**3. Methodologies for goat studies**

#### **3.1. Evaluation of phenotypic parameters**

Various phenotypic characteristics are measured in goats following treatment with supplements or feeds in a study. Usually, body weight, body condition score and FAMACHA score are recorded periodically as a measure of effect on growth and health. Body weights are taken before morning feeding using a portable scale [8]. Body condition is scored on a scale of 1–5 by physical examination of the goat's body as described by Villaquiran et al. [29]. Blood samples collected aseptically are evaluated for packed cell volume (PCV) and white blood differential cell counts. PCV is widely used as an indicator trait for anemia. White blood differential counts are measured using the procedure described by Schalm et al. [30]. Fecal samples are collected directly from the rectum and evaluated for the number of parasite egg counts. More specifically, the number of strongyle eggs and coccidia oocytes is measured using the modified McMaster method [31]. The fecal eggs counted are multiplied by 50, and resulting total is expressed as eggs per gram (epg) of fecal sample per animal [14].

#### **3.2. Molecular techniques**

The molecular effects of immunomodulators have been evaluated in goats at the gene transcription and protein levels using different techniques including real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).

#### **3.3. Real-time PCR**

processes which represent a large series of regulatory proteins of the immunologic system. Many cytokines are referred to as interleukins, a name indicating that they are secreted by some leukocytes and act upon other leukocytes. Two general patterns of cytokine secretion by such cells have been described. In the Th1 response, cytokines initiate cell-mediated reactions defined as the activation of macrophages to combat infectious pathogens by releasing IL-1, IL-2, IL-8, and IL-12 to activate inflammation [21]. In the Th2 response, T-helper cells activate B-cells; interleukins IL-4, IL-5, IL-6, IL-10 and IL-13 are released to counter infectious agents caused by extracellular organisms [21]. Studies have shown that the release of cytokines is

The Wingless (Wnt) signaling pathway is a conserved pathway in mammals. It involves Wnts, which are secreted glycoproteins that are associated with the Wnt-1 and Wingless gene products of Drosophila [22]. Activation of Wnt signaling happens when Wnt ligands binds to Frizzled receptors together with other receptors lipoprotein receptor-related protein (LRP) 5 and 6 [23–25]. About 19 Wnt ligands and 10 Frizzle receptors have been identified in metazoan mammals. The receptor-ligand interaction leads to downstream signal regulation which is categorized into two: canonical (Wnt/β-catenin) and noncanonical pathways. The former is dependent on β-catenin, but the latter is not. The noncanonical pathway is further subdivided into the planar cell polarity and the Wnt/Ca2+ pathways. The Wnt signaling pathway function in cellular processes includes cell proliferation, cell differentiation, cell migration, cell polarity and cell fate determination and has recently been implicated in stem cell renewal [26]. Wnt signaling has also been associated with various biological processes including adipogenesis, myogenesis, embryogenesis and meat quality. In addition, Wnt signaling has been associated with innate immune and inflammation responses via a cross talk with the TLR and NF-κB pathways [27, 28]. Therefore, a defective or deregulated Wnt signaling has detrimental effect on developing embryo (birth defects) and also affects a number of

Various phenotypic characteristics are measured in goats following treatment with supplements or feeds in a study. Usually, body weight, body condition score and FAMACHA score are recorded periodically as a measure of effect on growth and health. Body weights are taken before morning feeding using a portable scale [8]. Body condition is scored on a scale of 1–5 by physical examination of the goat's body as described by Villaquiran et al. [29]. Blood samples collected aseptically are evaluated for packed cell volume (PCV) and white blood differential cell counts. PCV is widely used as an indicator trait for anemia. White blood differential counts are measured using the procedure described by Schalm et al. [30]. Fecal samples are collected directly from the rectum and evaluated for the number of parasite egg counts.

essential for host survival from infection and is also required for tissue repair.

**2.4. Wingless pathway**

6 Goat Science

pathological disease conditions.

