extfieldsofstudy: Medicine Chemistry

text:
Dietary Stevioside Supplementation Alleviates Lipopolysaccharide-Induced Intestinal Mucosal Damage through Anti-Inflammatory and Antioxidant Effects in Broiler Chickens

The study was conducted to investigate the effects of dietary stevioside (STE) supplementation on the lipopolysaccharide (LPS)-induced intestinal mucosal damage of broiler chickens. A total of 192 one-day-old male Ross 308 broiler chicks were randomly divided into four treatments: (1) basal diet (CON); (2) basal diet supplemented with 250 mg/kg stevioside (STE); (3) basal diet + LPS-challenge (LPS); (4) basal diet supplemented with 250 mg/kg stevioside + LPS-challenge (LPS + STE). LPS-challenged groups received an intraperitoneal injection of LPS at 17, 19 and 21 d, whereas the CON and STE groups received a saline injection. The results showed that dietary STE supplementation normalized LPS-induced changes in protein expression of p-NF-κB and p-IκBα, mRNA expression of inflammatory genes (TLR4, NF-κB, and IFN-γ), tight junction-related genes (CLDN2, OCLN, and ZO-1), and antioxidant genes (Nrf2 and HO-1). LPS-induced decreases in serum diamine oxidase (DAO) level, villus height-to-crypt depth ratio, apoptotic index, and protein expression of proliferating cell nuclear antigen (PCNA) were reversed with dietary STE supplementation. Additionally, STE supplementation ameliorated the redox damage by reducing malondialdehyde (MDA) content and increasing total antioxidant capacity (T-AOC) and antioxidant enzyme activity. In conclusion, dietary stevioside supplementation could alleviate LPS-induced intestinal mucosal damage through anti-inflammatory and antioxidant effects in broiler chickens.

Introduction
The main function of intestinal mucosae is to maintain intestinal homeostasis and to prevent pathogenic microorganism invasion. In poultry production, intestinal mucosal damage is a common disease in broiler chickens. Intestinal mucosal damage is attributable to various pathogenic factors, including environmental stress, bacterial infection, immunological stress and oxidative stress [1,2]. Avian pathogenic E. coli (APEC) is a major pathogen in the poultry industry. Lipopolysaccharide (LPS), an endotoxin secreted by APEC, is able to activate toll-like receptor 4 (TLR4). The activation of TLR4 triggers the phosphorylation of nuclear factor kappaB (NF-κB), and eventually promotes the synthesis of pro-inflammatory cytokines and induces oxidative stress [3][4][5]. The immune response of LPS administration is proven to be similar to avian pathogenic E. coli infection on the epithelial cell of chickens [6]. Previous studies have shown that LPS challenge impairs the integrity, permeability,

Animals and Treatment
The animal experiments were performed in accordance with the Animal Care and Use Committee of Nanjing Agricultural University, Nanjing, China (PZ2019064). A total of 192 one-day-old male Ross 308 broiler chicks with similar hatching weights were purchased from a local commercial hatchery. Broilers were randomly allocated to four treatments. Each treatment contained six replicates (cages) of eight broilers per replicate in each group. The present experiment lasted for 21 d (from 1 to 21 d of age). The basal diet used in this study was according to National Research Council (1994) ( Table 1). The four experimental treatments were as follows: (1) non-challenged broilers fed a basal diet (CON); (2) non-challenged broilers fed a basal diet supplemented with 250 mg/kg stevioside (STE); (3) LPS-challenged broilers fed a basal diet (LPS); (4) LPS-challenged broilers fed a basal diet supplemented with 250 mg/kg stevioside (LPS + STE). Stevioside used in this study were purchased from Macklin Inc (Shanghai, China) with a purity of more than 98%. The supplemental stevioside level was optimized according to previous studies [11,14,19]. All broilers were housed in four-level cages in a temperature-and light-controlled room with continuous light in Nanjing Agricultural University. All broilers had ad libitum access to mash feed and water. The temperature of the room was maintained at 32 to 34 • C for a week, and it was then gradually decreased by 1 • C every 2 d until a final temperature of 26 • C was achieved. Furthermore, all broilers were inoculated with a Newcastle disease vaccine on 7 d and with an inactivated infectious bursal disease vaccine on 14 d. The experiment consisted of a 2 × 2 factorial design. The main factors were (1) Lipopolysaccharide (LPS)-challenge, injection with LPS or saline, and (2) diet, basal diet with 0 or 250 mg/kg stevioside. LPS from Escherichia coli (L2880, Sigma Aldrich Inc., St. Louis, MO, USA) was dissolved in 0.9% sterile saline solution. At 7:00 am of 17,19, and 21 d, LPS-challenged groups received an intraperitoneal injection of LPS solution at a dose of 1 mg/kg, whereas the CON and STE groups received a sterile saline injection. The dosage and injection of LPS were referred to as available findings [2,22]. At 17 d and 21 d, all broilers were weighed to calculate average daily gain (ADG). The feed consumption by the broilers in a replicate (cage) was recorded to calculate average daily feed intake (ADFI). The spilled feed was carefully collected and weighed in order to correct the final data of ADFI. Feed conversion rate (FCR) was defined as ADFI: ADG.

Sample Collection
Three hours after injection of LPS at 21 days of age, one broiler in each replicate (six broilers per treatment) with bodyweight close to the average body weight in the respective cage were selected. Blood samples were collected from the wing vein. The serum was then separated after centrifugation at 3000 g for 15 min at 4 • C, and it was stored at −20 • C for further analysis. After euthanasia by CO2 asphyxiation, the jejunum (anterior of Meckel's diverticulum) and ileum (posterior to Meckel's diverticulum) were then gingerly separated. Sections of approximately 2 cm in length were cut off from the middle of each jejunum and ileum. The jejunal and ileal sections were promptly fixed in 4% paraformaldehyde for histological analyses. The jejunal and ileal mucosae were gently scraped by a glass microscope slide from the rest of the jejunum and ileum. The mucosae were stored at −80 • C for the analysis of the oxidative status, gene expression, and protein expression.

Measurement of Serum Diamine Oxidase Activity
Diamine oxidase (DAO) activity in the serum was determined using a commercial reagent kit (Jin Yibai Biological Technology, Nanjing, China) according to the manufacturer's instructions. The intra-assay coefficient of variation (CV) was < 9%, and the inter-assay coefficient of variation was < 10%.

Intestinal Morphology Analysis
After fixation in 4% paraformaldehyde for 24 h, the jejuna and ilea were soaked through a graded series of ethanol and xylene, embedded in paraffin, and sectioned at 5 µm with a Lecia RM2235 microtome (Leica Biosystems Inc., Buffalo Grove, USA). The sections were deparaffinized with xylene and rehydrated through graded dilutions of ethanol, and stained with hematoxylin and eosin. The images of jejuna and ilea were acquired using an Olympus simon-01 microscope (Olympus Optical Co., Ltd., Beijing, China). The values of villus height (VH) and crypt depth (CD) were measured 5 times from different villus and crypts per section from each broiler using the Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Washington, USA). VCR was defined as villus height-to-crypt depth ratio.

Assessment of the Oxidative Status
The amount of 0.2 g frozen mucosa was precisely weighed and homogenized in 2 mL of ice-cold saline. After being centrifuged at 12,000× g for 10 min at 4 • C, the supernatants were collected to measure the oxidative status. The protein content of the supernatants was measured with a BCA Protein Assay Kit (P0010, Beyotime Biotechnology, Shanghai, China). We assessed malondialdehyde (MDA) content, total antioxidant capacity (T-AOC), catalase (CAT) activity, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity in the jejunal and ileal mucosae using commercial reagent kits (S0131, S0121, S0051, S0101 and S0056, Beyotime Biotechnology, Shanghai, China). All experimental procedures were performed according to the manufacturer's instructions. All results were normalized to protein concentration in each sample.

TUNEL Assay
Intestinal apoptosis was determined using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay with a TUNEL BrightGreen Apoptosis Detection Kit (A112, Vazyme Biotech, Nanjing, China) according to the manufacturer's instructions. First, the paraffin sections of jejuna and ilea were deparaffinized, rehydrated, and then incubated with Proteinase K (20 µg/mL) at room temperature for 20 min. Second, the sections were incubated with the TdT enzyme buffer containing double-distilled H 2 O, Equilibration Buffer, BrightGreen Labeling Mix and Recombinant TdT Enzyme at 37 • C for 60 min in the dark. Finally, the sections were stained with 4 ,6-diamidino-2-phenylindole staining solution (C1005, Beyotime Biotechnology, Shanghai, China) for 5 min in the dark. The negative control was performed as above, but without incubation of the TdT enzyme buffer to ensure that no non-specific reaction appeared in the experiment. The images were acquired through an LSM 700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). The numbers of apoptotic cells (green color) and total cells (blue color) were counted using the Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Washington, USA). The ratio of apoptotic cells to total cells represents the apoptotic index.

