**4. Discussion**

In this study the most dogs in the control group and CVL were mixed breed. The clinical symptoms of seropositive dogs (CVL) included lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, scaling, weight loss, and others. Dogs were classified into three clinical stages: stage I, mild disease; stage II, moderate disease; and stage III, severe disease. There was no statistical difference in the distribution between clinical stages and parasitic index. IL-6 and TNF-α concentrations increased in serum from infected dogs with a statistically significant difference between the clinical stages of CVL. Between the dogs with CVL and the control group, there was a statistical difference in the serum concentrations of cytokines IL-4 and TNF-α. IgG levels were elevated in the CVL group when compared to IgM levels. Antibody levels were positively correlated with IL-4 expression (rs = 0.5997; p = 0.0040). There was a significant positive correlation of IL-6 cytokine levels between stage I and stage III.

The clinical signs of CVL are important for the diagnosis. In the present study, the most prevalent clinical signs were lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, and scaling. However, prevalence is highly variable across

**109**

*Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis*

studies, but generally these clinical signs are the most commonly reported in the literature. These results corroborate the findings of several authors [25, 26].

Regarding gender, there was a greater predominance of males in infected dogs and females in dogs in the control group. Regarding age, it did not present large variations. This fact seems to be associated with the higher risk of male exposure. However, the study shows no statistically significant differences for age and gender

Bone marrow samples were taken from 21 dogs serologically positive for *L. infantum*. According to the clinical signs, dogs were classified as stages I, II, and III. Real-time PCR detected no parasite copies (×1010)/μl *L. infantum* DNA in all animals of the CVL group, distributed as follows: stage I mean (4.964), stage II average (4.63), and stage III (4.55). No statistically significant difference was found in the average amount of DNA copy number between the different clinical stages (p = 0.9467). In bone marrow samples from dogs that are cytologically positive, a

Previous studies report that quantitative PCR on bone marrow samples from positive dogs in conventional tests contained a higher number of *Leishmania* kDNA copies than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load [28]. This

PCR can be used for detection of *Leishmania* in naturally infected dog samples, and PCR-RFLP (restriction fragment length polymorphism) is sensitive for identification of *Leishmania* species [28]. In addition, qPCR is effective in quantifying *Leishmania* DNA loading in clinical samples [29]. The blood sample from dogs infected by *L. infantum* was found by real-time PCR to have a sensitivity of 100%

Most cytokines remain partially conserved between species; in this sense, the amino acid sequence of humans and canine cytokines shows 49–96% homology, suggesting a high probability of cross-reactivity between monoclonal antibodies; thus antibodies against human cytokines may be recommended as immunological biomarkers under pathological conditions by flow cytometry in human [31] and

In the present work, the serum concentration of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17) was compared between the control groups and the group with CVL. In addition, cytokine levels were compared within the CVL group with clinical staging I, II, and III. When comparing the groups, IL-4 and TNF-α were higher in infected dogs than in the control group, showing significant difference between IL-4 (p = 0.0469) and TNF-α (p = 0.0009) groups. In the group with CVL there were differences between stages I and III with significant differences

Elevated levels of IL-6 were found in serum from dogs with active leishmaniasis compared to healthy dogs [33]. These results corroborate the findings of this study. However, other authors reported that IL-6 production did not vary significantly between the groups studied [34]. On the other hand were described in the literature that elevated levels of IL-6 in dogs without clinical signs or symptoms in CVL dogs [35], and also highlights that, among other factors, it may indicate a balance between the parasite elimination effort and the active disease. Increased IL-6 levels suggest a restricted ability to control infection [36]. Even in the absence of clinical signs or symptoms, the animals showed granulomas on histopathological evaluation, suggesting chronicity and therefore a longtime course of infection [35]. Innate immune effector cells primarily neutrophils, monocytes, and macrophages produce and respond to IL-6, which may result in amplification of inflammation and a

*DOI: http://dx.doi.org/10.5772/intechopen.90573*

between healthy and sick dogs [27].

high parasitic index is detected [21].

and specificity of 96.4% [30].

dogs [32] as used in this study.

literary quote converges with the findings of this study.

only for cytokines IL-6 (p = 0.0350) and TNF-α (p = 0.0462).

change from an acute inflammatory state to a chronic state [37].

#### *Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.90573*

studies, but generally these clinical signs are the most commonly reported in the literature. These results corroborate the findings of several authors [25, 26].

Regarding gender, there was a greater predominance of males in infected dogs and females in dogs in the control group. Regarding age, it did not present large variations. This fact seems to be associated with the higher risk of male exposure. However, the study shows no statistically significant differences for age and gender between healthy and sick dogs [27].

Bone marrow samples were taken from 21 dogs serologically positive for *L. infantum*. According to the clinical signs, dogs were classified as stages I, II, and III. Real-time PCR detected no parasite copies (×1010)/μl *L. infantum* DNA in all animals of the CVL group, distributed as follows: stage I mean (4.964), stage II average (4.63), and stage III (4.55). No statistically significant difference was found in the average amount of DNA copy number between the different clinical stages (p = 0.9467). In bone marrow samples from dogs that are cytologically positive, a high parasitic index is detected [21].

Previous studies report that quantitative PCR on bone marrow samples from positive dogs in conventional tests contained a higher number of *Leishmania* kDNA copies than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load [28]. This literary quote converges with the findings of this study.

