Use of Humic Substances from Vermicompost in Poultry

*Jesús A. Maguey-González, Sergio Gómez-Rosales, María de Lourdes Ángeles and Guillermo Téllez-Isaías*

## **Abstract**

In recent years, there has been a surge in antibiotic resistance in both humans and animals, as well as increased public concern over medication residues in animal products. As a result, the use of antibiotics as growth promoters in chicken has been banned in the European Union, and consumer pressure is likely to lead to their removal in other countries. More recently, the United States of America adopted the same restriction in 2017. Different alternatives to antibiotics have been proposed as a measure to eliminate pathogens or to improve growth and feed conversion in poultry, such as probiotics, enzymes, bacteriophages and antimicrobial peptides, herbal compounds and organic acids. These substances exert their effects on the gastrointestinal biota and digestion processes, directly or indirectly. Humic substances (HS) in animal applications have shown improved live-weight, growth rates and feed intakes by improving immune functions and gut health. In poultry nutrition as an alternative to growth-promoting has been proven with promising results on the growth and health of birds. Additional research suggests that HS can increase gut integrity and performance when combined with good nutrition, management, and biosecurity policies. Therefore, recent results of HS extracted from vermicompost in poultry will be described in this chapter.

**Keywords:** humic substances, vermicompost, gut health, poultry

## **1. Introduction**

During the previous decade, the poultry business has been the most active and fastest-growing sector in the worldwide market for the production of meat directed to human consumption. The world's population is forecasted to double by 2050, and agricultural production is expected to double as well [1]. The supply of protein of animal origin in countries like Mexico is insufficient to fulfill the population's demand; nevertheless, the OECD-FAO Agricultural Outlook 2016–2025 projects that chicken meat output will expand at a pace of 2.9 per cent per year on average between average 2016 and 2025. As a result, national output and consumption will continue to rise [2].

Increased livestock output must be done with the most efficient use of resources as possible, without compromising environmental integrity or animal or human health. For achieving this, one of the most significant obstacles is the restriction to

the use of growth-promoting antibiotics (GPA) as feed additives due to concerns about pathogenic bacteria developing antibiotic resistance, which represents a threat to human and animal health [3]. As a result, non-pharmacological growth promoters have been studied for decades in an attempt to replace or alternate the application of conventional promoters [4].

Humic substances (HS) are one of the alternative additives that has been studied for several years as a means to promote animal health. HS has been employed in agriculture for many years to enrich the soils, but because of their many qualities, they have been recently resurrected in environmental sciences and the human biomedical sector [5, 6]. In veterinary medicine, HS have been used as antidiarrheal, analgesic, immunostimulatory, and antibacterial agents [7]. When HS has been combined with the correct ration, husbandry, and biosecurity procedures, has been shown to enhance intestinal integrity and performance in chickens [8–11].

The majority of HS used in these studies were commercial products generated from mineral sources such as lignites and leonardite Vermicompost, from which compost and leachates are produced, is one of the first HS sources employed by Gomez and Angeles [11, 12], they have been reported to have promising advantages in poultry production as growth promoters.

## **2. Humic substances**

HS are organic macromolecules that play an important role in biochemistry; they are a fraction of soil organic matter and have the highest density in soil and composts. They are produced by the biodegradation of organic matter, which involves physical, chemical, and microbiological processes [5], in which eukaryotes (worms and fungi) and prokaryotes (aerobic bacteria) further decompose organic matter [11]. HS are a natural component of streams, lakes, and oceans, containing the majority of the nutrients in the soil, accounting for approximately 80% of the carbon in soils and 60% of the dissolved carbon in the aquatic environment [13].

Based on their solubility, HS are classified as humic acids (HA), fulvic acids (FA) and humin. It has been stated that the isolating and characterizing of the organic components of soils is challenging due to the breakdown products of organic matter associated with other minerals [5]. For the first time, Senn and Kingman [14] characterized the molecular structure of HS, determining that the oxidized sites provide the molecule with a negative charge, allowing it to attach to mineral ions.

HA molecules have a wide range of weight and size, ranging from hundreds to thousands of atomic mass units, and are constituted of aromatic units, units linked by oxygen and nitrogen, functional groups mostly linked to carboxylic acids, phenols, and hydroxyl alcohol, ketone and quinone groups [15]. These chemical properties provide HA with the capacity to serve as a surfactant, binding to a variety of substances and generating hydrophobic and hydrophilic chemical complexes [16]. HS are excellent at transporting and binding organic and inorganic agents in the environment because of this function, which is combined with their colloidal characteristics [17].

The great electron transport capability of HS in oxidation–reduction processes is their principal attraction [6, 18]. HS also can develop bindings with ions such as Mg2+, Ca2+, Fe2+, and Fe3+ due to the presence of carboxylic groups and phenolates. Creating chelate compounds with one or more of these ions and controlling metal ion bioavailability [19].