Western Blot
The jejunal and ileal mucosae were homogenized in Radio Immunoprecipitation Assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) to extract the proteins. Equal amounts of proteins (40 µg) were electrophoresed in 10% (w/v) SDS-PAGE, and then transferred on to polyvinylidene After washing the membranes with TBST buffer for three times, the membranes were incubated with secondary antibody (1:3000, AS003, ABclonal Biotechnology Co., Ltd., Wuhan, China) for 60 min at room temperature. Finally, the blots were developed using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Wilmington, USA), and were visualized using a Luminescent Image Analyzer LAS-4000 (Fuji Film, Tokyo, Japan). The blots were normalized to β-actin. The intensities of the immunoreactive bands were quantified by ImageJ to estimate the protein expressions.

Statistical Analysis
Data were statistically analyzed by two-way ANOVA in a 2 × 2 factorial arrangement with LPS challenge and stevioside supplementation as the main effects and their interaction, using SPSS software (SPSS 20.0, SPSS, Chicago, USA). The Shapiro-Wilk test was used to assess the normality distribution of the data. When the interaction was observed to be significant, Tukey's multiple range tests were applied to examine the statistical differences among different treatments. Differences were considered to be statistically significant at p < 0.05, and 0.05 < p < 0.10 was considered to be a trend towards significance.

Growth Performance
The interaction between STE supplementation and LPS challenge notably affected average daily feed intake (p < 0.05). The LPS-challenged group had a significantly lower (p < 0.05) average daily feed intake than the other groups (Table 3). LPS challenge also reduced (p < 0.05) average daily gain of broiler chickens. No differences (p > 0.05) were observed in the feed conversion rate among any groups.

Intestinal Permeability and Morphology
We performed H&E staining to observe the effects of LPS challenge and STE supplementation on the intestinal morphology of broilers. Almost no damages were found in either the CON group ( Figure 1A,E) or the STE group ( Figure 1B,F). LPS injection could cause obvious hyperemia ( Figure 1C,G, triangles) and neutrophil infiltration ( Figure 1C,G, arrows) in jejunal and ileal mucosae. The LPS + STE group ( Figure 1D,H) had reduced hyperemia and neutrophil infiltration compared with the LPS group. gain of broiler chickens. No differences (p > 0.05) were observed in the feed conversion rate among any groups. Means (n = 6) with different letters within a row are significantly different (p < 0.05).

Intestinal Permeability and Morphology
We performed H&E staining to observe the effects of LPS challenge and STE supplementation on the intestinal morphology of broilers. Almost no damages were found in either the CON group ( Figure 1A,E) or the STE group ( Figure 1B,F). LPS injection could cause obvious hyperemia ( Figure  1C,G, triangles) and neutrophil infiltration ( Figure 1C,G, arrows) in jejunal and ileal mucosae. The LPS + STE group ( Figure 1D,H) had reduced hyperemia and neutrophil infiltration compared with the LPS group. The interaction between STE supplementation and LPS challenge notably affected serum DAO level (p < 0.05). The LPS-challenged group had a higher (p < 0.05) serum DAO level than the non-challenged groups (Table 4). STE supplementation reduced the serum DAO level of LPS-challenged broilers (p < 0.05).
As shown in Table 4, significant interactions between STE supplementation and LPS challenge were observed for CD and VCR in both jejunum and ileum (p < 0.05). LPS challenge significantly decreased VH and VCR, but increased CD in both jejunum and ileum (p < 0.05). Dietary The interaction between STE supplementation and LPS challenge notably affected serum DAO level (p < 0.05). The LPS-challenged group had a higher (p < 0.05) serum DAO level than the non-challenged groups (Table 4). STE supplementation reduced the serum DAO level of LPS-challenged broilers (p < 0.05).
As shown in Table 4, significant interactions between STE supplementation and LPS challenge were observed for CD and VCR in both jejunum and ileum (p < 0.05). LPS challenge significantly decreased VH and VCR, but increased CD in both jejunum and ileum (p < 0.05). Dietary supplementation of STE significantly reduced CD and increased VH and VCR in both jejunum and ileum (p < 0.05).

Protein Expression of NF-κB, p-NF-κB, IκBα and p-IκBα
We observed notable interaction (p < 0.05) between STE supplementation and LPS challenge on the protein expression of ileal p-IκBα, jejunal NF-κB, p-NF-κB, and p-IκBα in the intestinal mucosae of broilers ( Figure 2). LPS-challenged group exhibited extremely higher (p < 0.05) expression in jejunal NF-κB, p-NF-κB, and p-IκBα. But STE supplementation significantly reduced (p < 0.05) the expression of these proteins. The LPS challenge also increased (p < 0.05) the protein expression of p-IκBα in the ileal mucosa, while STE supplementation reduced it (p < 0.05). Moreover, LPS administration elevated (p < 0.05) the protein expression of p-NF-κB in the ileal mucosa, whereas STE supplementation had no protective effect on it (p > 0.05).

Tight Junction-Related Genes Expression
We observed a significant interaction (p < 0.05) between STE supplementation and LPS challenge on the mRNA expression of CLDN2, OCLN, and ZO-1 in the jejunal mucosa of broilers ( Table 6). The mRNA abundances of CLDN2, OCLN, and ZO-1 in the jejunal mucosa decreased (p < 0.05) in the LPS group, while no significant differences were observed in the LPS + STE group compared with the CON group (p > 0.05). LPS challenge resulted in decreased (p < 0.05) expression of OCLN, whereas STE supplementation had no significant effect in ileal OCLN expression (p > 0.05). There was no effect of either LPS challenge or STE supplementation on CLDN1, CLDN2, and ZO-1 expression in the ileal mucosa (p > 0.05).
Effects of stevioside supplementation on intestinal mucosal oxidative status of LPS-challenged broilers.

Antioxidant Gene Expression
As shown in Table 8, there was an LPS × STE interaction for the mRNA expression of SOD2 and Nrf2 in the jejunal mucosa of broilers (p < 0.05). LPS challenge significantly decreased the expression of SOD1, CAT, and HO-1 in both jejunal and ileal mucosae (p < 0.05). LPS-challenged broilers also showed lower expression of SOD2, GPX1 and Nrf2 in the jejunal mucosa (p < 0.05). In addition, supplementation with STE significantly increased the expression of SOD1, CAT, and Nrf2 in both jejunal and ileal mucosa (p < 0.05). The expression of jejunal HO-1 (p = 0.052) and ileal GPX1 (p = 0.086) tended to be increased by STE supplementation. LPS challenge had no effect on the mRNA expressions of ileal SOD2, GPX1, and Nrf2 (p > 0.05). There was no effect of STE supplementation on SOD2 expression in the jejunal mucosa (p > 0.05).

Apoptotic Index by TUNEL Assay
The apoptotic cells were primarily distributed to the apical region of the jejunal and ileal villus ( Figure 3A,B). A significant interaction (p < 0.05) between STE supplementation and LPS challenge was observed for the apoptotic index in both jejunal and ileal mucosae ( Figure 3C,D). LPS-challenged broilers exhibited a greater percentage in both jejunal and ileal mucosae than the other groups (p < 0.05). In contrast, the LPS + STE group had a lower apoptotic index in both jejunal and ileal mucosae than the LPS group (p < 0.05).

Protein and mRNA Expression of PCNA
We observed a significant interaction (p < 0.05) between STE supplementation and LPS challenge on the protein and mRNA expression of PCNA in the jejunal mucosa ( Figure 4). LPS challenge significantly decreased the protein and mRNA expression of PCNA in both jejunal and ileal mucosae (p < 0.05). Dietary STE supplementation increased the protein expression of PCNA in both jejunal and ileal mucosae (p < 0.05). There was no effect of STE supplementation on the mRNA expression of PCNA in the ileal mucosa (p > 0.05).

Protein and mRNA Expression of PCNA
We observed a significant interaction (p < 0.05) between STE supplementation and LPS challenge on the protein and mRNA expression of PCNA in the jejunal mucosa ( Figure 4). LPS challenge significantly decreased the protein and mRNA expression of PCNA in both jejunal and ileal mucosae (p < 0.05). Dietary STE supplementation increased the protein expression of PCNA in both jejunal and ileal mucosae (p < 0.05). There was no effect of STE supplementation on the mRNA expression of PCNA in the ileal mucosa (p > 0.05).