**3. Methodologies for goat studies**

**3.1. Evaluation of phenotypic parameters**

Quantitative real-time PCR is used to measure messenger RNA (MRNA) levels [77]. For realtime PCR analysis, total RNA is isolated from whole blood cell pellets using Trizol method or QuickRNA MiniPrep Kit (Zymo Research) as per manufacturer's procedure. The concentration and purity of the RNA are checked on NanoDrop Spectrophotometer (ND-1000; Thermo Fisher). Mostly, a pure RNA typically yields a 260/280 ratio of ~2.0 and this is considered ideal. A 260/280 ratio below 2.0 suggests protein contamination [82]. In addition, the integrity of the RNA (RNA integrity number (RIN)) can be measured with a bioanalyzer, and a RIN <7.0 indicates a good RNA. Since RNA is not stable, it is converted into more stable complimentary DNA (cDNA) using cDNA conversion kits containing oligo (DT) and random primers, reverse transcriptase, and other needed reagents as specified in the manufacturer's manual. Real-time PCR is performed with reaction mixture comprising of cDNA template, primers and SYBR Green [78]. Housing-keeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), *β*-actin (*ACTB*), ribosomal protein L32 (RPL32), TATA sequence binding protein (TBP) and cyclophilin are used as internal controls for normalization of the RT-PCR data obtained [32]. The RT-PCR data are analyzed by calculating fold change in the expression of the specific genes tested using statistical approaches including the comparative CT (also known as 2−ΔΔ*<sup>C</sup>* T or Livak's method).

#### *3.3.1. Enzyme-linked immunosorbent assay*

Enzyme-linked immunosorbent assay (ELISA) is a molecular assay used for analytical detection and quantification of specific antigens or antibodies in a given sample. It uses the concept of an antigen binding to its specific antibody which enables detection of antigens such as proteins, peptides and antibodies [33]. With ELISA, goat serum or plasma is evaluated for the levels of immune response and inflammation biomarkers such as cytokines, prostaglandin and immunoglobulins. Cytokines measured in goat serum following dietary supplementation include TNFα, IL-1β, IL-8, GCSF, GMCSF, Rantes and IFNγ [7, 8, 34]. The levels of secreted prostaglandin E, an eicosanoid and also an inflammation mediator have also been measured in goat serum and plasma with ELISA [34–36].

#### **3.4. Effect of pathogen-associated molecular patterns**

Goats rely on pasture as their main source of feed. Studies have been done to elucidate the effects of different PAMPs, microbe-associated molecular pattern (MAMP) and plant polyphenol metabolite in animal feed on goat health. Pathogen-associated molecular pattern evaluated in goats includes the following: probiotics, mushroom, plant polyphenols, cowpea, lipopolysaccharide (LPS), peptidoglycan, nystatin and Sericea lespedeza (**Table 1**).

#### *3.4.1. Probiotics*

Probiotics has been studied and considered as health beneficial microorganism which plays a role in maintaining homeostasis. Previous studies have shown the use of probiotics to modulate gastrointestinal health. Liong [37] reported the resistance to infectious diseases in the gastrointestinal tract as a result of probiotics. Probiotics as a supplement in animal feed has shown to have a beneficial effect on milk yield, fat and protein content [38]. Ekwemalor et al. [39] reported the release of proinflammatory cytokines in goats orally drenched with probiotics (*Coriolus versicolor* [CV]). Previous study conducted by our research team looked at the molecular impact of probiotic administration on physical health parameters and activation of genes involved in homeostasis and immunity in goat blood. We reported that genes associated with innate and adaptive immunity were modulated as a result of probiotics treatment. Genes that were expressed are associated with the host response to bacteria, virus, T-cell activation, cytokines and inflammatory response. **Table 2** shows genes modulated as a result of probiotic modulation.