### **2.1 Use of humic substances in animals**

HS have been used as an antidiarrheal, analgesic, immunostimulatory and antimicrobial agent in veterinary practices in Europe [7]. Furthermore, they have been shown to have a strong affinity for binding to heavy metals, mutagens, minerals, bacteria, and aflatoxins [20]. HS can be used orally in horses, ruminants, pigs and poultry for the treatment of diarrhoeas, dyspepsia and acute intoxications following the recommendations of the Committee for Veterinary Medicinal Products of the European Agency for the Evaluation of Medicinal Products [7].

*In vitro* studies of the antioxidant properties of HS in doses of 0.1 per cent in rat liver, the organ with the highest metabolic function and responsible for the metabolism of pharmacological compounds, found that HS aids in the elimination of free radicals and superoxide radicals, as well as maintaining the balance in the oxidation– reduction reactions of the mitochondria [21]. In rats with 2/3 of their liver removed, the administration of HS resulted in liver regeneration [18].

When rats were administered HS in their drinking water, Yasar [22] discovered a considerable rise in weight, as well as an increase in epithelial surface, intestinal villi length, and crypt depth.

#### **2.2 Use of humic substances in poultry**

The effects of different concentrations of HS in the diet on live weight, feed consumption, carcass characteristics, and gastrointestinal characteristics in broilers have been extensively studied [8, 23–26]. The addition of HS in the drinking water or feed improves most of the productive parameters, such as daily weight gain, in addition to enhancing the carcass yield of broilers.

According to Jin [27], the feed conversion does not change at 21 days but improves at 42 days, implying that the increase in weight gain and feed conversion efficiency might be due to the stimulating impact of HS in the digestive system and the nutrient utilization in metabolic processes. Adding HS to laying and broiler chickens can boost profitability by improving the production performance, reducing mortality, lowering feed conversion, and increasing egg output [28].

HS have been used to reduce mycotoxicosis in chicken due to its adsorbent capability [29–31]. Several studies have found that HS reduces ammonia emissions to the environment [9, 32, 33]. Similarly, in farms with a high density of chicken population, HS have been found to have a considerable anti-stress impact, reducing the negative effects of chronic stress in laying hens in production [34].

The mechanism through which HS affects poultry performance remains unclear. According to Shermer [35], HS might affect poultry performance by modifying the microbiota in the gastrointestinal tract, particularly in *Escherichia coli* populations, by altering the pH and promoting a greater activity of intestinal enzymes and feed digestibility. Several trace elements in HS can act as co-factors, increasing the activity of numerous enzymes involved in digestion and metabolic processes [36].

## **3. Humic substances derived from vermicompost**

Vermicomposting is a bio-oxidative process that involves the breakdown of organic materials by litter-dwelling detritivorous earthworm species. Earthworms are important because they fragment organic materials and increase surface area, but microscopic species in the earthworm stomach and their castings are responsible for the actual decomposition [37, 38]. These gut and cast-related processes are considered to have a significant impact on the properties of vermicompost. *Eisenia fetida*, *Eisenia andrei*, and *Dendrobaena veneta* are the earthworm species used in vermicomposting. *E. fetida* is one of the most frequently utilized earthworm species due to its high rate of organic matter digestion, tolerance to environmental conditions, fast reproductive rate and short life cycle, and tolerance to handling [39].

Two phases can be distinguished in vermicomposting: 1) an active phase in which earthworms process the waste through physical comminution, ingestion, and microbial decomposition, and 2) a maturation-like phase in which earthworms move to fresher layers of undigested waste and microbes provide additional decomposition [40].

Bacteria and fungi are the two primary kinds of microorganisms involved in the composting process. Bacteria degrade sugars and other easily accessible organic materials faster than fungus. They are, therefore, crucial in the early phases of composting, when the feedstocks contain large quantities of carbohydrates. More complex compounds, such as hemicellulose, starch, and even lignin, can be degraded by actinomycetes. They are more common in the later stages of composting, after the majority of easily degradable substrates have been used [41].

The fresh organic matter (animal manure, food wastes, green wastes, agricultural leftovers, etc.) is transformed into more stable humus-like substances, nutrients are recycled, and energy is created throughout the composting process [42]. The process should be aerobic, with a portion of it taking place under thermophilic circumstances. It minimizes phytotoxicity, removes pathogens, and stabilizes the material in terms of nitrogen and oxygen demand, avoiding N immobilization by soil biota, which competes with the plant for limited nitrogen resources [43].

#### **3.1 Establishment of vermicompost**

The establishment of our vermicompost took place in the greenhouse area, which has concrete and stone walls and a white polyethylene roof (**Figure 1**). The temperature inside the greenhouse is maintained throughout the year, with maximum temperatures of 35°C and minimum temperatures of 15°C, as well as relative humidity of 70–80%. *Eisenia Foetida* is used in vermicompost; and it is composed of 50% pastures from the area/50% sheep manure; with an internal temperature of 15–25°C, a pH of 6.5 to 7.5 and relative humidity of 80–90%.