Discussion
Intestinal health is essential for the growth of animals. Many pathogenic factors, including oxidative stress and pathogenic bacterial challenges, especially APEC, can damage the intestinal barrier and cause intestinal inflammation [7,23,24]. Ample pieces of evidence have proven that LPS is able to disrupt the intestinal barrier of broilers by bacterial challenge, which could cause a reduction in feed intake [25][26][27]. In addition, LPS challenge induced obvious hyperemia and more neutrophil infiltration. Mucosal hyperemia is the main change in chickens that responded with intestinal lesions [28]. Neutrophil infiltration is a prominent feature of inflammatory reactions. In keeping with our findings, Li et al. [8] found that a greater amount of neutrophils could be observed in the duodenum of LPS-challenged broilers. Additionally, several studies have demonstrated that LPS is able to induce gut inflammatory responses in broilers by increasing pro-inflammatory cytokine levels [2,7,8]. The release of pro-inflammatory cytokines requires the activation of TLR4 [29]. TLR4 serves as a receptor of LPS, and LPS can combine with it to activate a TLR4/MyD88/NF-κB signaling pathway in the intestine epithelial cell [30]. In this study, the transcription level of pro-inflammatory genes and the protein expression of p-NF-κB significantly increased in the intestinal mucosae of LPS-challenged broilers. NF-κB plays a crucial role in regulating inflammation

Discussion
Intestinal health is essential for the growth of animals. Many pathogenic factors, including oxidative stress and pathogenic bacterial challenges, especially APEC, can damage the intestinal barrier and cause intestinal inflammation [7,23,24]. Ample pieces of evidence have proven that LPS is able to disrupt the intestinal barrier of broilers by bacterial challenge, which could cause a reduction in feed intake [25][26][27]. In addition, LPS challenge induced obvious hyperemia and more neutrophil infiltration. Mucosal hyperemia is the main change in chickens that responded with intestinal lesions [28]. Neutrophil infiltration is a prominent feature of inflammatory reactions. In keeping with our findings, Li et al. [8] found that a greater amount of neutrophils could be observed in the duodenum of LPS-challenged broilers. Additionally, several studies have demonstrated that LPS is able to induce gut inflammatory responses in broilers by increasing pro-inflammatory cytokine levels [2,7,8]. The release of pro-inflammatory cytokines requires the activation of TLR4 [29]. TLR4 serves as a receptor of LPS, and LPS can combine with it to activate a TLR4/MyD88/NF-κB signaling pathway in the intestine epithelial cell [30]. In this study, the transcription level of pro-inflammatory genes and the protein expression of p-NF-κB significantly increased in the intestinal mucosae of LPS-challenged broilers. NF-κB plays a crucial role in regulating inflammation and cell death, and it is inhibited by IκB. The phosphorylation of serine residues on IκBα could degrade IκBα, which, in turn, induces phosphorylation and nuclear translocation of NF-κB [31]. Phosphorylated NF-κB activated by TLR4 or TNF-α signaling could subsequently lead to the synthesis and release of pro-inflammatory cytokines [32]. However, dietary STE supplementation was capable of alleviating the phosphorylation of IκBα and NF-κB, which resulted in normalizing the transcription level of pro-inflammatory cytokines in the intestinal mucosae of broilers. Similarly, Casas-Grajales et al. [11] reported that STE could prevent liver inflammation in rats by reducing the expression level of NF-κB, TNF-α and pro-inflammatory cytokines (IL-17a, IL-1β, and IL-6). Boonkaewwan et al. [16] demonstrated that STE inhibited LPS-induced synthesis of pro-inflammatory cytokines in human colonic epithelial cells. Alavala et al. [33] indicated that STE was beneficial for treating ulcerative colitis by inhibiting NF-κB and MAPK pathways. Diterpenoid moiety in the structure of STE may contribute to the anti-inflammatory activity in STE [34,35]. Thus, our results suggest that dietary STE supplementation could attenuate LPS-induced inflammation by attenuating the TLR4/NF-κB signaling pathway in the intestine of broilers.
Intestinal integrity is a key factor for preventing pathogenic microorganism invasion in broiler chickens. We found that the LPS challenge severely impaired the intestinal morphology in broilers, as indicated by decreased VH and VCR in both jejunum and ileum. The VH is related to the ability to absorb nutrients in the intestine. A high VCR is widely believed to be a good index of mucosal turnover, and it is associated with the strong ability of digestion and absorption [7,36]. LPS-induced structural damage of the intestinal mucosal barrier was reversed with dietary STE supplementation. It has been reported that natural extract with anti-inflammatory effect can restore the damaged intestinal morphology in LPS-challenged broilers [37,38]. The down-regulation of TLR4/NF-κB signaling and reduced mRNA expression of pro-inflammatory cytokines might be one of the reasons that intestinal integrity improved in the LPS + STE group. Our results indicate that dietary STE supplementation can ameliorate the impaired intestinal integrity in LPS-challenged broilers.
DAO is an intracellular enzyme produced by the intestinal epithelium and mainly exists in the intestinal mucosae [39]. Once the intestinal mucosal barrier is impaired, DAO is released into the systemic blood. Thus, serum DAO activity is a marker for intestinal permeability. Our result was accorded with the previous finding that LPS impaired the intestinal permeability [7,40]. Furthermore, intestinal mucosal barrier functions are mainly regulated by tight junctions [41]. The reduced expression level of tight junctions caused by LPS challenge could affect intestinal permeability and cause pathological states [42]. This result was consistent with increased serum DAO activity. Additionally, previous studies have demonstrated that increased IFN-γ and IL-1β would damage the tight junctions and disrupt intestinal permeability [43,44]. However, dietary STE supplementation repaired the intestinal permeability. The exact mechanisms for this effect of STE require further investigation. Our data reveal that STE administration could restore the disrupted intestinal permeability of LPS-challenged broilers.
The LPS challenge can not only induce inflammation but also induce oxidative stress [45,46]. Increased protein expression of p-IκBα and p-NF-κB also reflected that strong oxidative stress was induced by LPS challenge [47]. Oxidative stress is a pivotal factor for disrupted mucosal barrier function [48]. MDA is the chief oxidative degradation product, and it reflects the level of lipid peroxidation [49]. The antioxidant defense system mainly consists of T-AOC, CAT, SOD, and GSH-Px. T-AOC is one of the most important indices reflecting the total antioxidant capacity as a single measure [50]. CAT, SOD, and GSH-Px act as free radical scavengers to decompose H 2 O 2 . In this study, LPS induced lipid peroxidation damage in the intestinal mucosae. Our result of decreased MDA content by STE supplementation was consistent with the previous finding that STE significantly reversed the lipid peroxidation in the liver of the LPS-challenged rat [49]. Increasing evidence has shown that STE exerts potent antioxidant properties in vivo and in vitro [11,49,51,52]. In agreement with these findings, we also demonstrated that STE markedly increased the total antioxidant capacity, and reduced oxidative stress in the intestinal mucosae of LPS-challenged broilers. The transcription level of SOD1, SOD2, CAT, and GPX1 coincided with the data of antioxidant enzyme activity. Moreover, HO-1 is a major antioxidant enzyme, and its transcription is primarily under the control of Nrf2 [53]. It is well established that the Nrf2-HO-1 pathway is critical for redox balance. Our result of increased mRNA expression of Nrf2 and HO-1 suggested that STE could exert antioxidant effect via activation of Nrf2-HO-1 signaling. Similarly, a previous study has indicated that rebaudioside A, one of the steviol glycosides besides STE, considerably induces the Nrf2 cascade [54]. Casas-Grajales et al. [11] have also demonstrated that STE is able to prevent thioacetamide-induced liver damage through up-regulating Nrf2, thus preserving the normal redox status in rats. Based on these results, we hypothesize that STE exerts its antioxidant potential by inducing the Nrf2 cascade and enhancing the antioxidant enzyme activity. However, further functional studies are required to validate this hypothesis.
The balance of cell apoptosis and proliferation are important for maintaining the turnover of the intestinal mucosal epithelium [55]. Inflammation and oxidative stress can induce excessive apoptosis and declined proliferation, which could lead to intestinal barrier dysfunction [56,57]. In the present study, we observed apoptosis was promoted in the intestinal mucosae of LPS-challenged broilers using a TUNEL assay. Supportively, LPS increased pro-apoptotic Bax and Caspase-3 expression and decreased anti-apoptotic Bcl-2 expression in the duodenal mucosa of broilers [8]. However, enhanced apoptosis induced by LPS injection was alleviated by STE administration. Emerging evidence has indicated that STE has an anti-apoptotic effect on rodents [49,58,59]. Our results of decreased NF-κB expression level and reduced apoptosis were in agreement with a previous finding that STE could suppress apoptosis via a mechanism involving ERK1/2, STAT3 and NF-κB suppression [59]. The reduced apoptosis of LPS-challenged broilers might also be attributable to the anti-inflammatory and antioxidant effects of STE supplementation. On the other hand, both protein and transcription levels of PCNA (an indicator for cell proliferation) in the intestinal mucosae significantly declined after LPS injection. This decline of PCNA was also counteracted by STE supplementation. Similarly, a previous study showed that STE substantially increased cell proliferation in the LPS-stimulated T-and B-lymphocytes ex vivo [18]. STE administration could enhance macrophage function and resulted in modulating the T and B cell proliferation [18]. This increases the possibility that STE could modulate cell proliferation through immunomodulatory activity. Hence, our results suggest that STE can inhibit apoptosis and promote cell proliferation in the intestinal mucosae of LPS-challenged broilers.
Interestingly, our data showed that dietary STE supplementation had better effects on LPS-challenged jejunum than ileum on p-NF-κB expression, the transcription level of tight junction, and pro-inflammatory cytokines. A previous study has reported that the LPS challenge could cause numerous changes in microflora [60]. Since ileum has more abundant microflora, LPS might induce more irreversible damage to the ileal mucosa. Furthermore, stevioside is hydrolyzed to steviol by the intestinal microflora [61]. It has been shown that stevioside could suppress the activation of NF-κB and the release of TNF-α and IL-1β induced by LPS in THP-1 cells, whereas steviol did not have the same effect [62]. We presume that after long detention time in the intestine, more part of stevioside is hydrolyzed to steviol in the ileum. Stevioside might exert better anti-inflammatory and antioxidant effects on the intestinal mucosae than steviol, which could explain the differences in the effects of stevioside in different intestinal segments. This speculation requires further investigation.