Researchers have reported effects of probiotics in goats of which most effects have been attributed to an increase in the innate immune system and others in the acquired immune response. Leeber et al. [40] reported that probiotics have the properties to modulate host immune system through different signaling pathways of innate immune cells. The innate immune system functions by initiating a response to microorganisms or their components via pattern recognition receptors such as nucleotide-binding oligomerization domain-like receptors or TLR [41]. Previous studies conducted by Worku and Morris [42] and Worku et al. [7] have shown the

**Category Genes Reference**

TLR7, TLR8, TLR9

CXCL8, TNF

TLR6

**Table 2.** List of genes associated with innate and adaptive immunity.

Cytokines CCL2, CCL5, CSF2, CXCL10, IFNA1, IFNB1, IL18, 1L1A, IL1B, IL2,

Innate immunity genes APCS, C3, CASP1, CD14, CD4, CD40, CD40LG, CD8A, CRP, HLA-A,

Th17 markers CCR6, IL17A, RORC, STAT3 [8] T-cell activation CD80, CD86, ICAM1, IFNG, IL23A, 1 L6, SLC11A1 [8] Treg markers CCR4, CCR8, FOXP3, IL10 [8] Adaptive immunity genes CD40, CD40LG, CD8A, CRP, FASLG, HLA-A, IFNARI, IL1B, IL1R1, IRF3,

Inflammatory response APCS, C3, CCL5, CRP, FOXP3, IL1A, IL1B, IL4, IL6, MBL2, STAT3, TNF [8]

IL23A, IL6, NLRP3, TICAM1, TLR3, TLR7, TLR8, TYK2

TLR1, TLR3, TLR4, TLR6, TLR9, TNF

DDX58, NLRP3, NOD1, NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,

HLA-E, IL1R1, IRAK1, IRF3, IRF7, ITGAM, LY96, LTZ, MAPK1, MAPK8, MBL2, MPO, MX1, MYD88, NFKB1, NFKB1A, STAT1, TICAM1, TRAF6

CCR5, CD80, CXCR3, IFNG, IL18, IL23A, SLC11A1, STAT4, TBX21, TLR4,

IRF7, ITGAM, JAK2, MAPK8, MBL2, MX1, NFKB1, RAG1, STAT1

IFNB1, IFNG, IL23A, IL6, LYZ, MBL2, MYD88, NOD1, NOD2, SLC11A1,

CD4, CD40, CD86, CD8A, CXCL10, DDX58, HLA-A, IFNARI, IFNBI,

[8]

9

Molecular Genetics and Genome Biology of Goats http://dx.doi.org/10.5772/intechopen.72414

[8]

[8]

[8]

[8]

[8]

[8]

When ligands bind to TLRs, they trigger at least two most important cell signaling pathways. One of the pathways involves MyD88, an adaptor protein which is shared by most TLRs. When this pathway is triggered, it leads to the activation of the transcription factor NF-κB which then results in the release of proinflammatory cytokines [10, 43, 44]. Ekwemalor et al. [8] reported that probiotics modulated the expression of genes in myeloid differentiation antigen 88 (MYD88)-dependent or MYD88-independent system, TLR-mediated signaling induction pathway, nuclear factor κB (NF-κB), cytokine-mediated signaling pathways and Wnt signaling pathway. **Table 3** shows the different genes that were expressed in the Wnt signaling pathway involved in canonical Wnt signaling, planar cell polarity, negative regulation,

calcium signaling, cell growth and proliferation as a result of probiotics.

expression of TLRs in whole blood.

Pattern recognition receptors (PPRs)

Th1 markers & immune

Defense response to

Defense response to

bacteria

viruses

response

**Table 1.** List of immunomodulators tested on goats.


**Table 2.** List of genes associated with innate and adaptive immunity.

metabolite in animal feed on goat health. Pathogen-associated molecular pattern evaluated in goats includes the following: probiotics, mushroom, plant polyphenols, cowpea, lipopolysac-