PCR can be used for detection of *Leishmania* in naturally infected dog samples, and PCR-RFLP (restriction fragment length polymorphism) is sensitive for identification of *Leishmania* species [28]. In addition, qPCR is effective in quantifying *Leishmania* DNA loading in clinical samples [29]. The blood sample from dogs infected by *L. infantum* was found by real-time PCR to have a sensitivity of 100% and specificity of 96.4% [30].

Most cytokines remain partially conserved between species; in this sense, the amino acid sequence of humans and canine cytokines shows 49–96% homology, suggesting a high probability of cross-reactivity between monoclonal antibodies; thus antibodies against human cytokines may be recommended as immunological biomarkers under pathological conditions by flow cytometry in human [31] and dogs [32] as used in this study.

In the present work, the serum concentration of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17) was compared between the control groups and the group with CVL. In addition, cytokine levels were compared within the CVL group with clinical staging I, II, and III. When comparing the groups, IL-4 and TNF-α were higher in infected dogs than in the control group, showing significant difference between IL-4 (p = 0.0469) and TNF-α (p = 0.0009) groups. In the group with CVL there were differences between stages I and III with significant differences only for cytokines IL-6 (p = 0.0350) and TNF-α (p = 0.0462).

Elevated levels of IL-6 were found in serum from dogs with active leishmaniasis compared to healthy dogs [33]. These results corroborate the findings of this study. However, other authors reported that IL-6 production did not vary significantly between the groups studied [34]. On the other hand were described in the literature that elevated levels of IL-6 in dogs without clinical signs or symptoms in CVL dogs [35], and also highlights that, among other factors, it may indicate a balance between the parasite elimination effort and the active disease. Increased IL-6 levels suggest a restricted ability to control infection [36]. Even in the absence of clinical signs or symptoms, the animals showed granulomas on histopathological evaluation, suggesting chronicity and therefore a longtime course of infection [35]. Innate immune effector cells primarily neutrophils, monocytes, and macrophages produce and respond to IL-6, which may result in amplification of inflammation and a change from an acute inflammatory state to a chronic state [37].

*Parasitology and Microbiology Research*

*The results were expressed in mean and standard error.*

*rs, correlation coefficient of Spearman.*

*rs, correlation coefficient of Spearman.*

*serum from dogs of the CVL group.*

**Table 3.**

**Table 4.**

**Table 5.**

In this study, as shown in **Table 5**, the correlation of the evolution of clinical signs between the stages presented below was analyzed. There was a significant

*Correlation of IL-6 and TNF-α cytokine levels of dogs with canine visceral leishmaniasis by clinical staging of*

**Group Control CVL p***-***Value** IgG 636.94 ± 255.52 2288.04 ± 610.08 <0.0001 IgM 241.12 ± 51.81 282.42 ± 33.99 0.0773

*Immunoglobulin concentrations (IgG and IgM) in serum from dogs with canine visceral leishmaniasis.*

**Parameters rs p-Value** IL-4 0.5997 0.0040 TNF-α 0.4164 0.0603 Parasitic index −0.2243 0.3282

*Correlation between IgG concentrations with IL-4 and TNF-α and parasitic index of dogs infected with CVL.*

**Stage I and II I and III II and III**

IL-6 0.6031 0.0855 0.8469 0.0162 0.5630 0.1144 TNF-α 0.0350 0.9288 0.3784 0.4026 0.0168 0.9658

**rs p rs p rs p**

**IgG**

In this study the most dogs in the control group and CVL were mixed breed. The clinical symptoms of seropositive dogs (CVL) included lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, scaling, weight loss, and others. Dogs were classified into three clinical stages: stage I, mild disease; stage II, moderate disease; and stage III, severe disease. There was no statistical difference in the distribution between clinical stages and parasitic index. IL-6 and TNF-α concentrations increased in serum from infected dogs with a statistically significant difference between the clinical stages of CVL. Between the dogs with CVL and the control group, there was a statistical difference in the serum concentrations of cytokines IL-4 and TNF-α. IgG levels were elevated in the CVL group when compared to IgM levels. Antibody levels were positively correlated with IL-4 expression (rs = 0.5997; p = 0.0040). There was a significant positive correlation of IL-6 cytokine levels between stage I and stage III. The clinical signs of CVL are important for the diagnosis. In the present study, the most prevalent clinical signs were lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, and scaling. However, prevalence is highly variable across

positive correlation of IL-6 cytokine levels between stage I and stage III.

**108**

**4. Discussion**

IL-6 expression increases in dogs with active visceral leishmaniasis and may be a useful marker for active disease [33, 35]. Increased IL-6 production is not directly related to anti-*Leishmania* antibody titers, suggesting that other cytokines may be involved with hypergammaglobulinemia [33].

As shown in this work, it was observed that there was correlation of IL-6 expression between stages I and III of bone marrow aspirate of dogs infected with CVL. IL-6 production in dogs with active leishmaniasis appears to be associated with severe disease [33]. This statement converges with the findings in this study, as the dogs used in the control group were mostly stage II and III. IL-6 is essential for terminal B-cell differentiation and immunoglobulin production [38].

TNF-α concentration was higher in infected dogs than in the control group, as detected by de Lima et al. [33]. CVL susceptibility is closely associated with downregulation of key cytokines such as IFN-γ, TNF-α, and IL-17A, thus impairing iNOS activation and NO production and favoring parasite replication and disease development [39].

