Guava (Psidium guajava L.) leaf meal and cooked extract in post-weaning piglets’ diets improve production rates and control the incidence of diarrhea

W Caicedo^1,2, F N A Ferreira³, Y Arteaga¹, A Flores^2,4, C Buenaño⁴, M Pérez¹, C S Silva Neta³ and W M Ferreira⁵

¹ Departamento de Ciencias de la Tierra, Universidad Estatal Amazónica, Puyo, Pastaza, Ecuador
orlando.caicedo@yahoo.es
² Granja Agropecuaria Caicedo, Tarqui, Puyo, Pastaza, Ecuador
³ Technical Services Department, Agroceres Multimix, 1411 01JN St., 13502-741, Rio Claro, São Paulo, Brazil
⁴ Rancho Santa Rita, Tarqui, Puyo, Pastaza, Ecuador
⁵ Department of Animal Science, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, 31270-901, Belo Horizonte, Minas Gerais, Brazil

Abstract

At present natural phenolic compounds from plants are gaining a considerable interest as functional additives for their potential to improve animals’ health and productive performance. This study evaluated the antioxidant effect of the dietary supplementation of guava leaf ( Psidium guajava L.) meal and cooked extract on production rates and diarrhea incidence in piglets after weaning. Sixty neutered male and female piglets in equal 1:1 ratio, weaned at 30 days of age, with an average initial weight of 9.18 ± 1.14 kg, were randomly assigned to three dietary treatments: basal control diet without growth promoting antibiotics (CTL); GULM (CTL + inclusion of 1 g guava leaf meal/100 g DM); and GUCE (CTL + inclusion of 10 mL cooked guava leaf extract/100 g DM), with 20 piglets in each group. The results showed that the groups supplemented with guava leaf meal and cooked extract in the period (30-58 d) showed the best BW (p<0.0001), ADG (p<0.0001), ADFI (p <0.0001) and FC (p<0.0001), respectively. During the first week (30-37 d) there was incidence of diarrhea (p<0.001) for all treatments, control (59.29%), meal (3.57%) and extract (3.57%); and between the 2nd week (37-44d); 3rd week (44-51d); and 4th week (51-58d) there was only incidence of diarrhea (p<0.001) in the control treatment (64.29; 35.71 and 8.57%, respectively). In sum, dietary supplementation with leaf meal (1 g/100 g DM) and cooked extract (10 mL/100 g DM) in the period (30-58d) improved ADFI, ADG, FC, BW and controlled the incidence of diarrhea in post-weaning piglets.

Key-words: intestinal health, phytobiotics, post-weaning diarrhea, Psidium guajava

Introduction

In the Ecuadorian Amazon region, there is little information regarding the use of phytobiotic additives for feeding pigs after weaning. However, there is a wide range of plants with medicinal potential. Among these, there is guava (Psidium guajaba L.), a shrub that belongs to the Myrtaceae family. Guava leaves have been used as a popular herbal tea to treat diabetes for a long time in Asia and North America, its fruits and foliage have various bioactive compounds that help to significantly reduce blood sugar, total cholesterol, total triglycerides, glycosylated serum protein, creatinine, malonaldehyde, rheumatism, diarrhea, diabetes mellitus, cardiovascular diseases, mouth ulcers, bacterial and parasitic infections (Dakappa et al 2013; Morais-Braga et al 2016; Díaz-de-Cerio et al 2017; Luo et al 2019). The antioxidants in the guava foliage include essential oils, polysaccharides, pectins, minerals, vitamins, triterpenoid acid enzymes and alkaloids, steroids, glycosides, tannins, flavonoids and saponins, which can have potential use in functional feeds to improve the health of animals (Venkatachalam et al 2012; Naseer et al 2018).

Moreover, the prevalence of diarrhea caused by enterotoxigenic Escherichia coli in pre-weaning is around 17% while after weaning it can reach 24% (Van Breda et al 2017). Diarrhea due to enterotoxigenic Escherichia coli is an economically important disease in the production of pigs throughout the world, affecting piglets during the first 2 weeks after weaning and is characterized by sudden death or diarrhea, dehydration and growth delay in the surviving animals (Amezcua et al 2002; Fairbrother et al 2005; Rhouma et al 2017).

To prevent and control enteric problems and their impact on productive performance, companies producing balanced feed for pigs use antibiotic growth promoters (colistin, lincomycin) in their formulations to inhibit the growth of pathogenic microorganisms in the piglets’ gastrointestinal tract. On the other hand, the prolonged use of growth promoting antibiotics causes resistance to the pathogenic agents and possibly generates residues in meat (Rakotoharinome et al 2014; Briceño-Fereira et al 2015).