Probiotics has been studied and considered as health beneficial microorganism which plays a role in maintaining homeostasis. Previous studies have shown the use of probiotics to modulate gastrointestinal health. Liong [37] reported the resistance to infectious diseases in the gastrointestinal tract as a result of probiotics. Probiotics as a supplement in animal feed has shown to have a beneficial effect on milk yield, fat and protein content [38]. Ekwemalor et al. [39] reported the release of proinflammatory cytokines in goats orally drenched with probiotics (*Coriolus versicolor* [CV]). Previous study conducted by our research team looked at the molecular impact of probiotic administration on physical health parameters and activation of genes involved in homeostasis and immunity in goat blood. We reported that genes associated with innate and adaptive immunity were modulated as a result of probiotics treatment. Genes that were expressed are associated with the host response to bacteria, virus, T-cell activation, cytokines and inflammatory response. **Table 2** shows genes modulated as a result

charide (LPS), peptidoglycan, nystatin and Sericea lespedeza (**Table 1**).

**Modulator (s) Sample type (s) Cytokines Innate immune** 

Plant extract Whole blood — TLR2 [54]

IFNγ

Peptidoglycan Whole blood — TLR2 [54]

IL-1α, IP-10

TNFα, IL1α, ILβ, IL8 *IL10RA*, IL15, IP10, G-CSF, Rantes and

IFNr, Rantes and granulocyte colony stimulating factor (GCSF). granulocyte macrophage colonystimulating factor (GM-CSF)

IL1B, CCL3 and IL8, *CCL2*, *CXCL6*, *IL6*, *CXCL8*

*CXCL6, CXCL8, CCL5 PTGS2, IFIT3,* 

Probiotics Whole blood, serum IL2, IL5, IL10, IL8, IL18 TLR4, TLR6,

**response**

TLR7, TLR9

TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10

*PTGS2, IFIT3, MYD88, NFKB1, and TLR4*

*NFKB1, TLR4, and* 

*TOLLIP*

TLR2 [34, 69]

TLR2 and TLR4 [7]

**Reference**

[80, 81]

[8]

[54, 83]

[84]

*3.4.1. Probiotics*

8 Goat Science

of probiotic modulation.

Cowpea Whole blood, serum,

Mushroom Neutrophils, whole

Lipopolysaccharide Mammary epithelial

Lipoteichoic acid Mammary epithelial cells

**Table 1.** List of immunomodulators tested on goats.

plasma

blood, serum

cells, whole blood, blood leukocytes

Sericea lespedeza Whole blood, serum TNF-α, IFNr, GCSF, GMCSF,

Researchers have reported effects of probiotics in goats of which most effects have been attributed to an increase in the innate immune system and others in the acquired immune response. Leeber et al. [40] reported that probiotics have the properties to modulate host immune system through different signaling pathways of innate immune cells. The innate immune system functions by initiating a response to microorganisms or their components via pattern recognition receptors such as nucleotide-binding oligomerization domain-like receptors or TLR [41]. Previous studies conducted by Worku and Morris [42] and Worku et al. [7] have shown the expression of TLRs in whole blood.

When ligands bind to TLRs, they trigger at least two most important cell signaling pathways. One of the pathways involves MyD88, an adaptor protein which is shared by most TLRs. When this pathway is triggered, it leads to the activation of the transcription factor NF-κB which then results in the release of proinflammatory cytokines [10, 43, 44]. Ekwemalor et al. [8] reported that probiotics modulated the expression of genes in myeloid differentiation antigen 88 (MYD88)-dependent or MYD88-independent system, TLR-mediated signaling induction pathway, nuclear factor κB (NF-κB), cytokine-mediated signaling pathways and Wnt signaling pathway. **Table 3** shows the different genes that were expressed in the Wnt signaling pathway involved in canonical Wnt signaling, planar cell polarity, negative regulation, calcium signaling, cell growth and proliferation as a result of probiotics.