Conclusions
In conclusion, dietary stevioside supplementation could alleviate LPS-induced chicken intestinal mucosal damage by ameliorating inflammation and improving the antioxidant status of intestinal mucosae. Our results have shown that stevioside has anti-inflammatory and antioxidant effects. Therefore, stevioside can be used as a feed supplement in preventing intestinal inflammatory disease in broiler chickens.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Rice Collectors’ Attitude Towards Purchasing of Hybrid Rice in Myanmar

Hybrid rice is one of the most feasible options for increasing rice reproduction and Myanmar has area expansion under hybrid rice production. However, hybrid rice is a very new seed industry but social perceptions are not favorable. The objectives of the study were: 1) to study the characteristics of the rice collectors and their performance on purchasing of hybrid rice, 2) to identify the determinants for changing attitudes of willing to purchase by rice collectors. The study was conducted in Nay Pyi Taw area, Myanmar and used structured questionnaires to collect data from 61rice collectors. Data was analyzed by using descriptive statistics and Likert-type scale. This study revealed that majority 75.4% of respondents were rice brokers and 24.6% of them were miller collectors. Then, 82.0% out of respondents were male and respondents’ average age was 49 years. Next, 72.4% of rice collectors had formal education and 27.9% rice collectors possessed Bachelor degree. Besides, more than half 52.5% of rice collectors were above 10 years buying experience. Majority 91.8% of rice collectors bought above 836 tons in monsoon and over 2090 tons in summer paddy. Moreover, 63.9% of collectors received information to purchase from “hybrid rice seed companies”, less than half of respondents received information from “extension workers”, from “contract farming, from “fellow buyers” and accepted the information of “price less than local varieties”, those points were mentioned as “very important or important”. In addition, majority 82% of rice collectors stated “strongly agreed” or “agree” to the point of “need to grow hybrid rice for rice industry development”, more than half 65.5% of respondents expressed strongly agree or agree to the point of “hybrid rice is more profitable than inbred rice”, 86.9%of respondents mentioned strongly agree or agree to the point of “enhancing quality of hybrid rice varieties”, 70.5% of respondents pointed out to “more milling outcome and avoid chalkiness”, 85.3% of respondents strongly agree or agree to “hybrid rice definitely advantages of outputs and export easily” and 85.2% of rice collectors expressed strongly agree or agree to the point of “ I am willing to pay extra money if it is good in quality. Overall results of this study, rice collectors have desired to buy the unique paddy rice grain shape, color, chalkiness, white belly, and milling performance of grain translucency that drive the price and their decision for purchasing. This study highlighted the areas needed to improve such as establishment of a well-structured research programs to develop new consumers’ preferable hybrid rice varieties, public ICT system for agriculture, and advertising campaign persuaded by extension services and communication channels, marketing system and quality seed control system.

Introduction
This study was an integral part of the whole research of hybrid rice supply change and influential attitudes of rice collector's willingness to purchase hybrid rice and their perceptive to develop hybrid rice technology. DOA (2017) mention that in Myanmar total rice growing area was 7 million hectare, over 29.26 million tons of production and yield of 3.84 ton/ha in 2016-17 growing season [1]. IRRI (2017) reported rice is the staple food and main energy source of the Myanmar people. Rice consumption per capita is approximately 154 kilograms per year [2]. Denning et al (2003) reported Myanmar consumers are sensitive to eating depend on quality. People prefer the rice which is not soft and hard after cooking. Besides, there are two components to seed selection: choice of variety and quality of seed. The choice of variety is made by farmers based on: (1) adaptation to the growing environment, (2) eating/cooking preferences of the consumers, (3) market preference/price, and (4) cost of seed. Modern rice varieties (also known as HYVs) are variously reported to be used for 70-80% of the monsoon crop and for virtually all summer crops [3]. IRRI (1997) showed in Hybrid Rice Breeding Manual; hybrid rice has the potential of yielding 15-20% more than that of best inbred varieties grown under similar conditions [4]. DOA (2015) reported in Myanmar, hybrid rice cultivation was commercially initiated again in 2011, monsoon season and increased areas year by year up to 2.1% of total rice growing area. Besides, it could be successful 10 ton/ha (potential yield) if it had systematic and proper care and consequently resulting in alleviating of poverty and better living standard of Myanmar farmers. There is changing variety for superior output production, it is needed to choose for hybrid rice production in Myanmar [5].
In Myanmar, rice is generally collected by wholesalers and wholesalers contact to collectors and brokers who typically collect paddy, directly from farmers in small scale but in some regions famers directly sell their paddy go to the wholesalers or to the millers. Some wholesalers themselves are rice millers, some resell the purchased paddy to the rice millers. Rice millers delivered to retailer for the domestic consumption and exporters for international markets. Some wholesalers purchase rice from main regions and sell to retailers. Some exporting companies themselves are millers and wholesalers and they also purchase large amount, milled rice according to the grades and then export, the involvement of private sectors in milling, trading and processing also solves the problem of existing multi-layer and compacted distribution system that cause inefficiency in the marketing system. According to Nay (2012) mentioned in his paper that farmers are selling after harvest, directly to the millers without or reducing the use of brokers and local traders in some township. This is increasing return for farmers. This makes it difficult for Myanmar to ensure a stable position on the international market, and there is no consistent business flow from the producers through collectors to consumers/export [6]. MRF (1014) showed that the private sector composed of farmers, input suppliers, traders, millers, processors, and exporters, drive the extensive rice value chain. Individual entrepreneurs to large wholesalers and exporters are involved in production, collection, processing, marketing, and distribution [7]. Hazra (2001) supported grain shape and appearance, milling and head rice recovery, amylose content and gelatinization temperature (alkaline value) are some of the parameters that influence the cooking and eating qualities of rice. All hybrids released, including those marketed by the private sector, were evaluated for quality traits. A good quality hybrid has:1) head rice recovery of over 55%; 2) intermediate amylose content (20-25%); 3) intermediate alkaline value (4.0-5.0); and 4) translucent grains without chalkiness [8].
Therefore, rice collectors evaluated for quality characteristics of hybrids before purchasing rice which is used almost exclusively as a food product. Cooking and eating quality traits therefore assume special significance. For general, consumer acceptance is essential that the hybrids developed good quality traits as well as high yielding potential. Therefore, the hybrids included in development of parental lines have been critically screened for various quality parameters. Preferences for quality vary from region to region. A variety of hybrid liked in one region may not be liked in another. Therefore, it is imperative to bear in mind local preference when developing hybrids. Smith (1982) mentioned that attitudes are related to how people perceive the situations in which they find themselves. Also, attitudes vary in direction (either positive or negative), in degree (the amount of positiveness or negativeness), and in intensity (die amount of commitment with which a position is held, [9]. Smith and Ragan (1999) expressed the role of prior attitudes is in shaping attitude formation and change. They describe attitude as a kind of spectrum with "latitude of acceptance" surrounding a current attitude; a new position is more likely to be accepted if it falls within this latitude and less likely to be accepted if it does not [10].
Farmer complained to grow hybrid rice due to the market problem, low grain price and poor eating quality. On the other hand, rice collectors' attitudes to buy or collect hybrid rice grain according to their preferable characters and collect from farmers. Therefore, study objectives were as follows; (1) to study the characteristics of the rice collectors and their performance on purchasing of hybrid rice, (2) to identify the determinants for changing attitudes of willing to purchase by rice collectors Hybrid rice cultivation and production in Study area DOA (2016/17) reported that in Nay Pyi Taw area, rice has been grown two-times per year as monsoon rice and summer rice by farmers who were growing hybrid rice and inbred rice. Therefore, rice collectors have bought the inbred rice and also hybrid rice. There was (53876) total rice growing farmers representing (66266) hectares in Nay Pyi Taw area, in 2016-17. Out of which (51597) farmers were inbred rice growing farmers and (2279) farmers were hybrid rice growing farmers. Table 1 reported that there were grown maximum (5197) hectares in 2014-15 year but it was going down the cultivated area and also production, [11]. It was commonly grown hybrid rice varieties such as GW1, Long 8, Long 10 and Long 12, characteristics of these hybrid rice varieties were particularly translucent, low amylose and good eating quality, local people was not prefer due to so soft and chalkiness of rice although it has potential to get more milling outcome. On the other hand, local inbred rice varieties were Manathukha, Sinthukha, Thainankauk and Ayeyarmin which were usually collected by rice collectors whenever farmers sell to those varieties, Table 2.