The European Union has banned the use of antibiotics and growth promoters for animals of zootechnical interest as of 2003 (CORDIS 2003). This setting gave way to new investigations with organic acids, prebiotics, probiotics and, recently, to the use of plant extracts and meals (phytobiotics), that have the ability to act as promoters of natural growth since they improve immune development, prevent diseases, besides showing improvements in animal production indicators in several studies (Michiels et al 2010; Liu et al 2013; Liao and Nyachoti 2017; Pastorelli et al 2020). Therefore, this research was conducted to evaluate the antioxidant effect of dietary supplementation of guava (Psidium guajava L.) leaf meal and cooked extract on the productive indices and the incidence of diarrhea in weaned piglets.

Materials and methods

This research was conducted on the postweaning piglets’ premises of the "Rancho Santa Rita" located in the Tarqui area, Pastaza Region (01° 32'00" South and 78° 00'00" West), Ecuador. All the procedures were approved by the Code of Ethics in research with domestic animals and wildlife of the Universidad Estatal Amazónica, under protocol 25-01/2017.

Preparation of the Guava foliage’s meal and cooked extract

The foliage samples were collected from the Rica variety guava in a plantation established for three years, in the "Rancho Santa Rita". Six terminal leaves in each branch of the scrub were taken, immediately after harvest. The samples were transferred to the Chemical Laboratory of the Universidad Estatal Amazónica, washed, and placed to drain. For the production of the meal, the leaves were placed in a Barnstead model 3523 stove for 72 hours, at 65 ºC, ground in a Thomas Model 4 Wiley Mill, with a 1 mm sieve, and placed in sterile ziploc containers. To prepare the cooked extract, 50 g of fresh washed foliage/L of water were cooked in a water bath, brand MEMMERT WNE model 22, at 100 ºC, for 10 minutes, and left reposing for one hour before use.

Determination of total phenols and verification of antioxidant activity in samples of guava leaf meal and cooked extract

The determination of total phenols was performed by the Folin-Ciocalteu spectrophotometric method (Magalhaes et al 2006); and, to verify the total antioxidant activity, we used the FRAP techniques (Ferric Reducing Antioxidant Power) (Benzie and Strain 1996) and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) (Re et al 1999).

Animals, housing, experimental design and diets

The animals, housing, experimental design, and diets for this experiment followed the recommendations of Flores et al (2015). The study lasted 28 days and use a total of 60 piglets, neutered males and females in the same 1:1 ratio, of commercial crossing (Landrace x Duroc x Pietrain), weaned at 30 d, with an initial average live weight of 9.18 ± 1.14 kg, the pigs had no access to pre-weaning feed during lactation. At the time of weaning, the pigs were assigned according to their initial body weight to one of the three dietary treatments and housed in individual pens of 0.80 x 1.0 m (0.80 m²) built on a plastic floor with walls 1.40 m high and equipped with heating lamps to regulate the temperature (28 °C), a one opening feeding hopper and a nipple waterer. All animals had ad libitum access to feed and water throughout the experimental period. The cubicles were cleaned every day to ensure a sanitary environment for the piglets.

The experiment was carried out under a completely randomized design with 20 pigs for each treatment. The treatments consisted of a basal control diet without growth promoter antibiotic (CTL); GULM (CTL+ inclusion of 1 g of guava leaf meal/100 g DM, ensuring a supply of 45.61 mg gallic acid/g meal); and GUCE (CTL + inclusion of 10 mL of cooked guava leaf extract/100 g of DM, guaranteeing a supply of 4.9 mg gallic acid/10 mL of extract). The CTL was formulated according to the recommendations of Rostagno et al (2017) (Table 1). Before offering the feed, a homogeneous mixture was made with the meal or extract according to the treatment.

Productive indicators

The piglets were weighed individually every 7 days, while the leftover feed from each feeder was weighed daily before offering the new feed to the animals. The average daily feed intake (ADFI), average daily gain (ADG), feed conversion (FC) and body weight (BW) were verified weekly.

Fecal consistency and incidence of diarrhea

The pens were cleaned every day to guarantee a sanitary environment for the piglets as well as to better control fecal consistency. The feces from each pen were visually examined every morning at 07:00 h before the daily cleaning, for 28 d after weaning, and the evaluations were always performed by the same trained person. Fecal consistency was scored using a 3-point scale modified from the one described by Marquardt et al (1999), where 0 = solid (firm feces), 1 = semi-liquid (soft feces) and 2 = liquid (diarrhea).