Vermicompost is made by enclosing a 2 × 5 m rectangular space with bricks and covering it with polypropylene (**Figure 2**). To reduce the bacterial content, the organic material and manure are first pre-composted for a month. The components are then combined and placed in the compost's lower side; worms are then sewn on top, and the compost is watered. Finally, a part of the organic material is coated over the compost to protect it from light rays, covered with black polyethylene (**Figure 3**).

The compost is moistened once a week, vegetable matter is added once a week, and all parameters are checked regularly for upkeep. To collect the compost, the vermicompost must grow for four months. The worm harvest is conducted first, and then the compost is sifted to eliminate any unprocessed organic and inorganic elements. The compost was preserved after being oven-dried for 24 hours at 60°C.

**Figure 1.** *Greenhouse area.*

**Figure 2.** *Delimitation of the area with bricks and covered with polypropylene.*

## **3.2 Humic substances extraction/isolation**

HA, FA, and humin are the three main types of humic compounds found in soils and sediments [5, 13]. A strongly basic aqueous solution of sodium hydroxide or potassium hydroxide is used to extract HS from humus and other solid phases. The HS precipitate in this solution when the pH is adjusted to 1 with hydrochloric or sulfuric acid, leaving the FA in the solution (upper section) [19, 44]. This is the most important distinction: FA are insoluble in alkaline media, while HA are insoluble in acid media, and humin are insoluble at any pH (**Figure 4**).

#### **Figure 3.**

*Vermicompost is covered with organic matter and polyethylene.*

**Figure 4.** *Fractions of humic substances.*

In addition to alkaline solvents, chelating agents, organic solvents, and aqueous saline solutions have been proposed for the extraction of HS. Alkaline solvents are the most efficient and commonly utilized of them [44]. The extraction of HA with NaOH is a standard method for isolating HA, with an extraction efficiency of more than 80% of samples from soils (**Table 1**).

The isolation and extraction of HS from the earthworm compost were carried out using an alkaline extraction process. Sodium hydroxide (0.1 M NaOH) was used in a ratio of 5:1 parts of compost (mL/g); it was left to rest for 24 h at room temperature, and then filtered through a 125 μm mesh; sulfuric acid was added (H2SO4, 10%),


#### **Table 1.**

*Agents used for the extraction of humic substances.*


#### **Table 2.**

*Estimated chemical properties of the humic and fulvic acid molecules, extracted from vermicompost.*

#### **Figure 5.**

*The flat structure of a humic acid molecule with aromaticity (a) and flat structure of a fulvic acid molecule with aromaticity (b).*

rectifying a pH of 2. The solids and liquids were separated by decantation. The precipitate (HA) was washed two times with distilled water to remove sulfuric acid residues and between each washing it was centrifuged for 20 min at 5000 rpm. Then, to remove the remnants of sulfuric acid, in a rotary evaporator, the sample was dried at 60°C until it had a gel consistency. Finally, it was dried in an oven at 60°C. The result was a yellowish-brown powder with a pH of 10.

For the identification of functional groups, the extracted HS were analyzed using an infrared spectrophotometer with Fourier transformation with attenuated total reflectance (FTIR-ATR); the elemental analysis was carried out using energy dispersive X-Ray spectroscopy (EDS); and the crystal types were detected with X-ray diffraction (XRD). These results were used for the calculation of aromaticity and were published in a previous paper [45]. Additionally, the chemical properties (**Table 2**) and the flat structures of the HS molecules with aromaticity (**Figure 5**) using the chemistry software ACD Lab v.12 (Advanced Chemistry Development, Toronto, Canada) were estimated [46].

## **4. Use of humic substances from vermicompost in poultry**

Various *in vitro* and *in vivo* models for evaluating the behavior of different chemicals in chicken supplements have recently been established in our lab. The *in vitro* digestion model replicates broiler body temperature, peristaltic motions, enzymatic and pH conditions in each simulated compartment (crop, proventriculus, and small intestine) [47, 48]. In addition, *in vivo* models of intestinal inflammation in birds have been employed. Non-starch polysaccharide-rich diets [49, 50]; dexamethasone [51]; dextran sodium sulfate (DSS) [52, 53]; and 24 hours of feed restriction [54, 55]. With the help of these research models, we have been able to elucidate some effects of HA.

Firstly, two experiments were conducted to evaluate the effects of HA on recovery of *Salmonella* Enteritidis (SE) [56]; there were no effects of HA on SE recovery in an *in vitro* digestive system, or in Salmonella intestinal colonization, bacterial numbers in ceca, intestinal IgA, or serum FITC-d in neonatal broiler chicks.