The increased activity of TNF-α in the liver of infected dogs compared to healthy canines has been reported [37, 40]. Higher TNF-α levels in infected dogs indicate that the presence of *L. infantum* induces an immune response with relevant TNF-α expression when the protozoan is present [40].

Studies suggest that decreased survival of L. *infantum* in canine macrophages is associated with increased TNF-α and IFN-γ production and decreased IL-10 production [41].

In dogs naturally infected with *L. infantum*, increased hepatic TNF-α may be associated with increased parasite load on this organ [42]. The cytokines IL-2, IL-4, IL-10, IFN-γ, TNF-α, and IL-12 may be used as markers in epidemiological studies conducted in endemic areas to distinguish between different clinical forms of VL [15]. However, Lima et al. [33] indicate that TNF-α is not considered a good marker of active disease in dogs with VL.

A study has reported a significant relationship between bone marrow IL-4 detection in naturally infected dogs with and without clinical signs and disease severity, suggesting that IL-4 production is associated with pathology [43]. Increased expression of IL-4 cytokine is associated with both severe clinical signs and a high parasitic index on skin lesions [44]. In bone marrow aspirates, IL-4 was elevated in naturally infected dogs with more severe symptoms [43].

The study points to evidence that IL-4 cytokine polymorphism may contribute to innate immunity to *L. infantum* infection [45].

Antibody levels were positively correlated with IL-4 expression (rs = 0.5997; p = 0.0040). IgG is also linked to chronic infection in patients with VL, where high levels of IgG are predictive of the disease. This finding is in line with the study by Lima et al. [33] suggesting that other cytokines, such as IL-10 or IL-4, may be associated with hypergammaglobulinemia observed in dogs with CVL. Previous studies have detected increased serum IgG levels in symptomatic dogs compared with healthy dogs and are related to pathophysiological disorders and active disease [33].

Response to natural infection of *L. infantum* is linked to the presence of IgG [43] and *Leishmania*-specific IgM antibodies that can be detected in infected dogs [46]. Some studies have reported that increased total protein is frequent in dogs infected with visceral leishmaniasis due to increased antibody production [47, 48].

#### **5. Conclusion**

These results may contribute to a better understanding of the immune response in dogs infected with *L. infantum*. Antibody levels were positively correlated with

**111**

*Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis*

IL-4 expression. There was a significant positive correlation of IL-6 cytokine levels with the evolution of stages I and III. However, this cytokine can be used as a

This research received grants from the Mato Grosso Research Support Foundation (FAPEMAT No. 299032/2010) and from the National Council for Scientific and Technological Development (CNPq No. 447218/2014–0 and No.

The authors declare that there is no conflict of interest and nonfinancial

, Valéria Régia Franco Sousa1

1 Faculty of Veterinary Medicine, Federal University of Mato Grosso, Cuiabá, Mato

2 Institute of Biological and Health Sciences, Federal University of Mato Grosso,

© 2019 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,

,

, Adenilda Cristina Honorio-França2

*DOI: http://dx.doi.org/10.5772/intechopen.90573*

**Acknowledgements**

305725/2018–1), in Brazil.

**Conflict of interest**

competitors.

**Author details**

Grosso, Brazil

José Nivaldo da Silva1

and Eduardo Luzía França2

Arleana do Bom Parto Ferreira de Almeida1

Barra do Garças, Mato Grosso, Brazil

provided the original work is properly cited.

\*

\*Address all correspondence to<EMAIL_ADDRESS>
marker to distinguish between different clinical stages.

*Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.90573*

IL-4 expression. There was a significant positive correlation of IL-6 cytokine levels with the evolution of stages I and III. However, this cytokine can be used as a marker to distinguish between different clinical stages.


**5. Sharka (***Plum pox virus***) resistance in genetic diversity of apricot**

Sharka or Plum pox virus (PPV) is the most serious disease of Prunus trees. The disease has spreaded throughout many European countries, especially in Mediterranean countries. Resistant cultivars are limited and only some North American cultivars are known to be resistant to the disease. 'Stark Early Orange' (SEO), 'Goldrich', 'Harlayne', 'Stella', and 'Harcot' are the resistant cultivars mostly used as resistant source for breedings (Martinez-Gomez et al. 2000). The resent focus on the disease increased also cruiosty on the source of resistance of American cultivars. It is believed that North American cultivars originated from a limited number of European cultivars. The source of the resistance is unknown. However, recent studies shows that Central Asian apricots is the most likely source of resistant genes in the North American donors. *Prunus mandshurica* was first to be offered as PPV resistance into North American germplasm (Badenes et al. 1996). Zhebentyayeva et al. (2003) showed that 'Harlayne' and 'Goldrich' clusters with native Central Asian cultivars. 'Stark Early Orange', LE 2904, LE 3276, and 'Vestar' are also grouped with native Chinese material on the genetic diversity study (Zhebentyayeva et al. 2003). Hormaza (2002) also demonstrated that Chinese cultivars contributed to the pedigree of 'Stark Early Orange'. The recent research of Zhebentyayeva et al. (2008) shows that cultivars 'Harlayne', 'Goldrich', and 'Stark Early Orange' has genetic similarity with native Central Asian genotypes. The researchers also showed that *Prunus davidiana* alleles in 'Stark Early Orange' and *Prunus mume* alleles in 'Stark Early Orange' and 'Goldrich' pointed out a contribution of these species to PPV resistance as well (Zhebentyayeva et al. 2008).