Research Methodology
Study was conducted at Nay Pyi Taw area including Pyinmana, Lewe, Dekhinathiri, Zabuthiri, Tatkone, Pobbathiri, Zeyarthiri, and Ottarathiri Townships. Data was collected from Rice Miller Association and DOA Township officers' offices, Table 3 was Sixteen one rice collectors were randomly selected and collected the information in the survey which covered features of collectors such brokers and miller collectors were interviewed by using quaternaries between August to September, 2018.The collected data were demographic factors, purchasing experience and buying amount of rice, preferable type of rice, information sources, market situation, rice collectors' opinion on hybrid rice, and rice collectors' attitude to change to hybrid rice, determinants and their future perception to buy hybrid rice. Hogg and Vaughan (2005) pointed out that an attitude is "a relatively enduring organization of beliefs, feelings, and behavioral tendencies towards socially significant objects, groups, events or symbols" [12]. McLeod (2018) expressed that attitudes can serve functions for the individual as "Attitudes and behavior", [13]. Katz (1960) showed that outlines of four functional areas: The knowledge function is our need for a world which is consistent and relatively stable. This allows us to predict what is likely to happen, and so gives us a sense of control. Attitudes can help us organize and structure our experience, knowing a person's attitude helps us predict their behavior. Self / Ego-expressive function: The attitudes we express (1) help communicate who we are and (2) may make us feel good because we have asserted our identity. Self-expression of attitudes can be nonverbal too: Therefore, our attitudes are part of our identity and help us to be aware through the expression of our feelings, beliefs and values. Adaptive function: If a person holds and/or expresses socially acceptable attitudes, other people will reward them with approval and social acceptance. The ego-defensive function: attitudes that protect our self-esteem or that justify actions that make us feel guilty. The basic idea behind the functional approach is that attitudes help a person to mediate between their own inner needs (expression, defense) and the outside world (adaptive and knowledge). The basic idea behind the functional approach is that attitudes help a person to mediate between their own inner needs (expression, defense) and the outside world (adaptive and knowledge), [14].
In order to achieve the objectives, it was analyzed by simple descriptive and Likert scale to measure attitude [15]. The typical Likert scale includes a 5-7 point ordinal scale to rate the degree to which they agree or disagree with the statement by respondents. Data were ascertained utilizing a five-point "Likert-type" scale concerning one to five levels of "agreement": 1) "Strongly agree", 2) "Aagree",3) "Neutral") "Disagree" and d) "Strongly disagree".

Demographic Profile of Rice Collectors
In this study found out that two third of 75.4% of rice collectors were rice brokers and 24.6% of them were miller collectors. Majority 82.0% of respondents were male rice collectors and 18.0% of them were female rice collectors. Next, their average age was 49 years, ranging from a minimum of 30 years to a maximum age of 68 years. More than half 54.1% of rice collectors were under high school, 27.9% rice collectors possessed Bachelor Degree and a few 18.0% of rice collectors finished the high school. Besides, regarding the rice buying experience; 23.0% of respondents had 1 to 5 years buying experience and 24.6% of respondents had 6 to 10 years, while 8.2% of rice collectors had 11 to 15 years and 23.0% of rice collectors had 16 to 20 years and the remaining of 21.3% of rice collectors had above 21 years experiences. Moreover, majority 91.8% of rice collectors bought above 836 tons in monsoon and over 2090 tons in summer paddy, 3.3% of small collectors purchased 105-314 tons and 1.6% of rice collectors collected 315 to 627 tons, while 3.3% of collectors bought 628 to 836 tons yearly, (Table 4).

Important Information Sources of Rice Collectors
This study revealed that rice collectors connected to information sources for purchasing of rice fromfarmers. A few 3.3% and 16.4% of rice collectors showed 'very important and important' information source to "Farmer Economic Journal", while 32.8% and 41.0% of rice collectors mentioned less important and not important, 6.6% of rice collector did not know to this Journals. Next, rice collectors made friendship with extension agents who give the information to buy rice. Therefore, rice collectors were supported the information by "extension workers" that a few (6.6%) and 36.1% of rice collectors appreciated to extension agents who expressed "very important" and important, but 19.7% and 32.8% of rice collectors mentioned less important and not important, while 4.9% of rice collectors did not gain to access to extension agents. Besides, information from "hybrid rice seed Co., Ltds" to buy hybrid rice; most 42.6% and 21.3% of rice collators answered "very important, and important, while 26.2% and 4.9% of rice collectors demonstrated less important and not important, a few 4.9% of rice collectors did not communicate to seed companies. Moreover, Seed Co.,Ltds engaged rice contract farming with farmers. It was found out the information of rice purchasing from "contract farming"; 19.7% and 24.6% of collectors mentioned very important and important but 27.9% and 19.7% of rice collectors showed less important and not important, while 8.2% of rice collectors did not notice to contact farming practice. In addition, information from "fellow rice collectors"; almost 14.8% and 23.0% of collectors indicated very important and important, while 31.1% and 23.0% of collectors showed less important and not important, 8.2% of collectors directly bought from farmers. A few 6.6% and 1.6% of collectors disclosed very important and important, however more than half 54.1% of rice collectors responded less important and 39.3% of rice collectors responded not important to the point "market information" and 4.9% of collectors normally bought rice harvesting season. Furthermore, a few 6.6% and 8.2% of rice collectors mentioned very important and important to export market available, on the other hand, more than half 54.1% of collectors and 18.0% collectors declared not important, while 13.1% of rice buyers explained that this is their seasonal work so they were far away from export markets. Initially, rice collectors had opinion likely to buy hybrid rice the information was "price less than inbred rice"; almost 31.1% and 16.4% of collectors preferred to show very important and important but 16.4% and 21.3% of rice collectors mentioned less important and not important, while 14.8% of rice collectors did not mention because rice price was depend on the quality they responded (Table 5).  Table 6 indicated that 68.85% of collectors preferred to buy long grain rice, fragrant and good eating quality. Then 29.51% of respondents wished to buy normal moisture when they were buying and half of 50.81% of rice collectors selected to buy good grain color rice. Some rice collectors reported their opinion on willingness to purchase was depends on local markets or export potential. Some of them intended to buy hybrid rice to export china market. Local consumers appreciated that hybrid rice namely Long 8 and GW1 around after 6.-8 months stored, hybrid rice paddy grains were accepted to eat and upland people likely to eat mostly because they were pleased to have softening of cooked rice. Therefore, some collectors send the milled hybrid rice to hilly regions. As a result, hybrid rice grains begin acceptable to buy among the rice collectors.

Rice Collectors' Attitudes to Hybrid Rice Development
This study revealed that majority 82% of rice collectors showed "strongly agree" or "agree" to the point of "grow more hybrid rice for rice industry development", but (11.5%) were "disagree" and no one showed "strongly disagree", while (6.5%) of respondents did not know hybrid rice was important or unimportant for rice industry. Next, more than half 65.6% of respondents showed "strongly agree" or "agreed" to the point of "hybrid rice is more profitable than local varieties" and almost 31.2% of respondents answered "disagree" or "strongly disagree", the remaining (3.3%) of rice collectors did not mention to this point. Besides, half of (50.8%) of respondents expressed "strongly agree" or "agree", but 36.1% and 6.6% of rice collectors responded "disagree" and "strongly disagree", while (6.6%) of respondents did not mention to the point of "I always pay price more than inbred rice price". In addition, majority 86.9% of respondents were "strongly agree or agreed to that point of "enhancing quality of hybrid rice", while 1.6% and 3.3%, of respondents showed "disagree and strongly disagree" to that point. Most of 70.5% of the respondents answered strongly agree or agree to point of" more milling outcome and avoid chalkiness", 24.6% of rice collectors showed strongly disagree or disagree and 4.9% rice collectors did not remark to this point. Rice collectors' attitude to point of "Hybrid rice is normally less management of post harvest time"; 52.5% of rice collectors indicated strongly agree or agree, 40.9% of rice collectors reported strongly disagree or disagree and 6.6% of rice collectors did not mention their opinion. In regard to "hybrid rice is less labor used than inbred rice when buying"; 29.5% of rice collectors remarked strongly agree or agree, more than half 63.8% of rice collectors commented to strongly disagree or disagree, while a few 6.6% of respondents did not respond to this point. Furthermore, majority 85.3% of respondents were "strongly agree or agreed" to the point of "hybrid rice definitely advantage of output than local rice and export easily" and a few (6.6%) of respondents mentioned "disagree", while (8.2%) did not mention respectively. Lastly, regarding the point of "I am willing to pay extra money if I know it is good in quality", out of 85.2% of rice collectors stated strongly agree or agree, while (1.6%) and (11.5%) of respondents expressed strongly disagree and disagree, remaining of (1.6%) did not show their opinion, Table 7.