*a Data expressed as Mean ± SE.*

*b Intestinal viscosities evaluated in Log10 (in centipoise, cP = 1/100 dyne sec/cm2 ), n = 5 chickens/group.*

*c Serum (FITC-d) was evaluated in 20 chickens/group.*

*d Liver bacterial translocation was evaluated in 12 chickens/group.*

*e Data expressed as positive/total chickens (%).*

*f,gSuperscripts within columns indicate significant difference at P < 0.05.\* P < 0.001.*

#### **Table 3.**

*Evaluation of intestinal viscosity, serum FITC-d, and bacterial liver translocation in chickens consuming a corn-based diet with or without the inclusion of 0.2% of humic acids following 24 hours of feed restriction (FR) in broiler chickensa .*

#### *Use of Humic Substances from Vermicompost in Poultry DOI: http://dx.doi.org/10.5772/intechopen.102939*

Additionally, a second study was carried out [57], to evaluate direct or indirect impacts of HA on intestinal integrity because of their physical and chemical features. Using a 24-hour feed restriction model, the aim was to investigate how HA affected intestinal viscosity, leaky gut, and ammonia excretion in broiler chicks. The experimental group was given 0.2% HA had increased intestinal viscosity and showed lower levels of FITC-d, bacterial liver translocation, and ammonia in the excreta. It was confirming its advantages, enhancing the viscosity and the integrity of the intestine (**Table 3**).

The previous researches were the first to address the effects of HA from vermicompost on bacterial challenges with SE both *in vitro* and *in vivo* in chicks; furthermore, the mechanism of action of HA on the maintenance of intestinal integrity in poultry was demonstrated for the first time. In line with this finding, Mudronová [58] reported that HS positively regulates MUC-2 gene expression.

In further research in broilers fed an extract of HS from vermicompost, increased carcass yield and lactic acid bacteria (LAB) and reduced coccidian oocysts excretion were observed; but increased *Clostridium perfringens* (CP) counts were also seen compared to broilers fed diets supplemented with GPA (**Figure 6**) [45].

In a recent report, broilers kept in floor pens from 1 to 42 days of age and fed increasing levels of HS from vermicompost, showed lower feed intake (FI) and overall mortality, besides, a better feed conversion rate (FCR) compared to negative control birds not supplemented with HS and positive control birds added with antibiotics (**Table 4**) [59]. The greatest benefits of adding HS were observed in the last period of the trial, from 29 to 42; these findings closely resemble the observations in previous research. For the authors, it is unknown whether improved FCR from 29 to 42 days was dependent on, or independent of, the addition of HS from 1 to 14 and 15–28 days. This topic deserves further clarification in future research.

Although, HS have been proven to have prospective benefits in chicken production as growth promoters, data on their antimicrobial properties are inconsistent. Using an *in vitro* chicken digestive system, it was essential to examine the effect of HA isolated from vermicompost on the recovery of *Salmonella Enteritidis* (SE), *E. coli* (EC), *C. perfringens* (CP), *Bacillus subtilis* (BS), and *Lactobacillus salivarius* (LS). In general,

#### **Figure 6.**

*Counts of lactic acid bacteria (LAB) and Clostridium perfringens (CP), and the number of coccidian oocysts excreted in broilers fed with humic substances.*


*a SEM = estandard error of the mean.*

*b–dMeans with a different superscript in the same row differ significantly (P < 0.05).*

#### **Table 4.**

*Growth performance of broilers from 1 to 42 days of age fed increasing levels of humic substances extracted from vermicompost.*


*\* The initial inoculum of SE, EC and CP in the feed was 108 CFU/g.*

*\*\*Data are expressed as log10 CFU. \*\*\*Standard error of the mean.*

*a–fValues in columns with different letters differ significantly (P ≤ 0.0001).*

#### **Table 5.**

*Effect of humic acids on the counts of Salmonella Enteritidis, Escherichia coli and Clostridium perfringens\* under an in vitro poultry digestive model\*\*.*

the number of microorganisms counted increased in the final simulation, remarkably when the HA inclusion concentration increased (**Tables 5** and **6**).

HS may be utilized as substrates by bacteria since they are organic sources of carbon, nitrogen, phosphorus, and other nutrients. In this way, they can be used as prebiotics to enhance nutrient digestion.

As previously stated, HS have the potential to chelate minerals; when added to drinking water at concentrations of 345.0, 322.5 and 347.8 g/L, improvements in bone mineralization in broilers at 21 (**Table 7**) and 42d (**Table 8**) of age have been observed [46].

Subsequently, 28-year-old broilers were challenged to a radical change in diet, in addition to inducing immune stress when vaccinated against Newcastle, Infectious bronchitis and Salmonella, with the intention of causing damage to the intestinal mucosa. The results of this research [60] show that HS does not prevent intestinal mucosal atrophy but increase the number of goblet cells, and probably the mucus layer, compared to chickens that received the treatment without GPA.