#### *3.4.2. Mushrooms (Coriolus versicolor)*

Mushrooms have been studied and are known for their nutritional and medicinal properties. They contain bioactive compounds which are of medicinal importance. There are several types of mushroom of which *Coriolus versicolor* (CV) is one of the studied types of mushroom because of its medicinal properties. They contain active ingredients such as polysaccharide krestin (PSK) and polysaccharide peptide (PSP) [45]. Eliza et al. [46] reported that extracts of CV have the potential of boosting suppressed immune function, extending the survival rate and improving quality of life. They exert their therapeutic effects by modulating the host's immune response. Zhou et al. [47] demonstrated their effect in stimulating the immune system and inhibition of cancer growth. Lull et al. [48] also reported their effect in activating T and B lymphocytes, macrophages, natural killer cells, and lymphocyte-activated killer cells, as well as promoting the production of antibodies and various cytokines. Results from research team showed that mushroom extracts of CV modulated the expression of 10 TLR in neutrophils and modulation of innate immunity through differential regulation of the secretion of serum proteins including cytokines and prostaglandin E2 to impact goat health [8].

Peptidoglycan and lipoteichoic acids are the major stimulatory components of Gram-negative bacteria and are recognized by TLR2 [51]. Pathogen recognition receptors, such as TLRs, have

Molecular Genetics and Genome Biology of Goats http://dx.doi.org/10.5772/intechopen.72414 11

The linkage between PAMPs, TLR, activation of the prostaglandin pathway and the promotion of Wnt signaling in inflammatory response has been studied [52, 53]. Previous work by Asiamah et al. [54] also indicates that TLR2 and Frizzled receptors are increased in response to bacterial cell wall components (lipopolysaccharide, peptidoglycan). Nystatin is a lipid raft inhibitor derived from the bacterium *Streptomyces noursei*, and in addition, a proinflammatory agent was also found to modulate TLR2 and Frizzled receptor in goat blood. These findings open a window into the innate immune mechanism and inflammatory response mediated through TLRs and Wnt in goats and may provide more understanding about disease resis-

tance as well as aid in drug design and animal selection through breeding programs.

(*Lespedeza cuneata)* [62–64], herbs and fodder trees have been evaluated in goats.

Apart from plants being important feed resource for animal nutrition, they are also a rich source of polyphenol bioactive compounds that have beneficial health effects. Polyphenols (also known as phenolic compounds) are naturally occurring plant metabolites and are an integral part of both human and animal diet [55]. These compounds include flavonoids, tannins, phenolic acid and others [56]. Feed resources containing tannins have been reported to have both beneficial and detrimental effects on grazing animals. Tannin-rich plants have direct antiparasitic activity but might also act indirectly by increasing host resistance. These effects vary depending on the species of plant, parasite and host [57]. The antiparasitic potential of forage legumes (Fabaceae family), including sulla (*Hedysarum coronarium*) [58], sainfoin (*Onobrychis viciifolia*) [59], birdsfoot trefoil (*Lotus corniculatus*) [60], big trefoil (*Lotus pedunculatus)* [61] and Sericea lespedeza

The roles of polyphenol extracts from plants in the immune function have been reported on different cell types both *in vitro* and *in vivo* [65]. Polyphenol and other plant extracts demonstrated the ability to induce the release of both proinflammatory and anti-inflammatory cytokines, thus leading to the maintenance of the immune homeostasis in the host [66]. Epigallocatechin-3-gallate, a flavonoid found in green tea, has been shown to inhibit NF-κB activation induced by many proinflammatory stimuli [67]. Recently, there is a great interest in feed polyphenols due to their antioxidant capacity, inflammatory and immunomodulatory properties and their possible beneficial implications on animal health and production

Sericea lespedeza (*Lespedeza cuneata*) is a leguminous plant with high tannin content. It has been studied extensively for its possible anthelmintic potential especially in small ruminants [60]. A study by Worku et al. [7] demonstrated the impact of Sericea lespedeza (SL) diet on innate immune response mediators in goats. More specifically, Sericea diet increased serum level of proinflammatory cytokines TNF-α, IFNr, GCSF, GMCSF, IL-1α and IP-10 (*P* < 0.0002)

*3.4.4. Plant polyphenols*

[7, 54, 68, 69, 79].