#### **6.** *S***-genotyping in genetic diversity of apricot**

Like to other Prunus species, apricots show gametophytic self-incompatibility controlled by a single locus with multiple genes, *S*-haplotypes (De Nettancourt, 2001). The *S*-haplotype contains a female determinant, (*S-RNase*) encoding for a ribonuclease enzyme (McClure et al. 1989), and the recently identified male determinant, *S-haplotype-specific F-box* gene (Entani et al. 2003; Romero et al. 2004; Halasz et al. 2010).

The Irano – Caucasian group are usually self-incompatible whereas Europan apricots are mostly self-compatible (Halasz et al. 2005; Kostina, 1970). Mehlenbacher et al. (1991) stated Central Asian apricots are also mostly self-incompatible.

Cross-incompatibility between a pair of cultivars occurs frequently self-incompatible species. Cross-incompatibility was observed among the North American cultivars, Goldrich, Hargrand and Lambertin No.1 (Egea & Burgos, 1996), and also among giant-fruited Hungarian apricots (Szabo & Nyeki, 1991; Halasz et al. 2010). Halasz et al. (2010) determined total 12 cross-incompatiblity groups between Irano-Caucasian eco-geographical groups (Turkish apricots) and European eco-geographical groups (Hungarian and North American apricots) (Table 2).

Table 2. Cross-incompatibility groups of apricot (Halasz et al. 2010; Egea & Burgos, 1996; Halasz et al. 2005)

To date, 21 *S-RNase* alleles are known in European apricots, 20 of which (*S1-S20*) code for self-incompatibility and one (*Sc*) allowing self-compatibility (Burgos et al. 1998; Halasz, 2007; Halasz et al. 2005, 2007) and recently it was confirmed that *SC* haplotype is a pollen part mutant of *S8* haplotype (Halasz et al. 2007). Beside, some additional *S*-alleles have been also identified in Chinese cultivars (Wu et al. 2009; Halasz et al. 2010).

A gradually decreasing allele number was detected in apricot landraces from China to Western Europe, with some allelic exclusivity occuring in certain geographic areas (Halasz, 2007; Halasz et al. 2010).

contains a female determinant, (*S-RNase*) encoding for a ribonuclease enzyme (McClure et al. 1989), and the recently identified male determinant, *S-haplotype-specific F-box* gene (Entani

The Irano – Caucasian group are usually self-incompatible whereas Europan apricots are mostly self-compatible (Halasz et al. 2005; Kostina, 1970). Mehlenbacher et al. (1991) stated

Cross-incompatibility between a pair of cultivars occurs frequently self-incompatible species. Cross-incompatibility was observed among the North American cultivars, Goldrich, Hargrand and Lambertin No.1 (Egea & Burgos, 1996), and also among giant-fruited Hungarian apricots (Szabo & Nyeki, 1991; Halasz et al. 2010). Halasz et al. (2010) determined total 12 cross-incompatiblity groups between Irano-Caucasian eco-geographical groups (Turkish apricots) and European eco-geographical groups (Hungarian and North

I Goldrich, Hargrand, Lambertin No.1 S1S2 II Cologlu, Kadioglu, Seftalioglu, Cegledi orias, Ligeti orias

II Iri Bitirgen, Moniqui S2S6 IV Artvin PA, Priana S2S7 V Alyanak, Ziraat Okulu S2S8 VI Dortyol-4, Sebbiyiki S2S19 VII Sakit-3, Tokaloglu Izmir S3S19 VIII Cataloglu, Ozal, Soganci S6S9 IX Zerdali No.1, XI Zerdali S6S12 X Ordubat, X2 Zerdali S7S12 XI Adilcevaz-5, Hacihaliloglu, Kabaasi, Kamelya, Zerdali No.2

XII Shalakh (Aprikoz), Voski S11S13 XIII Levent, Sakit-1 S6S19 XIV Cekirge 52, X3 Zerdali S9S20

Sam (ScS2), Yerli Izmir (ScS7) Table 2. Cross-incompatibility groups of apricot (Halasz et al. 2010; Egea & Burgos, 1996;

identified in Chinese cultivars (Wu et al. 2009; Halasz et al. 2010).

To date, 21 *S-RNase* alleles are known in European apricots, 20 of which (*S1-S20*) code for self-incompatibility and one (*Sc*) allowing self-compatibility (Burgos et al. 1998; Halasz, 2007; Halasz et al. 2005, 2007) and recently it was confirmed that *SC* haplotype is a pollen part mutant of *S8* haplotype (Halasz et al. 2007). Beside, some additional *S*-alleles have been also

A gradually decreasing allele number was detected in apricot landraces from China to Western Europe, with some allelic exclusivity occuring in certain geographic areas (Halasz,

Canakkale (ScSc), Ethembey (ScS8), Karacabey (ScS2), Mektep (ScS8), Pasa Mismisi (ScS8),

Cultivars *S*-genotype

S8S9

S9S13

et al. 2003; Romero et al. 2004; Halasz et al. 2010).

American apricots) (Table 2).

Cross-incompatibility groups

0: Universal pollen

Halasz et al. 2005)

2007; Halasz et al. 2010).

donors

Central Asian apricots are also mostly self-incompatible.