On the other hand, when compared to a commercial zeolite, the adsorption capacity of HA against AFB1 when added to the feed in a 1% concentration exhibited the maximum effectivity of capture (**Figure 7**).

*Use of Humic Substances from Vermicompost in Poultry DOI: http://dx.doi.org/10.5772/intechopen.102939*


*\* The initial inoculum of BS and LS in the feed was 108 CFU/g.*

*\*\*Data are expressed as log10 CFU. \*\*\*Standard error of the mean.*

*a−e Values in columns with different letters differ significantly (P ≤ 0.0001).*

#### **Table 6.**

*Effect of humic acids on the counts of Bacillus subtilis and Lactobacillus salivarius\* under an in vitro poultry digestive model\*\*.*


*a n = 12, using six tibias per replicate..*

*b SEM = standard error of the mean.*

*d,eMeans with a different superscript in the same row differ significantly (P < 0.05).*

*f,gMeans with a different superscript in the same row differ significantly (P < 0.01).*

#### **Table 7.**

*Dry matter, ashes, calcium and phosphorus content of the tibia of 21 days old broiler chickens added with increasing levels of humic substances in the drinking watera .*



*a n = 12, using six tibias per replicate.*

*b SEM = estandard error of the mean.*

*d,e Means with different superscript in the same row differ significantly (P < 0.05).*

*f,g Means with different superscript in the same row differ significantly (P < 0.01).*

#### **Table 8.**

*Dry matter, ashes, calcium and phosphorus content of the tibia of 42 days old broiler chickens added with increasing levels of humic substances in the drinking watera .*

#### **Figure 7.**

*Adsorption capability of humic acids (HA), humic acids purified (HAP) and zeolite (ZEO) against AFB1 using an in vitro poultry digestive model.*

Finally, the addition of HS extracted from vermicompost in piglet feed at weaning [61] improved the weight gain and feed efficiency during 1–42 days post-weaning. The final body weight at 42 days, weight gain and feed efficiency were higher with the inclusion level of 0.50% HS. Most of the productive responses showed an increasing linear pattern as the addition of HS increased.

## **5. Conclusions**

Although, the antibacterial activity of HS/HA is unclear, they might be regarded as viable alternatives to replace or alternate the use of antibiotics in poultry. As was documented, HS extracted from vermicompost improve performance, increase intestinal viscosity and intestinal integrity in poultry. These benefits have been attributed to the macro colloidal structure of HS, which ensures effective protection on gastric and intestinal mucous membranes, as well as the promotion of mucin synthesis in the gut. However, their applications depend on the source and the way they are extracted. HS stimulates the growth of microorganisms, suggesting that they can be used as prebiotics.

*Use of Humic Substances from Vermicompost in Poultry DOI: http://dx.doi.org/10.5772/intechopen.102939*

In addition, multiple doses were investigated, which might have contributed to the differences in response variables among our investigations. It can also be argued that whether in a more challenging environment, the effects of HS may be better. Finally, our working team is focusing on elucidating the mechanism by which HS exert their effects on the intestinal mucosa and the intestinal microbiota.

## **Acknowledgements**

This research was supported by the Arkansas Biosciences Institute under the project: Development of an avian model for evaluation early enteric microbial colonization on the gastrointestinal tract and immune function.

CONACYT provided funding to carry out several experiments described in this chapter through the project "Efficacy of an extract of humic substances as a growth promoter, reducing infectious diseases and increasing the profitable production of meat of birds and pork" (PDCPN 2017/4777).

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Jesús A. Maguey-González1 \*, Sergio Gómez-Rosales2 , María de Lourdes Ángeles2 and Guillermo Téllez-Isaías3

1 National Autonomous University of Mexico (UNAM), Postgraduate Unit, University City, Coyoacán, Mexico City, Mexico

2 National Center of Disciplinary Research in Animal Physiology and Genetics, National Institute of Research in Forestry, Agriculture and Livestock, Ajuchitlan, Queretaro, Mexico

3 Department of Poultry Science, University of Arkansas, Fayetteville, AR, USA

\*Address all correspondence to: magueyjesus@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] La CA, La AY, Todos NP. El Estado Mundial de la Pesca y la Acuicultura. ROMA: FAO; 2016

[2] Fideicomisos Instituidos en Relación con la Agricultura (FIRA). Panorama agroalimentario, avicultura carne; 2016

[3] Van Vuuren M. Antibiotic resistance with special reference to poultry production. Conference OIE. 2001:135-146

[4] Huyghebaert G, Ducatelle R, Van Immerseel F. An update on alternatives to antimicrobial growth promoters for broilers. The Veterinary Journal. 2011;**187**:182-188

[5] Peña-Méndez EM, Havel J, Patočka J. Humic substances-compounds of still unknown structure: Applications in agriculture, industry, environment, and biomedicine. Journal of Applied Biomedicine. 2005;**3**:13-24