Addition Information
Rice collector reported that they preferred to buy the hybrid rice varieties of long and slender grain type, bright yellow color and good eating quality. As a result, Department of Agricultural Research (DAR) and other Research Institutions needto develop these types of rice varieties from breeding programs. Besides, Seed Co.,Ltds also produce seeds which persuades the farmers and consumers. Rice collectors reported that the additional information as good seed quality and color, recommended fertilizers rate for hybrid rice, resistance varieties and adaptable to weather condition, increase productivity and export market availability, standard weight, prohibit growing mixed F 1 and F 2 seed. Nevertheless, farmers' choices were important and stable market is more important for development of hybrid rice. On the other hand, this study indicated that rice collectors explained why they were not to buy the hybrid rice because of farmers did not grow their buying area so they have not chance to buy. They reported that a few farmers grew F 2 and some grew mixed F 1 seeds and F 2 . Therefore, they were afraid of inadequate purity of rice. Moreover, rice collector pointed out their perceptions that farmers faced the problem such as labors, investment, and cost which were more needed for hybrid. So far, farmers need credit to supports more and knowledge for systematic cultivation, method demonstration through contact farmers. High price of hybrid seed, poor seed quality were also reasons for unwillingness to buy hybrid rice seeds seasonally. Due to susceptible to pests and diseases of hybrid rice, farmers want to get resistant varieties to solve these problems. In addition, farmers need medium duration varieties for monsoon and some farmers need short duration varieties with high yield for summer season.
MRF (2014) reported that the current loan program of the Myanmar Agricultural Development Bank (MADB) for rice farmers allows them to borrow 250 $/ha and provides credit for six months only. Their access to additional financing is also very limited. This means that farmers have to sell their entire rice stock immediately after harvesting when the prices are at their lowest to repay their debts [7].
Besides, Seed Co.,Ltds produced hybrid varieties such as long 8 and long 10 and long 12 and GW1. Therefore, these will be needed to check for adequate purity before delivering seeds to farmers. This means, there are seed law enacted but weak in implementation so there is need to more solve to inadequate seed purity and related problems. Ginigaddara et al, (2017) indicated that eating quality related factors and nutritional factors are the most important factors predicting consumer preference towards consuming traditional rice. Other important factors are marketing factors and cooking quality related factors. Further, it was found that awareness on traditional rice is the main factor influencing on deciding to purchase of traditional rice. Urban consumers purchase traditional rice more compared to rural consumers. When consumer is educated, there is a higher purchasing decision making power than a non-educated consumer. Overall results imply that traditional rice has a good perception in general among rice consumers in Kegalle district in Sri Lanka, [16]. Spielman et al, (2013) supported that although hybrid rice has immense potential for increasing productivity and improving overall welfare for the poor in India, the challenges are not insignificant. Important challenges include increasing both seed and grain quality and customizing varieties to various agro ecological conditions and consumer preferences. Addressing the challenges of grain quality and customizing hybrids to consumer preferences have important implications for the output prices that farmers receive for their grains. At present, the price penalty on hybrid rice at the farm gate places it at 10-20% less than coarse grain rice in India. Although breeders have made progress in customizing hybrids to consumers' preferences, the new hybrids coming on the market will need to overcome this price penalty to encourage adoption, [17]. Uddin (2014) reported that the market price of hybrid rice grain has not yet gained its positive impact in comparison to inbred rice grain. General consumers in Bangladesh preferred long slender grains with high amylose percent and better cooking and keeping quality. Local OP variety, due to superior taste, gives higher market price compared to hybrids, [18]. Akhter et al, (2004) mentioned in his report that hybrids imported so far are of inferior grain quality that is unacceptable for farmers due to lower prices in local and international market, [19]. Sivagnanam (2004) found out that demand for the hybrid rice is relatively low in the market and they discourage these varieties by offering low price. Lower price is the major challenge the farmers are facing in selling the hybrid variety. The other perceptions prevailing about the hybrid varieties include lack of consumer demand for these varieties, poor cooking and preservation and getting broken during the milling stage, [20].

Conclusion and Recommendation
This study revealed that rice collectors pleased to buy good eating quality, good color, and long grain and fragrant. Then, this study resulted positively agreed the sources of information to collect the rice were from "extension workers", "hybrid rice seed Co.,Ltds" and "contracted farming". Next, this study highlight that rice collectors' attitudes to hybrid rice development related factors positively agreed to; "farmers need to grow hybrid rice more for rice industry development" and "hybrid rice is more profitable than inbred rice". Besides, they want to do "I always pay price more than inbred rice price" and positively pointed to "enhancing quality of hybrid rice varieties", should get "milling outcome than inbred and avoid the chalkiness and white bally". Moreover, some farmers also granted the hybrid rice normally "less management of post harvest time and labor less than inbred rice when buying". In addition, they positively perceived that hybrid rice is definitely "advantages of outputs than inbred rice and export easily" and they want to "pay extra money if they know it is good in quality". Overall results of this study highlighted that, rice collectors have desired to buy the unique paddy rice grain shape, color, chalkiness, white belly, and mill performance of grain translucency that drive the price and their decisions for purchasing.
Recommendations for the development of hybrid rice are as follows; 1. Hybrid rice researchers should prioritize to conduct research of resistant to pest, diseases and evaluation of good eating of the hybrid rice varieties and should be encouraged and prove those values are high in hybrid rice varieties, it is vital to popularize it. Fragrant value added hybrid for market persuasion such as Basmati hybrid rice. 2. Government consider again the credit program for farmers to store long time when price growing up, to get more profits. 3. A public information dissemination system on proven benefits of hybrid rice varieties should be established an advertising campaign should be carried out by using extension and communication channels. 4. Marketing activity should be well structured and regularly monitored by an authorized institute which supplies labels and certification. The capacity of the private sector to effectively and successfully compete in the international rice market therefore it is needs to be improved by laid down short-and long-term strategies. 5. Stringent rules and regulations should be imposed to prevent mixing with inbred rice and also inadequate seed quality.


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Performance evaluation of newly developed variety of menthol mint at farmer’s field – A case study of mint cultivation in Central Uttar Pradesh

The present study for performance evaluation newly developed varieties of CIM –Kranti and other varieties of menthol mint cultivation has been carried out at farmers’ field of central Uttar Pradesh. Mints are commonly used as the source of
 fragrance, flavor and pharmaceuticals industry. During the study period 2017-18, 100 farmers cultivating CIM-Kranti and other varieties have been selected from the region of central Uttar Pradesh. The primary data were collected from the selected
 farmer’s field on profitability comparison between CIM-Kranti and other varieties under cultivation. The highest area and production has been observed during 2012 and 2013. Simple statistical tools and techniques have been used for data analysis of
 the cost of cultivation and profitability. It has been observed during the study that CIM-Kranti gives higher returns (.98491/- ha/year) over other varieties (.70977/-ha/year). However, the input cost of CIM-Kranti is higher than other varieties of
 the crop but the net return of CIM-Kranti was more profitable than other varieties. The benefit cost ratio has been observed 1.45 and 1.74 of other varieties and CIM-Kranti respectively. The new variety “CIM-Kranti” of menthol mint is cold and frost
 tolerant and has the potential to produce 10-15% more oil i.e. 145-160 kg/ha in summer season as compared to all other popular commercial cultivars of menthol mint.It is suggested from the study that maximum profit is generated through CIM-Kranti
 cultivation followed by other varieties crop.

INTRODUCTION
Mints belong to the genus Mentha (family Lamiaceae) varying in their aroma and have been known as useful plant species from the time immemorial. Mints are commonly used as the source of fragrance, flavor and pharmaceuticals especially for culinary preparations. Although many species of mints are being cultivated all over the world , among them only four species are predominantly cultivated in India. These include Menthol mint (MenthaarvensisL.var.piperascens), Peppermint (M.piperita), Bergamot mint (M.citrata) and Spearmint (M.spicata). India is a leading supplier of Menthol mint oil in the world and a large number of farmers in India are being benefitted by its cultivation. Generally, the crop is cultivated during January to July either directly by suckers or by transplanting plantlets for production of mint oil.The cultivation of menthol mint in this country dates back to about 48 years. Prior to 1960s, the requirement of menthol in India was met by import. It was introduced as a crop in India through the efforts of CSIR's Central Indian Medicinal Plants Organization (now CIMAP) and Regional Research Laboratory, Jammu (now IIIM). The project on the Mentha cultivation was taken up at the CIMAP's Pantnagar Research Centre which was established in 1962 near Haldwani in Uttarakhand state. As a result of transfer of technology through this centre large areas were brought under this crop especially in terai region, i.e. Kashipur, Moradabad, Rampur, etc. Some companies like Richardson Hindustan Ltd (now P&G) and Bhavana Chemicals Ltd. also organized cultivation and processing of the Japanese mint crop for its oil and menthol in terai UP. The cultivation of mint became popular progressively since then and spread gradually in vast areas of Uttar Pradesh, and small to large areas of Punjab, Haryana, Madhya Pradesh and Bihar (Khanuja et.al., 2005). About five year back few multinational companies (e.g. Symrise, BASF) have also started production of menthol through synthetic route posing a serious threat to the natural menthol. Besides this, climate change may also affect the cropping pattern and yield potential of the existing cultivars. It is, therefore, imperative to steer the research for breeding improved and high yielding varieties which can be grown in adverse climatic conditions and improvised agro-technology for cultivation of this industrial crop with minimum inputs. The diversified usage of menthol has shown that this commodity will be required consistently in large volume to meet the domestic as well as global requirements. Recently the new variety CIM-Kranti of menthol mint was released by CSIR-CIMAP to make the sustainability in production and to compete with synthetic menthol.

MATERIALS AND METHODS
Three districts of Uttar Pradesh viz. Barabanki, Sitapur and Raebareli were selected purposively for the study where the new cultivar is adopted by farmers. The data were collected randomly from the 100 farmers in these districts, out of which 48were cultivating other varieties and rest 52 were cultivating Kranti variety. The data were collected through personal interview using a pre-tested questionnaire, while the secondary data were collected from the publication of government and other agencies. To study the economics of menthol mint, simple cost accounting method was followed.

RESULTS AND DISCUSION
The last 24 years data from 1994 to 2018 has been shown in the graph on area and production of menthol mint. The highest area and production has been observed during 2012 and 2013. After introduction of synthetic menthol in the market the area was reduced drastically from 3.25 lakhs hectares in 2013 to 2.50 lakhs hectares in 2015. Due to the introduction of new cultivars CIM Kranti and new scientific production techniques at farmers field, the cultivation of this crop shown and increasing trend in area and production. The economics of new cultivar over existing cultivars as discussed in the paper. The trend is shown in following graph. Cropping patterns: As per the data obtained from the study area, it can be understood that farmers are shifting their focus towards the medicinal and aromatic crops from the traditional crops. About 29.14 percent farmers are growing medicinal and aromatic crops and remaining farmers are cultivating the traditional crops like paddy, wheat, potato, mustard and sugarcane etc. (Table 2).