**Table 5.**

*Goats (Capra) - From Ancient to Modern*

Sinhal varies in its coat color from black to white, where gray, black, and white mixed are the common coat colors of this breed. Average adult body weight of Sinhal is reported as 35 kg for male and 29 kg for doe, with 69 cm body length,

**Parameters Cluster A (46) Cluster B (70) Cluster C (73)** Body length (cm) 69.3 ± 0.3a 66.5 ± 0.2b 64.9 ± 0.2c Wither height (cm) 66.9 ± 0.2a 64.7 ± 0.1b 59.2 ± 0.2c Heart girth (cm) 69.9 ± 0.3a 66.5 ± 0.2b 65.9 ± 0.2c Flank girth (cm) 81.7 ± 0.2a 80.1 ± 0.2b 71.4 ± 0.1c Flank height (cm) 68.9 ± 0.2a 67.3 ± 0.1b 61.1 ± 0.2c Ear length (cm) 15.6 ± 0.2a 13.3 ± 0.2b 13.3 ± 0.1b Horn length (cm) 16.7 ± 0.6a 10.7 ± 0.5b 11.3 ± 0.3b Adult weight (kg) 38.6 ± 0.8a 31.8 ± 0.4b 27.7 ± 0.5c *Note: Number in parenthesis indicates the number of observations. Cluster A, goats from midwestern region (Salyan and Surkhet); Cluster B, goats from west (Lumle and Bandipur); and Cluster C, goats from east (Sindhuli and*

Chyangra goats are the mountain goat originating from Tibet reared in trans-Himalayan region along with Bhyanglung, a type of sheep in high mountain and trans-Himal region 2500 meters above sea level. They have been reared in situ condition by farmers themselves. They are suitable for meat and pack and are popular for high value as well as fine quality called *Chyangra* fiber known as *Pashmina* [20]. Their population is declining and hence needs attention. They have been characterized at phenotypic and mitochondrial levels. Chyangra fiber has high market potentials, as they have unique blend and qualities and hence are popular within and outside the country. Chyangra population is estimated to be around 1% of the total goat population, i.e., 0.11 million heads in Nepal [6]. Body color varies from pure white to pure black with mixed patch of different colors. Its compact body weighs around 30 kg with body length of 62 cm and chest girth of 71 cm. Wither height is 62 cm. Body weight varies from 29 to 32 kg for females and 35–40 kg for males. The morphological variation of four indigenous

Owing to the remoteness, harsh climatic condition and transhumance system of management Chyangra goat are not getting due consideration from public and private institutions. Thus, productivity and population of this breed is dwindling rapidly. The goat is used for meat and for pack purpose in mountain terrace where road facilities are seldom. The breed is also used for production of precious *pashmina* (Chyangra cashmere). Besides their incomparable contribution in mountain farming system, the breed has not been understood completely in the sense of their quantitative and qualitative attributes. Limited information are available on morphological characteristics and growth traits. The breed is not fully utilized for its Chyangra cashmere production, and this is an area of importance where focus is required [25].

Apart from above indigenous breeds, there are niche specific breed studied at inner Terai region, that is, Chitwan District of Nepal, referred to as Chitwan local

78 cm heart girth, and 59 cm wither height on an average.

*Pakhribas) of Nepal. Means with different superscripts differ significantly. Source: [14].*

*Khari goats from different clusters of eastern, western, and midwestern regions of Nepal.*

breeds of goats in Nepal is presented hereunder (see **Table 5**).

*4.1.4 Chyangra*

**Table 4.**

**62**

*4.1.5 Chitwan local goats*

*Comparative morphometric measurements of indigenous breeds of goat (values are means in cm ± standard errors).*

goats. Very limited information is available about this breed to date. These breeds have a medium-sized body having heavier body weight than Khari and Terai goat breeds with dominant white color with distribution of brown, black, and mixed color. It has been reported that the age at first conception, age at first kidding, gestation length, kidding interval, and postpartum estrus were 211, 356, 147, 236, and 87 days, respectively, for adult does of Chitwan local goats. The adult does of Chitwan local (31.1 kg) were significantly heavier (p < 0.001) than that of Terai goat (25.9 kg) and Khari goats (23.5 kg). It had been observed that the mean birth, weaning, and postweaning (8 months) weight for these breeds of goats were 2.48, 13.22, and 17.64 kg, respectively. Apart from above findings, it is also reported that the mean litter size at birth were 2.0, 1.53, and 1.48 kids per doe and at weaning were 1.94, 1.49, and 1.28 kids per doe for Chitwan local, Terai, and Khari goats, respectively [26, 27].

### **4.2 Popular exotic breeds of goats in Nepal**

### *4.2.1 Jamnapari*

Jamnapari is a breed of goat originating from the Indian subcontinent. It is a dual-purpose breed kept for both milk and meat. Jamnapari goats were mainly introduced in Nepal to upgrade and improve body weight of local Khari and Terai goats. Adult Jamunapari goat has a body weight of 45 kg for bucks and 38 kg for does along with wither height between 75 and 78 cm, body lengths of 75–77 cm, and heart girth of 76–79.5 cm (**Table 6**). Yearling weight of these goats in research stations was about 21 kg. The average age at first kidding and kidding interval in Jamnapari goats is 770 and 428 days, respectively.

### **Table 6.**

*Comparative productive performance of exotic breeds of goat in Nepal.*

### *4.2.2 Barbari*

The Barbari goats are a meat-type breed that is found in Mathura District of Uttar Pradesh, in addition to Gujrat, Jhelum, and Sargodha districts in Punjab Province. Barbari goats are popular for its compact and small body with average adult weight ranging from 23–36 kg (**Table 6**). Body color is mainly white with brownish red spots and coat is short. The head is small, with small upward-pointing ears and small curled horns. Does have good reproductive performance and produce well in intensive system and at dry areas. Average age at first kidding and kidding interval in Barbari goats is 588 and 274 days, respectively. Triple kidding and early maturity are common features of these goats.