[6] Aeschbacher M, Graf C, Schwarzenbach RP, Sander M. Antioxidant properties of humic substances. Environmental Scince Technology. 2012;**46**:4916-4925. DOI: 10.1021/ es300039h

[7] EMEA. Committee for Veterinary Medical Products. Humic Acids and their Sodium Salts. 1999. Available from: http:/www.emea.europa.eu/pdfs/vet/ mrls/055499en.pdf

[8] Karaoglu M, Macit M, Esenbuga N, Durdag H, Turgut L, Bilgin ÖC. Effect of supplemental humate at different levels on the growth performance, slaughter and carcass traits of broilers. International Journal of Poultry Science. 2004;**3**:406-410

[9] Ji F, McGlone JJ, Kim SW. Effects of dietary humic substances on pig growth performance, carcass characteristics, and ammonia emission. Journal of Animal Science. 2006;**84**:2482-2490

[10] Ipek H, Avci M, Iriadam M, Kaplan O, Denek N. Effects of humic acid on some hematological parameters, total antioxidant capacity and laying performance in Japanese quails. Arch Geflugelk. 2008;**72**:56-60

[11] Gomez-Rosales S, de Angeles ML. Addition of a worm leachate as source of humic substances in the drinking water of broiler chickens. Asian-Australas Journal of Animal Science. 2015;**28**: 215-222. DOI: 10.5713/ajas.14.0321

[12] Gómez-Rosales S, Angeles M, Maguey-González J. Productive responses and nitrogen balance in broilers fed with humic substances in the drinking water. Abanico Veterinario. 2021;**11**:e120. Epub 08 de noviembre de 2021. DOI: 10.21929/ abavet2021.30

[13] Lehmann J, Kleber M. The contentious nature of soil organic matter. Nature. 2015;**528**:60-68. DOI: 10.1038/ nature16069

[14] Senn TL, Kingman AR. A review of humus and humic acids. Research Series. 1973;**145**:1-5

[15] Saar RA, Weber JH. Complexation of cadmium (II) with water-and soilderived fulvic acids: Effect of pH and fulvic acid concentration. Canadian Journal of Chemistry. 1979;**57**:1263-1268

[16] Gaffney JS, Marley NA, Clark SB. Humic and fulvic acids and organic colloidal materials in the environment. In: Gaffney JS, Marley NA, Clark SB, editors. Humic and Fulvic Acids. Washington, DC: ACS Publications; 1996. pp. 2-16

*Use of Humic Substances from Vermicompost in Poultry DOI: http://dx.doi.org/10.5772/intechopen.102939*

[17] Piccolo A. The supramolecular structure of humic substances: A novel understanding of humus chemistry and implications in soil science. Advances in Agronomy. 2002;**75**:57-134. DOI: 10.1016/ S0065-2113(02)75003-7

[18] Maśliński C, Fogel WA, Andrzejewski W. The influence of Tołpa peat preparation (TPP) on rat liver regeneration. Acta poloniae pharmaceutica. 1993;**50**(4-5):413-416

[19] Stevenson FJ. Humus Chemistry: Genesis, Composition, Reactions. New York: John Wiley and Sons; 1982

[20] Islam KMS, Schuhmacher A, Gropp JM. Humic acid substances in animal agriculture. Pakistan Journal Nutrition. 2005;**4**:126-134

[21] Vašková J, Veliká B, Pilátová M, Kron I, Vaško L. Effects of humic acids *in vitro*. In Vitro Cellular and Developmental Biology—Animal. 2011;**47**:376-382. DOI: 10.1007/ s11626-011-9405-8

[22] Yasar S, Gokcimen A, Altuntas I, Yonden Z, Petekkaya E. Performance and ileal histomorphology of rats treated with humic acid preparations. Journal of Animal Physioly and Animal Nutrition. 2002;**86**:257-264

[23] Kocabagli N, Alp M, Acar N, Kahraman R. The effects of dietary Humate supplementation on broiler growth and carcass yield. Poultry Science. 2002;**81**:227-230

[24] Ceylan N, Ciftci I, Ilhan Z. The effects of some alternative feed additives for antibiotic growth promoters on the performance and gut microflora of broiler chicks. Turkish journal of veterinary and animal. Science. 2003;**27**:727-733

[25] Rath NC, Huff WE, Huff GR. Effects of humic acid on broiler chickens. Poultry Science. 2006;**85**:410-414

[26] Ozturk E, Ocak N, Coskun I, Turhan S, Erener G. Effects of humic substances supplementation provided through drinking water on performance, carcass traits and meat quality of broilers. Journal of Animal Physiology and Animal Nutrition. 2010;**94**(1):78-85

[27] Jin LZ, Ho YW, Abdullah N, Jalaludin S. Growth performance, intestinal microbial populations, and serum cholesterol of broilers fed diets containing Lactobacillus cultures. Poultry Science. 1998;**77**:1259-1265