Statistical analysis

The experiment was carried out using a completely randomized design with three treatments, 20 repetitions and an animal that constitutes the experimental unit. The assumptions of the normality and variance homogeneity model were examined using the Shapiro-Wilk and Levene tests, respectively. The results were statistically analyzed by a one-way ANOVA, with dietary treatment as the main factor. Responses were considered significantly different when p<0.05 and the means were compared using Tukey's 5 % test. The results are presented as mean ± standard error (SEM). All statistical analyses were performed using the R software (R Core Team 2019). For the statistical analysis of the diarrhea incidence values, the 5 % Chi-square test was used.

Results

The meal has a high content in total phenols (4561.29 mg gallic acid/100 g DM) and considerable antioxidant activity by the FRAP method (158.64 mg TROLOX/100 g DM), and a lower activity by ABTS (12.03 mg TROLOX/100 g DM). The cooked extract has a moderate content in total phenols (48.96 mg gallic acid/100 mL of extract) and antioxidant activity by the FRAP technique (22.82 mg TROLOX/100 mL of extract), and less activity with ABTS (12.24 mg TROLOX/100 mL of extract) (Table 2).

In this study there was no animal mortality during the experiment. Table 3 shows the effects of the inclusion of guava foliage meal or cooked extract on the ADFI, ADG, FC and BW of the piglets after weaning. In all the evaluated periods (30-37 d; 37-44 d; 44-51 d; and 30-58 d) the animals supplemented with meal and extract presented the best BW (p <0.0001), ADG (p<0.0001), ADFI (p<0.0001) and FC (p<0.0001), respectively.

In the period of 30 to 37 days it is noted that meal and extract treatments are on average 9.81% higher BW at 37d than the control group. The same behavior can be observed for ADG, where meal and extract treatments are, on average, 68.81% higher than the control group. On the other hand, for ADFI we observed that extract treatment, on average, was 21.13% higher than control treatment and 2.09% higher than meal treatment. The FC of the meal and extract treatments were on average 68.02% better than that obtained in the control group.

In the period of 37 to 44 days, it is possible to verify that the meal and extract treatments are, on average, 16.16 % better than the control group. The same behavior can be observed for ADFI, where meal and extract treatments are, on average, 20.37% better than the control. However, for ADG observed that extract treatment, on average, was 77.70% better than control treatment and 4.37 % better than meal treatment.

From 44 to 51 days, the meal and extract are better compared to the control, so that, on average, for weight at 51 days, 21.51% higher. ADG and ADFI, on average, were higher than the control, 20.82% and 3.28%, respectively. For FC and GP variables treatments were on average 49.67% higher than the control group. In the total period, meal and extract treatments are better compared to the control group for all performance variables.

In this study there was no animal mortality during the experiment. In the 1st week after weaning (30-37d), the control treatment had the highest diarrhea occurrence in relation to the treatments with guava leaf meal and cooked extract. In the 2nd week (37-44d); 3rd week (44-51d); and 4th week (51-58d) there was no occurrence of diarrhea for the treatments with guava leaf meal and cooked extract, differing significantly from the control treatment (Table 4).

Discussion

Table 2 indicates despite the fact that the meal is high in total phenols, its antioxidant activity is 28 times less with FRAP and 380 times with ABTS; this is because not all the total phenolic compounds in the meal have antioxidant activity (Ramírez et al 2016). Nonetheless, the antioxidant activity was relatively higher in the cooked extract with respect to the total phenol content, 2 times less in FRAP and 4 times in ABTS. A similar effect was reported by Caicedo et al (2019) who correlated the total phenols vs. the antioxidant activity of the foliage of Piper auritum in meal and cooked extract; obtaining a negative correlation (Y = -0.0767x + 0.7199; R² = 0.98) for the meal, and positive (Y = 0.0036x + 0.1199; R² = 0.98) for the cooked extract. When using the cooking technique, secondary metabolites can be eliminated in greater quantity, compared to controlled drying (Dos Reis et al 2015; Paciulli et al 2018), however, there are antioxidants that support boiling and steaming without losing their effect on health (Silva et al 2018; Chena et al 2019).