### *4.2.3 Sirohi/Ajmeri*

The Sirohi and Ajmeri goats are a meat-type breed that is found in Sirohi District of Rajasthan. This breed also extends to Palanpur in Gujarat. Mature males weigh around 50 kg but females weigh only 25–30 kg (**Table 6**). These are compact, medium-sized animals. Coat color is predominantly brown, with light or dark brown patches; a very few individuals are completely white. Most animals are wattled. Ears are flat and leaf-like and medium-sized and have a drooping ear length of 18.8 cm. Both sexes have small horns, curved upward and backward. Tail is medium in length and curved upward. Udder is small and round, with small teats placed laterally. Some commercial farms and lead farmers have imported this breed and crossbred with Khari and Terai goats, but the information regarding the productive and reproductive performance is still to come.

### *4.2.4 Boer*

The Boer is an improved breed with some infusion of European, Angora, and Indian goat breeding developed in South Africa in the early 1900s. The Boer goat is primarily a meat goat with several adaptations to the region in which it was developed. It is a horned breed with lop ears and showing a variety of color patterns. The most common color of this breed is white body with red head and large, muscular frame. The Boer goat is being popular for its browsing ability and limited impact on the grass cover. Producing weaning rates in excess of 160%, the Boer goat doe is a low-maintenance animal that has sufficient milk to rear a kid that is early maturing. The mature buck weighs between 110 and 135 kg and does between 90 and 100 kg. Performance records for this breed indicate exceptional individuals are capable of average daily gains over 200 g/day in feedlot. More standard performance would be 150–170 g/day.

**65**

**Figure 2.**

*Breeds and Breeding System of Indigenous and Crossbred Goats in Nepal*

an extended breeding season making possible three kids every 2 years.

*Different exotic goat breeds available in Nepal (photo courtesy: Animal Breeding Division, NARC).*

The ovulation rate for Boer goats ranges from one to four eggs/doe with an average of 1.7. A kidding rate of 200% is common for this breed. Puberty is reached early, usually about 6 months for the males and 10–12 months for the females. The Boer goat also has

It was introduced in Nepal from a private sector to improve growth performance of local goats. Recently, projects funded by the World Bank (WB) and International Fund for Agriculture Development (IFAD) implemented by the Ministry of Agriculture and Livestock Development specifically Agriculture Food Security Project (AFSP) and Kisan Ka Lagi Unnat Bui Bijan Karyakram (KUBK), respectively, are working on producing crossbreds with the local Khari/Hill goat, government, and breeders' farmers

*DOI: http://dx.doi.org/10.5772/intechopen.82821*


*3.2.3 Correlation of soil pH, carbon and available P with heavy metal concentrations*

Soil pH was strongly correlated with soil C and available P (**Table 4**). The soils treated with sewage sludge had total metal concentrations in the order Cr > Zn > C u > Pb > Ni > Cd = As. Soil Cr, Zn and Cu strongly correlated with soil pH, while Cd weakly correlated with soil pH. The correlation of pH with As was weak (significant) and negative. There was no correlation of pH with Pb and Ni. Total soil C was strongly correlated with the available P. Soil C was strongly correlated with all the metals except Pb, which was weakly correlated, and As, for which the correlation was not significant. The available P was correlated strongly with Cr, Zn, Cu and Ni, weakly with Pb and not correlated with As and Cd.

*Heavy Metals in Soils Following 50 Years of Sewage Sludge Application DOI: http://dx.doi.org/10.5772/intechopen.110009*

*Soil C = total soil carbon, Avail P = available phosphorus.\* Correlation is significant at p = 0.05 level. \*\*Correlation is significant at p = 0.01 level.*

**Table 4.**

*Correlation coefficients (r) of soil pH, total C and available P with soil-heavy metal concentrations.*


**2. Materials and Methods**

The focus of the SCI model was to create a lesion that mimics human SCI without clinical impairments. The experimental methods to create the lesion are described in detail in previous publications [4,7], but are presented here briefly. The subjects, six adult Macaca fasicularis monkeys, underwent two surgical procedures. The first to insert a small transmitter (PhysioTel D70, Data Sciences International, St. Paul, MN, USA) with attached fine-wire intramuscular electrode pair into the lower back of the subjects. The electrodes were implanted into the left and right flexor cauda longus and brevis muscles (tail muscles). The EMG signals were measured using the fine-wire electrodes with 10 mm inter-electrode distance, 10 mm exposed wire length, 1000 Hz sampling frequency, and common ground.