*3.4.5. Sericea lespedeza*

evolved to recognize these PAMPs and detect invading disease microbes [49].

#### *3.4.3. Lipopolysaccharide, peptidoglycan and nystatin*

Bacteria produce molecules such as lipopolysaccharide (LPS), lipoproteins, peptidoglycan and lipoteichoic acids (LTAs), and this serves as specific molecular signatures for different classes of bacteria [49]. Lipopolysaccharides (LPSs), also known as lipoglycans, are the main surface membrane of Gram-negative bacteria. LPSs comprise poly- or oligosaccharide region and lipid A, which is the main immunostimulatory part of LPS [50]. Lipopolysaccharide is recognized by TLR4 assisted by CD14 proteins [49].


**Table 3.** Differentially expressed genes on the Wnt signaling pathway in response to modulators.

Peptidoglycan and lipoteichoic acids are the major stimulatory components of Gram-negative bacteria and are recognized by TLR2 [51]. Pathogen recognition receptors, such as TLRs, have evolved to recognize these PAMPs and detect invading disease microbes [49].

The linkage between PAMPs, TLR, activation of the prostaglandin pathway and the promotion of Wnt signaling in inflammatory response has been studied [52, 53]. Previous work by Asiamah et al. [54] also indicates that TLR2 and Frizzled receptors are increased in response to bacterial cell wall components (lipopolysaccharide, peptidoglycan). Nystatin is a lipid raft inhibitor derived from the bacterium *Streptomyces noursei*, and in addition, a proinflammatory agent was also found to modulate TLR2 and Frizzled receptor in goat blood. These findings open a window into the innate immune mechanism and inflammatory response mediated through TLRs and Wnt in goats and may provide more understanding about disease resistance as well as aid in drug design and animal selection through breeding programs.

#### *3.4.4. Plant polyphenols*

*3.4.2. Mushrooms (Coriolus versicolor)*

10 Goat Science

*3.4.3. Lipopolysaccharide, peptidoglycan and nystatin*

recognized by TLR4 assisted by CD14 proteins [49].

Proliferation Probiotics DAB2

Nystatin

WNT calcium signaling Probiotics

Canonical WNT signaling

Wnt signaling target

WNT signaling negative regulation

Cell growth and proliferation

genes

Mushrooms have been studied and are known for their nutritional and medicinal properties. They contain bioactive compounds which are of medicinal importance. There are several types of mushroom of which *Coriolus versicolor* (CV) is one of the studied types of mushroom because of its medicinal properties. They contain active ingredients such as polysaccharide krestin (PSK) and polysaccharide peptide (PSP) [45]. Eliza et al. [46] reported that extracts of CV have the potential of boosting suppressed immune function, extending the survival rate and improving quality of life. They exert their therapeutic effects by modulating the host's immune response. Zhou et al. [47] demonstrated their effect in stimulating the immune system and inhibition of cancer growth. Lull et al. [48] also reported their effect in activating T and B lymphocytes, macrophages, natural killer cells, and lymphocyte-activated killer cells, as well as promoting the production of antibodies and various cytokines. Results from research team showed that mushroom extracts of CV modulated the expression of 10 TLR in neutrophils and modulation of innate immunity through differential regulation of the secretion of serum proteins including cytokines and prostaglandin E2 to impact goat health [8].

Bacteria produce molecules such as lipopolysaccharide (LPS), lipoproteins, peptidoglycan and lipoteichoic acids (LTAs), and this serves as specific molecular signatures for different classes of bacteria [49]. Lipopolysaccharides (LPSs), also known as lipoglycans, are the main surface membrane of Gram-negative bacteria. LPSs comprise poly- or oligosaccharide region and lipid A, which is the main immunostimulatory part of LPS [50]. Lipopolysaccharide is

**Category Modulator Genes Reference**

WNT7B, WNT8A

Planar cell polarity Probiotics DAAM1, MAPK8, VANGL2, [80]