[28] Yoruk MA, Gul M, Hayirli A, Macit M. The effects of supplementation of Humate and probiotic on egg production and quality parameters during the late laying period in hens. Poultry Science. 2004;**83**:84-88

[29] Van Rensburg CJ, Van Rensburg CE, Van Ryssen JB, Casey NH, Rottinghaus GE. *In vitro* and *in vivo* assessment of humic acid as an aflatoxin binder in broiler chickens. Poultry Science. 2006;**85**:1576-1583

[30] Gahhri H, AHbibian R, Fam MA. Effect of sodium bentonite, mannan oligosaccharide and humate on performance and serum biochemical parameters during aflatoxicosis in broiler chickens. Global Veterinaria. 2010;**5**:129-134

[31] Arafat RY, Khan SH, Saima. Evaluation of humic acid as an aflatoxin binder in broiler chickens. Annals of Animal Science. 2017;**17**:241-255. DOI: 10.1515/aoas-2016-0050

[32] Henderson PA. The growth of tropical fishes. In: Val AL, Vera MF, Randall DJ, editors. The Physiology of Tropical Fishes. Vol. 21. New York: Academic Press; 2005. pp. 85-100

[33] Zralý Z, Písaříková B, Trčková M, Navrátilová M. Effect of humic acids on lead accumulation in chicken organs and muscles. Acta Veterinaria Brno. 2008;**77**:439-445. DOI: 10.2754/ avb200877030439

[34] Cetin E, Guclu BK, Cetin N. Effect of dietary humate and organic acid supplementation on social stress induced by high stocking density in laying hens. Journal of Animal and Veterinary Advances. 2011;**10**:2402-2407. DOI: 10.3923/javaa.2011.2402.2407

[35] Shermer CL, Maciorowski KG, Bailey CA, Byers FM, Ricke S. Caecal metabolites and microbial populations in chickens consuming diets containing amined humate compound. Journal of Science Food and Agriculture. 1998;**77**:479-486

[36] Hayirli A, Esenbuga N, Macit M, Lacin E, Karaoglu M, Karaca H, et al. Nutrition practice to alleviate the adverse effects of stress on laying performance, metabolic profile, and egg quality in peak producing hens: I. The humate supplementation. Asian Australas. Journal of Animal Science. 2005;**18**:1310-1319

[37] Lazcano C, Gómez-Brandón M, Domínguez J. Comparison of the effectiveness of composting and vermicomposting for the biological stabilization of cattle manure. Chemosphere. 2008;**72**(7):1013-1019

[38] Diaz LF, De Bertoldi M, Bidlingmaier W. Microbiology of the composting process. In: Diaz LF, de Bertoldi M, Bidlingmaier W, editors. Compost Science and Technology. Elsevier; 2011. pp. 25-48

[39] Ryckeboer J, Mergaert J, Vaes K, Klammer S, De Clercq D, Coosemans J, et al. A survey of bacteria and fungi occurring during composting and self-heating processes. Annals of Microbiology. 2003;**53**(4): 349-410

[40] Domínguez J, Gómez-Brandón M. The influence of earthworms on nutrient dynamics during the process of vermicomposting. Waste Management & Research. 2015;**31**(8):859-868. Ryckeboer. 2003

[41] Raviv M. Production of high-quality composts for horticultural purposes—A mini-review. HortTechnology. 2005;**15**:52-57

[42] Bernal MP, Alburquerque JA, Moral R. Composting of animal manures and chemical criteria for compost maturity assessment. A review. Bioresource Technology. 2005;**100**(22):5444-5453

[43] Baglieri A, Ioppolo A, Negre M, Gennari M. A method for isolating soil organic matter after the extraction of humic and fulvic acids. Organic Geochemistry. 2007;**38**(1):140-150

[44] De Souza F, Bragança SR. Extraction and characterization of humic acid from coal for the application as dispersant of ceramic powders. Journal of Materials Research and Technology. 2018;**7**(3):254-260

[45] Dominguez-Negrete A, Gómez-Rosales S, de Angeles ML, López-Hernández LH, Reis-de Souza TC, López-Garc'ia Y, et al. Effect of the addition of humic substances as growth promoter in broiler chickens under two feeding regimens. Animals, Multidisciplinary Digital Publishing Institute. 2019;**9**(12):1101

*Use of Humic Substances from Vermicompost in Poultry DOI: http://dx.doi.org/10.5772/intechopen.102939*

[46] Lourdes-Angeles M, Gómez-Rosales S, López-García YR, Montayo-Franco A. Growth performance and tibia mineralization of broiler chickens supplemented with a liquid extract of humic substances. Brazilian. Journal of Poultry Science. 2021:1450 (In press)

[47] Annett CB, Viste JR, Chirino-Trejo M, Classen HL, Middleton DM, Simko E. Necrotic enteritis: Effect of barley, wheat and corn diets on proliferation of *Clostridium perfringens* type A. Avian Pathology. 2002;**31**:598-601