Baseline pre-lesion EMG data from the fine-wire electrode pair were collected during voluntary movement of NHPs within their home enclosures for 30 days. The data were collected from Monday to Friday (excluding holidays) for approximately 1 h per day. After 30 days, the subjects underwent a second surgical procedure. A small laminotomy was completed at the L5 vertebral level. An epidural balloon catheter was inserted and advanced approximately 10 cm cranial to the level of the lower thoracic spinal cord. The lesion was created by inflation of the balloon with air. The balloon remained inflated for 60 s and then was deflated. The catheter was then removed, and the surgical incision closed. The lesions initially disrupted the grey and white matter at the site of lesion creation. The histopathological features, that were created by this method are similar to human TSCI [4]. To mimic the time frame between human injury and administration of emergency medical treatment, the subjects remained under anesthesia for one hour. Four subjects that received the experimental combination treatment for 90 days while two subjects did not receive any treatment. Combination treatment consisted of a bolus 0.2 mg/kg bolus of thyrotropin releasing hormone (TRH), followed by a continuous infusion of 0.2 mg/kg per hour for 1 h. These subjects were also treated with 60 mg of selenium and 80 IU of vitamin E daily. TRH is a tripeptide produced by the hypothalamus; selenium and Vitamin E are antioxidants. The combination of these agents may modulate the physiological sequelae of TSCI [40]. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard University and the University of Wisconsin at Madison. As such, EMG data from the tail is analogous to EMG data obtained from the limb of a human being with TSCI EMG data were collected from all six subjects. However, in one subject, the data from one side was not recorded due to a technical error. As such, raw data in this paper represents the experience of three subjects that received the combination therapy, and two subjects who did not receive treatment. Figure 1 illustrates a sample of the recorded raw EMG data.

**Figure 1.** Graphical representation of the raw electromyography (EMG) signal.

In this NHP model, the induced lesion produced a TSCI limited to the upper motor neuron tracts that supply the lower motor neurons of the tail muscles [7]. Lesion of such nature result in a perturbation in the MUs discharge properties; i.e., abnormalities in the inter-discharge interval, firing rate, and floating serial correlation coefficient [14,41]. These abnormalities have been typically characterized in non-physiological experimental conditions such as low force levels and isometric contraction frequency [14,37–39]. In this work, the EMG data were collected when the subjects engage in physiological activities (i.e., locomotion in a cage). The proposed EMG analysis method consists of the following three steps:

	- 1. The EMG signals were decomposed into seven sub-bands, one approximate coefficient (cA6), and six detail coefficients (cD1, ... , cD6).
	- 2. The EMG signal was then reconstructed at each level using inverse discrete wavelet transform, and seven EMG reconstructed signals (A6, D1, ... , D6) were obtained from their coefficients (cA6, cD1, ... , cD1). Table 1 shows the frequency ranges of the seven EMG sub-bands.

$$RP(\%) = \frac{SBP}{TP} \,\, ^\prime \tag{1}$$

where:

*RP:* the relative power of the desired sub-band. *SBP:* the power of the desired sub-band (e.g., A6, D1, ... or D6). *TP:* the total power of all the sub-bands (A6 + D1, ... , + D6).

**Table 1.** The frequency ranges of the seven EMG reconstructed sub-bands.

**Figure 2.** Wavelet analysis; decomposition and reconstruction steps.

The goal was to address the research questions by analyzing specific subsections of the data and testing specific effects. To test the lateralization effect (attributed to limb dominance), only the pre-lesion data were considered for the two sides (left and right). Then, the lesion effect was tested using the data from both pre-and post-lesion periods, and two separate models were fitted one for each side. To test the treatment effect, two separate models (left and right) were fitted using only the post-lesion data. The data in this work is considered a clustered longitudinal dataset with three levels; days are nested in frequency sub-bands, which are in turn nested within subjects (day: is level 1, sub-bands: is level 2, and subject: is level 3). As a result of such hierarchical structure as in Figure 3, the non-independence problem of the observations would arise which requires an appropriate statistical analysis method. A mixed model is a statistical method that was developed particularly to address the non-independence (correlated, or repeated measurements) issue by including the random effect term in its model. The developed mixed models controlled for the within-subject correlation by including a random effect for the subject and the nested sub-bands within each subject, with a variance-covariance structure and restricted maximum likelihood estimation. The models were implemented in the statistical software R using the (lme4) package. The significance level was set to (α = 0.05). The *RP* for any given subject at (day) *i* for (sub-band) *j* nested within (subject) *k* denoted as *RPijk*, is represented in the following equation (see a summary of parameters in Table 2):

 $RP\_{ijk} = \beta\_0 + \beta\_1$
 $Day\_{ijk} + \beta\_2$
 $EFFECT\_{jk} + \beta\_3$
 $EFFECT\_{jk}$
 $Day\_{ijk} + \beta\_4$
 $Freq\_{jk} + \beta\_5$
 $Freq\_{jk}$
 $Day\_{ijk} + \beta\_6$
 $Freq\_{jk}$
 $EFFECT\_{jk} + \beta\_7$
 $Freq\_{jk}$
 $EFFECT\_{jk}$
 $Day\_{ijk} (fixad) + \beta\_6$
 $a\_{0k} + a\_{1k}$
 $Day\_{ijk} + a\_{0jk} + a\_{1jk}$
 $Day\_{ijk} + \varepsilon\_{ijk}$
 $(random)\_i$

**Table 2.** Parameters used in Equation (2).

**Figure 3.** Data Structure: The data in this work was collected from the left (L) and right (R) side of the tail for five subjects during multiple experiment days (d1, d2, ... , dn) for (pre- and post-lesion period). The data of each day was decomposed into seven frequency sub-bands (D1, D2, D3, ... , A6). Three of the subjects received a combination of treatment (Treatment group); the remaining two subjects did not receive any treatment post-lesion (Control group).