**Table 3.** Differentially expressed genes on the Wnt signaling pathway in response to modulators.

Probiotics APC, AXIN2, CSNK1A1, DVL2, FZDI, FZD7, FZD8,

GSK3A, GSK3B, LEF1, LRP5, NKD1, PORCN, RUVBL1, SFRP4, TCF7, TCF7L1, WIF1, WNT1, WNT2, WNT3A,

Probiotics CCND2, WISP1 [80]

Probiotics FBXW4, FBXW11, FRZB [80]

Probiotics FOXN1, JUN, MMPZ, PPARD [80],

NFATC1, WNT5B, WNT5A [35, 80]

[80]

Apart from plants being important feed resource for animal nutrition, they are also a rich source of polyphenol bioactive compounds that have beneficial health effects. Polyphenols (also known as phenolic compounds) are naturally occurring plant metabolites and are an integral part of both human and animal diet [55]. These compounds include flavonoids, tannins, phenolic acid and others [56]. Feed resources containing tannins have been reported to have both beneficial and detrimental effects on grazing animals. Tannin-rich plants have direct antiparasitic activity but might also act indirectly by increasing host resistance. These effects vary depending on the species of plant, parasite and host [57]. The antiparasitic potential of forage legumes (Fabaceae family), including sulla (*Hedysarum coronarium*) [58], sainfoin (*Onobrychis viciifolia*) [59], birdsfoot trefoil (*Lotus corniculatus*) [60], big trefoil (*Lotus pedunculatus)* [61] and Sericea lespedeza (*Lespedeza cuneata)* [62–64], herbs and fodder trees have been evaluated in goats.

The roles of polyphenol extracts from plants in the immune function have been reported on different cell types both *in vitro* and *in vivo* [65]. Polyphenol and other plant extracts demonstrated the ability to induce the release of both proinflammatory and anti-inflammatory cytokines, thus leading to the maintenance of the immune homeostasis in the host [66]. Epigallocatechin-3-gallate, a flavonoid found in green tea, has been shown to inhibit NF-κB activation induced by many proinflammatory stimuli [67]. Recently, there is a great interest in feed polyphenols due to their antioxidant capacity, inflammatory and immunomodulatory properties and their possible beneficial implications on animal health and production [7, 54, 68, 69, 79].

#### *3.4.5. Sericea lespedeza*

Sericea lespedeza (*Lespedeza cuneata*) is a leguminous plant with high tannin content. It has been studied extensively for its possible anthelmintic potential especially in small ruminants [60]. A study by Worku et al. [7] demonstrated the impact of Sericea lespedeza (SL) diet on innate immune response mediators in goats. More specifically, Sericea diet increased serum level of proinflammatory cytokines TNF-α, IFNr, GCSF, GMCSF, IL-1α and IP-10 (*P* < 0.0002) and decreased (*P* < 0.0001) IL-8 and RANTES. In addition, results from gene expression analyses showed increased mRNA transcripts of cell surface receptors TLR2 and TLR4, and the cytokines IL-8, IL-10, IL-2 and INF-γ. Previous work by Asiamah et al. [54] also demonstrated that transcription of TLR2 and Frizzled receptor in goat blood is variably responsive to Sericea lespedeza. In summary, goats respond to plant extracts and may have an effect on the expression of innate immune markers. This may offer an avenue for the exploitation of plant-derived tannins to regulate inflammatory response and enhance goat innate response.

**Author details**

**References**

\*Address all correspondence to: worku@ncat.edu

contortus. Veterinary Parasitology. 2011;**178**(1):192-197

Ecosystems & Environment. 2002;**90**(2):139-153

cultural Science. 2016;**8**(3):21

Archives of Biological Sciences. 2009;**61**(1):135-140

phils. Journal of Molecular Biology Research. 2016;**6**(1):71

Immunology and Immunopathology. 2011;**140**(3):252-258

innate immunity. Science. 1999;**284**(5418):1313-1318

immune system. Science. 2002;**296**(5566):298-300

Kingsley Ekwemalor, Sarah Adjei-Fremah, Emmanuel Asiamah and Mulumebet Worku\*

North Carolina Agricultural and Technical State University, Greensboro, North Carolina, USA