Cost of cultivation:
The cost of cultivation of menthol mint in the study area shown in Table 3. The cost of cultivation was observed higher 56509/-per hectare per year in CIM Kranti than the existing varieties which is 49023/-per hectare per year. The major portion of costs in both the cultivars was shared by irrigation, manures and fertilizers, intercultural operations, transplanting and harvesting. It can be concluded from the table that cost of cultivation in CIM-Kranti is higher than the existing varieties.
Comparative economics of other varieties and cimkranti: The data shown in Table 4 indicate that the new variety CIM-Kranti came out as a beneficial enterprise for the farmers. The yield of CIM-Kranti was more than other varieties under cultivation. Hence, the net profit of CIM-Kranti was found 38.76 percent higher than the other varieties. It is observed that CIM-Kranti is more profitable over variable cost than other varieties under cultivation in the study areas.
Difference between other varieties of menthol mint and cim-kranti: CIM-Kranti variety has several superior characters than other varieties of mentha. The cost of cultivation of CIM-Kranti was higher than other varieties but at the same time, CIM-Kranti variety has another excellences over other varieties like it is tolerant to cold and frost, suitable for kharif season, Number of cuttings which can be taken is twice the other varieties and also the oil yield, oil percentage and menthol percentage is higher than other varieties of mint under cultivation.

CONCLUSION
The new variety "SIM Kranti" of menthol mint is cold and frost tolerant and has the potential to produce 10-15% additional oil i.e, 145-160 kg/ha in summer season as compared to all other popular commercial cultivators of menthol mint. It is also observed from study that during winter season (September-January) when all other mint varieties suffer senescence due to cold and frost condition, variety "SIM Kranti" remains green in the field and grow vigorously to yield two or three times more essential oil (100gm/ha) have with sukers production (200-250 q/ha). The new variety "CIM Kranti" of menthol mint has widened its scope of cultivation in khariff seasons also and thus reducing the cost of cultivation in terms of less irrigation requirement. However, newly developed variety is suitable for commercial cultivation of menthol mint to generate additional income to farmers without any additional input for cultivation during winter besides its usual cultivation as a summer crop.


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Influence of Mineral Fertilization on the Capacity of Nodulation of Three Species of Legumes (Groundnut, Cowpea and Soybean)

Groundnut (Arachis hypogaea), cowpea (Vigna unguiculata) and soybean (Vigna radiata) are the main food legumes grown in Côte d’Ivoire and the localities of Haut-Sassandra. These legumes are highly valued for their nutritional, agronomic and economic value. Groundnut, cowpea and soybean are the main food legumes grown in Côte d’Ivoire and in the localities of Haut-Sassandra. On the other hand, Ivorian soils are characterized by a ten-dency towards acidification and a decrease in the content of nutrients such as phosphorus and nitrogen which have a direct impact on the products. To re-store soil fertility, farmers use mineral fertilization. However, this fertilization has an influence on the nodulation of these legumes. The study took place at Jean Lorougnon University. We adopted a completely random experimental block device. The parameters measured included the number of nodules, the length and weight of the plant, the length and weight of the root system. Our results showed that mineral fertilization reduced the nodulation capacity of legumes. However, a small dose of phosphorus (<2 g) would be required for early growing legumes.

Introduction
In rotational cropping systems, legume-fixed nitrogen can be used first by the legumes and then by subsequent crops [1]. Legumes are considered as plants that can protect soils from degradation [2]. Despite their recognized role in terms of improved performance, legumes occupy a marginal place in cropping systems in Côte d'Ivoire in favor of the coffee-cocoa duo. But today, in the face of the population growth and imperatives of food self-sufficiency, food crops such as sustenance legumes are becoming important. These crops now play a key role in intercommunity trade. In addition, they produce a wide range of agricultural products that can improve household diets. Moreover, they can be associated with industrial crops and the revenues from the marketing guarantee the economic accessibility to other foodstuffs. Among these legumes, cowpea (Vigna unguiculata (L) walp), groundnut (Arachis hypogaea) and green soybean (Vigna radiata) are the most important in most African communities where they are grown [3]. These plants also play a major role in human nutrition because of the quality of the proteins contained in their seeds.
Despite their nutritional and agricultural importance, legumes' yields are still low in our country. This low yield can result from the control of the quality of the sufficient information, the quality of cultural practice and especially the decline in fertility [4]. Also, mineral fertilization has an impact on the nodulation capacity of legumes. High soil nitrogen levels have been shown to reduce nodulation and nitrogen fixation [5]. Some studies have shown that phosphorus, dolomite and manure increase nodulation [6]. Similarly, the lack of phosphorus in soil reduces the number and mass of nodules as well as the activity of nitrogenase in legumes [7]. Yet, other studies have shown that NPK mineral fertilization, especially chicken manure, does not influence the number and weight of nodules [8]. The overall objective of this study is to promote a cultural practice that can improve the nodulation capacity of legumes. The study consists of fertilizing at different doses the soil with three species of legumes grown alone or in combination to check if: • fertilization influences the agronomic parameters of legumes; • there are fertilizer doses to increase the nodulation of the three legumes alone or in combination.

Study Site
The study was carried out on an experimental plot in the locality of Daloa. The city of Daloa is located in the Upper Sassandra region of west-central Côte d'Ivoire between 6˚ and 7˚ North latitude and 7˚ and 8˚ West longitude. This region has an area of 15,200 km 2 for an estimated population of 1,430,960 inhabitants. The department of Daloa is bounded in the North by the departments of Vavoua and Zuénoula, in the South by those of Issia and Sinfra, in the west the county districts of Duékoué and Bangolo and in the East by that of Bouaflé. It covers an area of 5423 km 2 , comprising 137 localities and 6 sub-prefectures ( Figure 1). The climate has four seasons. The long rainy season starts from April to mid-July, the short dry season from mid-July to mid-September, the short rainy season from mid-September to mid-November and the long dry season from December to March. The wet and dry seasons alternate with temperatures ranging from 24.65˚C to 27.75˚C on average. Rainfall, temperature and average humidity characterizing the study site in the test periods from May to August 2016 corresponding to the major rainy seasons are respectively: 142.81 mm; 26.42˚C and 83.7 g/m 2 .

Plant Material
The plant material used in this study consisted of three legume species from the Jean Lorougnon Guédé University collection. It is between the other Vigna unguiculata, Vigna radiata and Arachis hypogaea ( Figure 2). The choice of these  three species is due to the fact that they are more cultivated and consumed in the Upper Sassandra region.

Experimental Apparatus
The experimental device used is that of completely randomized blocks with three repetitions. A plot of 5 m 2 was divided into 3 blocks distant from each other by 0.5 m. Each block consists of 28 elementary batches spaced 0.05 m apart. Each lot consists of 5 pots. These pots are actually bags of black polyethylene 80 microns thick, 25 cm high and 12 cm wide flat. They are filled with earth 7.5 cm in diameter and 21 cm high, a volume of one liter and a weight of about 1.2 kg. The lots are arranged in 4 rows per block. Each row corresponds to a single dose of nitrogen, phosphorus or nitrogen-phosphorus mixture (0 g, 2 g, 4 g or 6 g) ( Figure 3). Seedlings were carried out on January 6, 2018 which is a season of light rains. The rows were arranged in groups. The first group (G1) consists of lots with only one species in each pot (peanut, cowpea or soy). The second group (G2) consists of lots with the association of two species in each pot (peanut-cowpea, peanut-soy or soybean-cowpea). The third group (G3) consists of the lots comprising the association of the three species in each pot (peanut-cowpea-soya). To increase the chances of germination, three seeds of each species were sown per pot. All seeds were sown 1 cm deep. Watering was done every morning and evening with tap water. The removal was done ten days after sowing in order to preserve the best plants per pot. An insecticide treatment with cypercal 50 EC (cypermethrin 50 g/l) was carried out 3 weeks after sowing.
A: groundnut without treatment, AN: groundnut associated with cowpea without fertilizer, AS: groundnut associated with soy without fertilizer, ANS: groundnut associated with cowpea and soy without treatment, AT: groundnut with treatment, AST: groundnut associated with soy with treatment, ANT: groundnut associated with cowpea under treatment, ANST: groundnut associated with cowpea and soya under treatment.

Collection of Data
All data were collected six weeks after sowing corresponding to the flowering period. They concerned the number of nodules, the length of the plant and that of the root system, the weight of the plant and that of the root system. All measurements were taken on three feet of each lot taken at random for each species. However, care must be taken to avoid the cutting of secondary roots and the loss of nodules detached from the roots by ridding the plants of the bags. The root of the plants is soaked in water to better see and count the nodules measurements began with the counting of nodules which consisted in giving the number of nodules on the root system of the plant. The length of the plant consisted in measuring the distance of the main stem from the neck to the most extreme leaf. That of the root system from the neck to the end of the root. They were measured using a tape measure. The root system was separated from the plant and its weight was determined using a precision Howell scale 5 kg × 1 g. The weight of the plant was also determined.

