82a | 4.37b | 0.16 | 0.027 |
| Yolk height, mm | 11.48 | 11.45 | 11.52 | 0.31 | 0.569 |
| Haugh units | 88.59b | 91.41a | 89.38ab | 0.77 | 0.044 |
| 35 weeks | |||||
| Egg weight, g | 11.26 | 11.30 | 11.29 | 0.27 | 0.994 |
| Shape index, % | 87.02 | 88.93 | 87.35 | 1.00 | 0.189 |
| Dense white height, mm | 3.88b | 4.24a | 3.84b | 0.04 | 0.019 |
| Yolk height, mm | 10.48 | 10.35 | 10.49 | 0.34 | 0.284 |
| Haugh units | 86.25b | 88.34a | 85.98b | 0.80 | 0.020 |
a,b Means with different superscripts in the same line differ at P<0.05
The height of the white and Haugh unit were the only indicators that changed due to the tested treatments (table 4) and, in turn, those are related to egg freshness (Martínez et al. 2021). Other studies with medicinal plants have shown that these indicators (height of white and Haugh unit) are the most susceptible to the inclusion of secondary metabolites in laying birds (Más et al. 2017). Keener et al. (2006) reported that the amount and height of albumen depends on the balance of amino acids provided by the diet and their absorption in the intestinal lumen. It appears that the beneficial secondary metabolites, provided by the addition of 0.5 % of J. curcas leaf powder, improved intestinal health and, in turn, the absorption of protein amino acids.
A higher addition (1.0 %) of the medicinal powder reduced the height of the dense white, compared to the treatment with 0.5 % (table 4). Perhaps a higher intake of J. curcas (0.32 g/bird/d) and secondary metabolites, such as tannins, decreased the availability and absorption of some amino acids (mainly sulfur amino acids) (Savón et al. 2007). Dietary supplementation with J. curcas leaf powder did not modify yolk height in laying quails (table 4). Other studies in quails and laying hens showed that the use of several phytobiotics did not influence on this internal quality indicator (Más et al. 2017 and Zeweil et al. 2019).
Table 5 shows that the addition of 0.5% of J. curcas leaf powder in the diets of laying quails reduced (P <0.05) glucose and creatinine with respect to the rest of treatments. In addition, the highest inclusion of J. curcas (1 %) increased (P <0.05) aspartate aminotransferase concentration, compared to other treatments. No differences among treatments were detected for the concentration of uric acid, alanine aminotransferase and total cholesterol in laying quails.
Table 5 Effect of J. curcas leaf powder on blood chemistry of laying quails (35 weeks)
| Items, (mmol/mL) | J. curcas leaf powder (%) | SE ± | Value of P | References (mmol/mL) * | ||
|---|---|---|---|---|---|---|
| Control | 0.5 | 1.0 | ||||
| Glu | 9.16a | 6.08b | 10.56a | 0.67 | 0.001 | 6-12 |
| AST | 3.98b | 4.10b | 6.20a | 0.42 | 0.032 | 3-6 |
| ALT | 98.20 | 97.58 | 102.36 | 2.61 | 0.656 | 90-120 |
| Crea | 42.34a | 29.58b | 39.88a | 1.87 | 0.001 | 25-45 |
| AU | 409.14 | 412.22 | 410.42 | 2.84 | 0.682 | 180-450 |
| CT | 5.86 | 5.98 | 5.30 | 0.88 | 0.331 | 3.07-6.80 |
a,b Means with different superscripts in the same line differ at P<0.05. Glu: glucose; Crea: creatinine; AST: aspartate aminotransferase; ALT: alanine aminotransferase; AU: uric acid; CT: total cholesterol
Biochemical parameters have always been used as health indicators in humans and animals. Studies of the influence of medicinal plants and their secondary metabolites on some indicators of blood biochemistry in laying quails are scarce. However, in laying poultry, it has been demonstrated that small concentrations of phytobiotics reduce serum glucose concentration, due to the astringent effect of these metabolites, which causes a slow intestinal release and maintenance of dietary glucose. In addition, there is a reduction of glucose in the animals, which can reduce blood circulation of harmful lipids (Martínez et al. 2012). This result shows that whole powder (0.5 %) of J. curcas leaves and some secondary metabolites could have a hypoglycemic effect on laying quails.
Contradictorily, the inclusion of 1 % of J. curcas leaves did not reduce serum glucose concentration (table 5). Apparently, a higher ingestion of secondary metabolites caused some asymptomatic liver damage, because the animals of this treatment showed the highest concentration of aspartate aminotransferase (AST) (table 5). A higher concentration of this enzyme (AST) in the blood has been related to liver problems (inflammation and/or irritation) in humans and animals (Saeed et al. 2017). Likewise, the concentration of alanine aminotransferase (ALT) did not show changes among treatments (table 5). This result shows that quails had no apparent diseases, since a high concentration of this enzyme is associated with microbial infections, cancer and liver diseases in humans and animals (de Oliveira et al. 2021).
Creatinine concentration decreased with the inclusion of 0.5 % of J. curcas leaf powder (table 5). A higher concentration of creatinine has been associated with kidney damage in humans and animals (Hajare et al. 2020). Apparently, the use of small concentrations of this medicinal plant (0.5%) can improve kidney function without apparent visible signs, which is important for the filtering process and chemical balance of blood (Mnisi and Mlambo 2018).
In laying quails, the experimental treatments (table 5) did not affect the concentration of uric acid, which is the final product of the metabolism of purines and some proteins (Lin et al. 2014). This result shows that the animals were adequately nourished (table 1), since feeding factors are the most probable causes of hyperuricemia in poultry. Similarly, total blood cholesterol was not different, due to the experimental treatments (table 5). However, authors such as Sawarta and Suryani (2019) and Zeweil et al. (2019) reported a decrease in low-density lipoprotein (mainly cholesterol) and total cholesterol, when they used a phytobiotic mixture (cinnamon and turmeric) and a medicinal powder (licorice), respectively, due to the hypocholesterolemic effect of some secondary metabolites. Apparently, the concentration of J. curcas powder in laying quail diets was not sufficient to change serum cholesterol concentration (table 5).
Jatropha curcas leaf powder showed a wide qualification of secondary metabolites, which influence directly on the biological response of laying quails. The inclusion of 0.5% of J. curcas leaf powder in diets for laying quails is suggested to improve laying, egg quality and some indicators of blood biochemistry.