The use of guava foliage in the form of meal and cooked extract as a supplement for animal feed is scarce. However, the best results in terms of BW, ADG, ADFI and FC for the animals supplemented with meal and cooked guava extract during the evaluation periods (30-37 d; 37-44 d; 44-51 d; and 30-58 d, Table 3) could be explained by the antioxidant effect of the meal and the cooked extract of the guava foliage (Table 2). In this sense, Más Toro et al (2016) carried out a study with a dietary supplementation of 1% of Anacardium occidentale and Psidium guajava leaf meal and achieved an increase in BW, ADG, ADFI and FC in post-weaning pigs. Similarly, Aroche-Ginarte et al (2017) performed dietary supplementation with mixed meal of A. occidentale, M. oleifera, M. citrifolia and P. guajava at levels of 0.5; 1 and 1.5% and recommended dietary supplementation of 1 % meal as it is the most effective on increases in BW, ADG, ADFI and FC in post-weaning pigs. Likewise, Caicedo et al (2019) using meal and cooked extract of Piper auritum Kunth foliage obtained improvements on the BW, ADG, ADFI and FC for the treated groups, results that are consistent in relation to those obtained in the present investigation.

The additives from the plants influence organoleptic characteristics such as smell, color and taste of the food, inducing a higher ADFI of the animals which directly influences the ADG, FC and BW (Patience et al 2015; Chiari-Andréo et al 2017; Collins et al 2017; Caicedo et al 2019). However, the animals supplemented with guava leaf meal and cooked extract had lower diarrhea incidence compared to the control group. Incidence was only verified in the period (30-37d) with 3.57 % for both treatments, respectively (Table 4). In this regard, the incidence of the control group piglets' diarrhea was probably caused by coliforms and may have directly affected the lowest ADFI, ADG, FC and BW of the piglets (Dong and Pluske 2007; Sørensen et al 2009; Luppi et al 2016).

Table 4 reflects the occurrence of diarrhea in pigs post-weaning during the 28 d of research. Post-weaning enteric colibacillosis is an important cause that can even lead to the death of weaned pigs and often occurs within the first 2 to 3 weeks post-weaning (Luppi 2017).

The appearance of post-weaning diarrhea between (30 and 37 d) for the 3 treatments: control (59.29 %), meal (3.57 %) and cooked extract (3.57 %) shows that the piglets during post-weaning transition are vulnerable to nutritional, physiological and psychological stressors; this leads to an microbial intestinal imbalance caused by an increase in bacteria of the Enterobacteriaceae family, involving the vast majority of coliform bacteria (Gresse et al 2017; Patil et al 2019). The impact of these factors in establishing the intestinal microbiota of the piglets by weaning includes effects on the diversity, structure and intestinal microbial sequence of the piglet (Guevarra et al 2019). Almeida et al (2020) state that the highest amount of coliforms (p<0.01) found in the ileum on day 7 compared to day 28 after weaning probably reflects the interruption of the intestinal microbiota during the early period after weaning, perhaps as a result of changing from a milk-based to a vegetable-based diet. Although it is evident that any enteric disease or condition may arise as a result of Gastrointestinal Tract (GIT) disturbances, in fact, intestinal health represents the result of GIT in response to its ability to respond and adapt to the changes and challenges that the animal faces (Almeida et al 2017; Pluske et al 2018).

On the other hand, in the 2nd week (37-44d); 3rd week (44-51d); and 4th week (51-58d) the animals of the meal and extract treatments did not show diarrhea; however, in the control treatment there was incidence of diarrhea (64.29; 35.71 and 8.57 %, respectively). This is probably due to the fact that the basal diet did not include growth-promoting antibiotic and that the animals of the control treatment were exposed to the proliferation of coliforms. However, in the treatments with meal and extract, it is probable that the antioxidant metabolites of the guava foliage exerted a beneficial effect on the intestinal health of the animals.

This fact was confirmed by previous studies by Más Toro et al (2016); Aroche-Ginarte et al (2017), and Caicedo et al (2019), who recommended using meals and plant extracts from P. guajava,A. occidentale, M. oleífera, M. citrifolia and Piper auritum to reduce the incidence of diarrhea in post-weaning piglets. Also, in studies conducted in humans, the literature reports that the use of P. guajava foliage extracts can combat diarrhea and dysentery without requiring antibiotics (Chiari-Andreo et al 2017; Naseer et al 2018).

Conclusions

• The use of meal and cooked extract of guava foliage reduces the use of growth-promoting antibiotics in feed for post-weaning piglets.
• Dietary supplementation with meal (1 g/100 g DM) and cooked extract (10 mL/100 g DM) of guava foliage improved ADFI, ADG, FC, body weight, and controlled the incidence of diarrhea in post-weaning piglets.

Conflict of interest

The authors declare that there is no conflict of interest associated with this publication.

Acknowledgements

This research was supported by the Universidad Estatal Amazónica – through the financing of the Biopreprados Funcionales Amazónicos en Beneficio de la Salud project, code (AGR-003-2015) – as well as by the technical staff of Rancho Santa Rita.

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