Statistical Analysis
The data collected for each of the 5 variables were entered with the Excel spreadsheet version 97-2003 and then processed taking into account the species and the fertilizer using the STATISTICA version 7.1 software through analysis of variance (ANOVA). The significance of the test was determined by comparing the probability (P) associated with the threshold statistic α = 0.05. When a significant difference was observed between the characters, the ANOVA was supplemented by the Smallest Significant Difference (LSD) test. SDPP allows you to see homogeneous groups, since it tells us where this significant difference is. This analysis of variance makes it possible to note the significant differences between the species and the mineral fertilizers considered.

Effect of Mineral Fertilization on Agronomic Parameters of Groundnut
The results of the analysis of variance indicate that agronomic parameters were influenced by mineral fertilization. Significant differences were observed in all fertilizer rates applied to groundnut for all agronomic parameters (Table 1). In fact, the highest number of nodules was obtained in groundnut grown without the use of mineral fertilizers (59.83 ± 24.2) (Figure 4(a)), when the smallest number of nodules was obtained at groundnut grown with 6 grams of nitrogen (2.00 ± 6.9) (Figure 4(b)).
Measurements made on the length of the plant showed that groundnuts grown with 2 g of nitrogen recorded the highest average (16.75 ± 21.6), while that cultivated with 4 g of nitrogen had the highest average low average (6.41 ± 5.2). The length of the plant is significantly identical for groundnut grown with 6 g of nitrogen, phosphorus (2 g, 4 g and 6 g), the nitrogen-phosphorus mixture (2   g, 4 g and 6 g) and of that cultivated without the use of mineral fertilizers. As for the weight of the plant, the highest average was obtained in groundnut cultivated without the use of mineral fertilizers (60.08 ± 32.3), while the lowest average was recorded at the level of 4 g. nitrogen (18.15 ± 13.6). Groundnuts grown with 2 g of nitrogen, phosphorus (2 g, 4 g and 6 g) and 2 g of nitrogen-phosphorus are significantly identical. In terms of the length of the root system, peanuts grown with 2 g of nitrogen gave the highest average (36.08 ± 15.3), while the lowest average was obtained in groundnut cultivated with 4 g of nitrogen (15.66 ± 12.3). The length of the root system is significantly identical for groundnuts grown with 2 g of phosphorus, 6 g of phosphorus, 2 g of nitrogen-phosphorus and that grown without the use of mineral fertilizers. The weight of the root system showed no significant difference for peanuts grown with or without mineral fertilizers.

Effect of Mineral Fertilization on the Agronomic Parameters of Cowpea
Variables such as number of nodules, plant length, plant weight, length of root system and root system weight in cowpea were measured and analyzed in relation to mineral fertilization. Results from statistical analyze showed a significant difference between the different doses of mineral fertilization (Table 2). In fact, cowpea grown without the use of mineral fertilizers recorded the highest number of nodules (35.91 ± 27.4) (Figure 5(a)), yet the smallest number of nodules was obtained at the 2 g level nitrogen-phosphorus (0.00 ± 0.0) ( Figure 5(b)). The results of the analyze showed that cowpea grown without mineral fertilizers had the highest plant length (16.69 ± 3.3), while that grown with 2 g of nitrogen-phosphorus had the shortest length of the plant. The plant (4.91 ± 3.5). The length of the plant is significantly identical for cowpea grown with 6 g of nitrogen and 6 g of nitrogen-phosphorus as well as for that grown with 6 g of phosphorus and 4 g of nitrogen-phosphorus. The cowpea grown with 6 g of nitrogen, 4 g of nitrogen-phosphorus and 6 g of nitrogen-phosphorus are significantly identical for the weight of the plant, as is that cultivated with 2 g of nitrogen, 2 g of phosphorus and 6 g of phosphorus. Cowpea grown with 2 g of phosphorus nitrogen expressed the highest average (47.81 ± 6.1), at the time when the lowest average was obtained in cowpea grown with 4 g of phosphorus (6.81 ± 5.2) Measurements made along the length of the root system have shown that cowpea grown without the use of mineral fertilizers has a high average (28.75 ± 10.2), or that grown with 6 g of nitrogen-phosphorus has the lowest average (11.66 ± 7.6). The length of the root system is significantly identical for cowpea   grown with 2 g of nitrogen, 6 g of nitrogen, 2 g of nitrogen-phosphorus, 4 g of nitrogen-phosphorus and that grown with phosphorus (2 g, 4 g and 6 g). With regard to the weight of the root system, cowpea grown with 2 g of nitrogen was distinguished by its high average (11.35 ± 9.2) while that cultivated with 2 g of nitrogen-phosphorus recorded the lower average (2.03 ± 1.2). The weight of the root system is significantly identical between cowpea grown with 2 g of nitrogen, 6 g of nitrogen, 2 g of phosphorus and 4 g of nitrogen-phosphorus.

Effect of Mineral Fertilization on the Agronomic Parameters of Soybeans
The result of the statistical analysis of the data presented in Table 3 showed that variables such as the number of nodules, the length of the plant, the weight of the plant, the length of the root system and the root system weight of the soybeans were influenced by mineral fertilization. In fact, the measurements made on the number of nodules and the length of the plant showed that the highest averages were recorded for soybeans grown without mineral fertilizers respectively (22.08 ± 16.91 and 23.25 ± 4.88) (Figure 6(a)) while the lowest averages were obtained   at soybean grown with 2 g of nitrogen-phosphorus respectively (0.16 ± 0.57 and 7.89 ± 6.28) (Figure 6(b)). The number of nodules is statistically identical for soybeans grown with nitrogen (2 g, 4 g and 6 g), 6 g of phosphorus and nitrogen-phosphorus (4 g and 6 g). As for the length of the plant, it is significantly identical between the soybean grown with 2 g of nitrogen, 4 g of nitrogen, as well as 4 g of nitrogen, 2 g of phosphorus, and 6 g of phosphorus. Soybeans grown with 4 g of nitrogen, 2 g of phosphorus and 4 g of phosphorus are significantly identical for the length of the root system. However, soybean grown without mineral fertilizers obtained the highest average (28.16 ± 9.42), while that grown with 4 g of nitrogen-phosphorus expressed the lowest average (10.50 ± 11, 16). The weight of the plant and that of the root system were not influenced by mineral fertilization.

Discussion
The results obtained in our study showed that nodulation is influenced by mineral fertilization (nitrogen, phosphorus and nitrogen-phosphorus). The number of nodules depends on the type of fertilizer applied. However, our results are in contradiction with those of Yeboah [8] who found that NPK mineral fertilization in particular and chicken droppings in no way influence the number and weight of nodules. This difference would be due to the fertilizer doses applied. In summary, our data indicate low nodulation in the presence of nitrogen fertilization.
Indeed, according to Uddin and al. [9], the number and size of nodules are greatly reduced in the presence of nitrogen in the soil. This decrease in the number of nodules for nitrogen fertilization may be due to the fact that the increase in the number of legume nodules is a function of the lack of nitrogen in the soil. When the soil is low in nitrogen, the activity of nitrogen fixation is intense, which favors the increase in the number of nodules to achieve this activity.
On the other hand, when the soil is already rich in nitrogen, the legume prefers to use this available nitrogen in the soil. It follows a decrease in the fixation of atmospheric nitrogen, thus a significant reduction in the number of nodules.
These results corroborate those of Zahran [5]. For this researcher, high soil nitrogen levels reduce nodulation and nitrogen fixation. Our results showed that the number of nodules decreases with phosphate fertilization. These results are contrary to those obtained by Bado [6]. In his work, he has proved that phosphorus, dolomite and manure increase nodulation Qiao and al. [7] [11], phosphorus intake increases the amount of nitrogen fixed by nodules and its concentration in the plant. This difference would be due to the high concentration of phosphorus applied in our study. For nitrogen-phosphorus ferti-lization, our results also showed a decrease in the number of nodules. These results corroborate those of Qiao and al. [7]. For these authors, high nitrogen-phosphorus concentrations have an antagonistic effect on nitrogen inhibition. Nitrogen fixation parameters, number of nodules, dry matter of nodules and specific nitrogenase activity were six times higher in high nitrogen and high phosphorus plants than in high nitrogen plants low phosphorus. In our study, the most important plant length and plant weight were obtained in fertilized soils. These results are consistent with those of Dugje and al. [12]. For these researchers, an excess of nitrogen fertilizer will lead to abundant vegetative growth but low seed production. Regarding the length and weight of the root system the most important were obtained at unfertilized soils. These results are supported by Skene [13] who argues that plants have developed strategies to assimilate Pi (inorganic phosphorus) with greater efficiency in soils depleted in this element.
These strategies consist of an increase in root growth and then the establishment of specialized bodies.

Conclusion
The general objective of our study was to promote cultural practices that can improve the nodulation capacity of legumes. In this study, it was shown that mineral fertilization of legumes negatively affects nodulation. Thus, growing legumes without adding mineral fertilizers gave a high number of nodules. However, a dose of less than 2 g of phosphorus at the beginning of growth of legumes is important. Therefore, the development of legume crops with a low dose of phosphorus would be a way to enrich the soil with nutrients in order to benefit the crops that will succeed.