## **3. Results**

The results were presented in the form of answers to the three research questions that were stated in the introduction. They are as follows:

*(1) Is the intramuscular fine-wire electrode pair capable of detecting limb dominance in the subjects prior to lesion?*

To test the symmetry of tail muscle activity on both sides, the *RP* of the EMG signals during the *pre-lesion* period were analyzed using a linear mixed model. The interaction of the frequency sub-band and the side variable in the statistical model demonstrated a significant effect (*p*-value < 0.001). Given that the (side\*frequency) interaction is significant, Tukey's mean comparisons were generated to identify what means differ significantly. These results suggested that side variable had a significant effect on the *RP* value of the D1, D2, D4, and D5 sub-bands. The estimated mean of the *RP* for the D1, D2 and D4 sub-bands of the *left side* were significantly higher than that of the *right side.* On the contrary, the estimated mean of the *RP* for the D5 sub-band on the *left side* was significantly lower than that of the *right side.* The estimated mean for the *RP* across the different frequency sub-bands for both sides is illustrated in Figure 4. Taken together, these results showed that the left and right tail muscles have asymmetry activation.

**Figure 4.** The estimated mean of the relative power (*RP*) of seven reconstructed EMG sub-bands prior to the creation of traumatic spinal cord injury (TSCI) for the left and right side of the tail. Of note for each band, there is a difference in *RP* value when compared to the left and right side of the tail. The D2 sub-band of the left and right side has the maximum RP, and the significant difference between the two sides is at the D1, D2, D4, and D5 sub-bands. The star indicates a significant difference.

*(2) In the post-lesion period, is there a change in the EMG activity attributed to the experimental spinal cord injury and how it could be characterized in terms of RP?*

To answer this question, the effect of the created lesion was analyzed using a linear mixed model. The lesion effect was studied by testing the difference between the EMG characteristics during the *pre-* and *post-lesion* period. The interaction of the frequency sub-band and the lesion variable in the statistical models of both sides demonstrated a significant effect (*p*-value < 0.001). Given that the (lesion\*frequency) interaction was significant, Tuckey's means comparisons were generated to identify which means differ significantly. Figures 5 and 6 summarized the estimated mean of the *RP* values for different frequency sub-bands of the *pre-* and *post-lesion* group for the two sides. On the *left side*, the estimated mean of the *RP* for the D4, D6 and A6 sub-bands was significantly higher in the *post-lesion* period compared to the *pre-lesion* period. On the *right side*, the estimated mean of the *RP* for the

D4, D6, and A6 sub-band was also higher significantly in the *post-lesion* period. On the other hand, the estimated mean of the *RP* for the D1 and D2 sub-bands was significantly lower in the *post-lesion* period compared to *pre-lesion* period. The results suggested that the created lesion had a clear effect on the discharge properties of MUs, and with this technique, changes in discharge properties can be detected even when there is no clinical evidence.

**Figure 5.** The estimated mean of the *RP* of seven reconstructed EMG sub-bands prior and post to the creation of TSCI for the *left side* of the tail. Of note, the *RP* values for the frequency sub-bands (D4, D6, and A6) are significantly higher in the post-lesion period. The star indicates a significant difference.

**Figure 6.** The estimated mean of the *RP* of seven reconstructed EMG sub-bands prior and post the creation of TSCI for the *right side* of the tail. Of note, the *RP* values for the lower frequency sub-bands (D4, D6, and A6) are significantly higher in the post-lesion period, while the higher frequency sub-bands (D1 and D2) are significantly lower in the post-lesion period. The star indicates a significant difference.

*(3) What is the di*ff*erence in the EMG activity between the control and the treatment group in the post-lesion period (Treatment e*ff*ect)?*

The *post-lesion data* were utilized to generate two separate mixed models, one for each side. The potential effect of the treatment was analyzed using the *RP* of the left and the right sides incorporating an analysis which compared the treatment and the control groups. The interaction of frequency sub-band and treatment variables in the models of both sides demonstrated a significant effect (*p*-value < 0.001). Given that the (treatment\*frequency) interaction was significant, Tukey's mean comparisons were generated to identify what means differ significantly. Figures 7 and 8 summarize the estimated mean of the *RP* values for different sub-bands of the control and treatment group for the two sides. On the *left side*, the D1, D2, D3, and D6 sub-bands have a significant difference, while on the *right side* the effect was significant in all the sub-bands except the D2. The results suggested that there is a significant difference in the discharge properties of MUs of the treatment and the control groups during the post-lesion period.

**Figure 7.** The estimated mean of the *RP* of seven reconstructed EMG sub-bands post-lesion for the treatment (Tr) and the control (Ctrl) groups of the *left side*; of note, the *RP* values for the frequency sub-bands (D1, D2, D3, and D6) are significantly different. Subjectively, the distribution of the *RP* in the treatment group is similar to the *RP* distribution in the pre-lesion period for all the subjects. The star indicates a significant difference.

**Figure 8.** The estimated mean of the *RP* of seven reconstructed EMG sub-bands post-lesion for the treatment (Tr) and the control (Ctrl) group of the *right side*; of note, the *RP* values for frequency sub-bands (D1, D3, D4, D5, D6, and A6) are significantly different. Subjectively, the distribution of the *RP* in the treatment group is similar to the *RP* distribution in the pre-lesion period for all the subjects. The star indicates a significant difference.


*4.2. Cases Studied*

In this paper, three different cases studied are performed for each system, and these cases are described as follows:

Case 1: this is considered the basic system condition without adding DGs units.

Case 2: in this case, the MG is operated without considering the optimal control settings of BGs, WT, and PV with daily load variations.

Case 3: this case develops the optimal operation of the MG with optimal control settings by using the EO algorithm for BGs, WT, and PV, simultaneously with considering the daily load variations.