[48] Latorre JD, Hernandez-Velasco X, Kuttappan VA, Wolfenden RE, Vicente JL, Wolfenden AD, et al. Selection of *Bacillus* spp. for cellulase and xylanase production as direct-fed microbials to reduce digesta viscosity and *Clostridium perfringens* proliferation using an in vitro digestive model in different poultry diets. Frontiers in Veterinary Science. 2015;**2**(25). DOI: 10.3389/fvets.2015.00025

[49] Tellez G, Latorre JD, Kuttappan VA, Kogut MH, Wolfenden A, Hernandez-Velasco X, et al. Utilization of rye as energy source affects bacterial translocation, intestinal viscosity, microbiota composition, and bone mineralization in broiler chickens. Frontiers in Genetics. 2014;**5**:339. DOI: 10.3389/fgene.2014.00339

[50] Tellez G, Latorre JD, Kuttappan VA, Hargis BM, Hernandez-Velasco X. Rye affects bacterial translocation, intestinal viscosity, microbiota composition and bone mineralization in Turkey poults. PLoS One. 2015;**10**:e0122390. DOI: 10.1371/journal.pone.0122390

[51] Vicuña EA, Kuttappan VA, Galarza-Seeber R, Latorre JD, Faulkner OB, Hargis BM, et al. Effect of dexamethasone in feed on intestinal permeability, differential white blood cell counts, and immune organs in broiler chicks. Poultry Science. 2015;**94**:2075- 2080. DOI: 10.3382/ps/pev211

[52] Kuttappan VA, Berghman L, Vicuña E, Latorre JD, Menconi A, Wolchok J, et al. Poultry enteric inflammation model with dextran sodium sulfate mediated chemical induction and feed restriction in broilers. Poultry Science. 2015;**94**:1220-1226. DOI: 10.3382/ps/pev114

[53] Menconi A, Hernandez-Velasco X, Vicuña EA, Kuttappan VA, Faulkner OB, Tellez G, et al. Histopathological and morphometric changes induced by a dextran sodium sulfate (DSS) model in broilers. Poultry Science. 2015;**94**:906- 911. DOI: 10.3382/ps/pev054

[54] Kuttappan VA, Vicuña EA, Latorre JD, Wolfenden AD, Téllez GI, Hargis BM, et al. Evaluation of gastrointestinal leakage in multiple enteric inflammation models in chickens. Frontiers in Veterinary Sciemce. 2015;**2**:66. DOI: 10.3389/ fvets.2015.00066

[55] Vicuña EA, Kuttappan VA, Tellez G, Hernandez-Velasco X, Seeber-Galarza R, Latorre JD, et al. Dose titration of FITC-D for optimal measurement of enteric inflammation in broiler chicks. Poultry Science. 2015;**94**:1353-1359. DOI: 10.3382/ps/pev111

[56] Maguey-Gonzalez JA, Michel MA, Baxter MF, Solis-Cruz B, Hernandez-Patlan D, Merino-Guzman R, et al. Effects of humic acids on recovery of Salmonella enterica serovar Enteritidis. Annals of Animal Science. 2018;**18**(2):387-399. Sciendo

[57] Maguey-Gonzalez JA, Michel MA, Baxter MF, Tellez G Jr, Moore PA Jr, Solis-Cruz B, et al. Effect of humic

acids on intestinal viscosity, leaky gut and ammonia excretion in a 24 hr feed restriction model to induce intestinal permeability in broiler chickens. Animal Science Journal. 2018;**89**(7):1002-1010

[58] Mudroňová D, Karaffová V, Pešulová T, Koščová J, Maruščáková IC, Bartkovský M, et al. The effect of humic substances on gut microbiota and immune response of broilers. Food and Agricultural Immunology. 2020;**31**(1):137-149

[59] Dominguez-Negrete A, Gómez-Rosales S, de Angeles ML, López-Hernández LH, Reis-de Souza TCY, Latorre-Cárdenas JD, et al. Addition of different levels of humic substances extracted from worm compost in broiler feeds. Animals. 2021;**11**:3199. DOI: 10.3390/ani11113199

[60] López-García YR, Gómez Rosales S, Angeles ML. Efecto de la adición de sustancias húmicas sobre la histología y número de células caliciformes en la mucosa intestinal de pollos de engorda. In: LVI Reunión Nacional de investigación pecuaria (RNIP); 10-12 November 2021; CDMX, México: Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP); 2021. pp. 410-412

[61] Agredo-Pelechor JA. Adición de sustancias húmicas extraídas de lombricomposta como promotor del crecimiento en cerdos al destete. In: LVI Reunión Nacional de investigación pecuaria (RNIP); 10-12 November 2021; CDMX, México: Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP); 2021. pp. 413-415

## **Chapter 8**