Molecular Genetics and Genome Biology of Goats http://dx.doi.org/10.5772/intechopen.72414 13

[1] McGuire, S. FAO, IFAD, and WFP. The state of food insecurity in the world 2015: Meeting the 2015 international hunger targets: Taking stock of uneven progress. Rome: FAO, 2015. Advances in Nutrition: An International Review Journal. 2015:**6**(5):623-624 [2] Joshi BR, Kommuru DS, Terrill TH, Mosjidis JA, Burke JM, Shakya KP, Miller JE. Effect of feeding Sericea lespedeza leaf meal in goats experimentally infected with Haemonchus

[3] Schiere JB, Ibrahim MNM, Van Keulen H. The role of livestock for sustainability in mixed farming: Criteria and scenario studies under varying resource allocation. Agriculture,

[4] Worku M, Franco R, Baldwin K. Efficacy of garlic as an anthelmintic in adult Boer goats.

[5] Worku M, Franco R, Miller JH. Evaluation of the activity of plant extracts in Boer goats.

[6] Adjei-Fremah S, Asiamah EK, Ekwemalor K, Jackai L, Schimmel K, Worku M. Modulation of bovine Wnt signaling pathway genes by cowpea phenolic extract. Journal of Agri-

[7] Worku M, Abdalla A, Adjei-Fremah S, Ismail H. The impact of diet on expression of genes involved in innate immunity in goat blood. Journal of Agricultural Science. 2016;**8**(3):1 [8] Ekwemalor K, Asiamah E, Worku M. Effect of a mushroom (*Coriolus versicolor*) based probiotic on the expression of toll-like receptors and signal transduction in goat neutro-

[9] Afacan NJ, Fjell CD, Hancock RE. A systems biology approach to nutritional immunology—Focus on innate immunity. Molecular Aspects of Medicine. 2012;**33**(1):14-25 [10] Raja A, Vignesh AR, Mary BA, Tirumurugaan KG, Raj GD, Kataria R, Kumanan K. Sequence analysis of toll-like receptor genes 1-10 of goat (*Capra hircus*). Veterinary

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#### *3.4.6. Cowpea*

Cowpea (*Vigna unguiculata,* L.Walp) is a highly nutritious legume plant used as human food and feed for animals. It has been utilized as a supplement feed to enhance feed intake and improve productivity in ruminants fed low-quality roughage diets [70, 71]. Cowpea also contains polyphenol compounds including phenol acids, flavonoids and tannins [72]. Polyphenolic extract of cowpea has been shown to have potential impact on ruminant health via antioxidant capacity [68], anti-inflammatory properties [34, 73] and modulating expression of genes associated with immunity and homeostasis [6, 43, 69]. Treatment with cowpea extract downregulated the expression of proinflammatory cytokine TNFα (fold change (FC; treatment/control) = −43.39), IL1α (FC = −6.19), ILβ (FC = −3.62) and IL8 (FC = −1.25). Also, CPE modulated the expression of *IL10RA* (a receptor for IL10, an anti-inflammatory cytokine) and IL15 [73].

A study by Adjei-Fremah et al. [69] demonstrated the impact of cowpea forage grazing, particularly Mississippi Silver variety on growth, internal parasite burden, and markers of immunity in goats. Their study results showed a modulation in cytokines levels, TNF-α, IL-8, and IP10 decreased, whereas an increase in G-CSF, Rantes and IFNγ was observed. The total antioxidants in plasma also increased in the cowpea-grazed goats [34, 73]. Cowpea diet may therefore stimulate innate immune response in goats, and this will help the animals fight against infectious pathogens and diseases. The immunomodulatory potential of cowpea feed may be due to at least in part their polyphenols [68, 74, 75]. Phenolic compounds in animal feeds have antioxidant properties that prevent the damaging effect of free radicals and their metabolic by-products [76] and stimulate an immune response in animals [75].
