African ostrich chicks (Struthio camelus) were divided into six groups, and each received different levels of boric acid (source of boron) in the drinking water (0, 40, 80, 160, 320, and 640 mg/L respectively) to examine the histological, apoptotic, biochemical, and transcriptomic parameters. Morphological analysis in different groups was assessed by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and terminal deoxynucleotide transferase dUTP Nick-End Labeling (TUNEL) assay. The biochemical profile was evaluated spectrophotometrically. Detailed RNA-Seq of the data was performed using the transcriptomic method. H&E staining showed well-developed liver structure up to the 160 mg/L boric acid (BA) supplement groups, while BA doses (320 mg/L and 640 mg/L) caused changes in hepatocytes and portal triads. PAS staining showed that glycogen levels were optimal in the 80 mg/L BA dose group, but a reduction in glycogen levels was observed after this group, particularly in the 640 mg/L BA supplement group. Cellular apoptosis showed a biphasic pattern, and the BA dose above 160 mg/L enhanced cell death. In addition, serum analysis showed that doses of 80–160 mg BA were beneficial for ostrich liver. Then, the transcriptome analysis of the 80 mg dose also showed mainly positive effects on the liver. These results demonstrated that chronic BA exposure (320–640 mg) can cause significant histological, apoptotic, and biochemical changes in African ostrich liver, while the adequate dose of supplementation (particularly 80 mg BA) promotes liver growth.

Background: Ghrelin is widely distributed in an animal’s body, playing important biological roles. In mammals, Ghrelin can stimulate food intake and weight gain, but in poultry, it inhibits food intake and drinking. We aimed to determine the effect of Ghrelin on feeding, drinking, body weight and growth hormone (GH) content of African ostrich chicks. Methods: Sixteen 40-day-old African ostrich chicks were randomly divided into four groups and injected intravenously with saline (control) or Ghrelin (10, 50 and 100 µg/kg) for 7 days. The feeding, drinking and growth hormone (GH) levels using measurement method and radioimmunoassay were determined. Result: The results show that exogenous Ghrelin increased the feed intake rate, the drinking rate and the secretion of GH in the ostriches.

Background: The African ostrich is the largest herbivorous bird in the world, which has high economic value. However, the brooding period of African ostrich is long. During this period, the various organs of the body are not fully developed, the resistance is low and it is easy to get sick, thus affecting the growth and development. Therefore, it is of great significance to enhance the digestion and absorption capacity of ostrich chicks and improve its growth speed in shortening the incubation period of ostrich, improving the survival rate of ostrich and the economic benefits of ostrich breeding. Methods: Sixteen 40-day-old African ostrich chicks (male or female) were randomly divided into four groups and injected intravenously of Metatarsal vein with saline (control) or ghrelin (10, 50 and 100 µg/kg) for 6 days. The morphology, gastrin and pepsin levels in the duodenum were measured using stereology, HE staining and radioimmunoassay. Result: The 10, 50 and 100 µg/kg of ghrelin administered groups showed lower duodenal length and length index than the control group (P less than 0.05). Villus length increased in the 50 µg/kg and 100 µg/kg ghrelin administered groups (P less than 0.05). Crypt depth decreased in the 10 µg/kg ghrelin administered group (P less than 0.05). The ratio of villus length to crypt depth in each group increased and the difference was significant in the 50 µg/kg and 100 µg/kg ghrelin administered groups (P less than 0.05). Pepsin content in the duodenum slightly increased in the 10 µg/kg ghrelin administered group (P greater than 0.05) and it significantly decreased in the 50 µg/kg and 100 µg/kg ghrelin administered groups (P less than 0.05). Gastrin content in the duodenum increased significantly (P less than 0.05) in the 10 µg/kg ghrelin administered group and decreased in the 100 µg/kg ghrelin administered group (P less than 0.05).

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Effect of boron on development of testis in the ostrich was studied by observing the changes in the microstructure and ultrastructure. Apoptotic status of testicular cells and hormone level were examined. Compared with the control group, the experimental groups exhibited different degrees of change in the histological structure of the testis on addition of boron in different quantities. Cell apoptosis could be detected in testicular tissues of both control and experimental groups, mainly in Leydig cells and spermatocytes. Level of the follicle stimulating hormone (FSH), estradiol (E-2), luteinizing hormone (LH) and testosterone (T) of the serum in the experimental groups also varied compared with those in the control group. In the present study, 100 mg/l of boron when supplemented with drinking water was helpful for the development of testis in the ostrich. It was also observed that high doses of boron significantly inhibited cell development in the testis of the ostrich.

This study aimed to investigate the effect of exogenous ghrelin on pancreatic growth and development in African ostrich chicks. Sixteen 40-day-old African ostrich chicks (male or female) were randomly divided into four groups and injected intravenously metatarsal vein with saline (control) or ghrelin (10, 50, and 100 mg/kg) for 6 days. Body and pancreas weight were determined, structural characteristics were observed using HE staining, somatostatin-immunopositive cells were detected using immunohistochemistry. The results were as follows: 1. The 50 and 100 mg/kg groups showed lower relative pancreas weight than the control group (P < 0.05). 2.The islet area per unit area of the pancreas was higher in the 10, 50, and 100 mg/kg groups than in the control group (P < 0.05). The number of islets per unit area of the pancreas was lower in the 10 mg/kg group than in the control (P < 0.05) and slightly higher in the 50 and 100 mg/kg groups than in the control (P > 0.05. Moreover, compared with the control, the islet cells in treatment groups were loosely arranged and showed reduced cytoplasm. In the exocrine pancreas, the volume of acinar cells in the 10, 50, and 100 mg/kg groups all decreased to varying degrees. 3. Somatostatin immunopositive cells were mainly located around the periphery of the islets and sporadically distributed in the center. The density of the somatostatin immunopositive cells in the 10, 50, and 100 mg/kg groups was higher than that in the control (P < 0.05). These findings suggest that exogenous ghrelin increases the area and number of islets and number of somatostatin immunopositive cells but reduces relative pancreas weight and effects the morphological and structural development of the pancreas, which may inhibit the pancreatic growth and development in African ostrich chicks.

The present study aimed to explore the effects of supplemental boron on osteogenesis of tibia and to investigate the possible relationship between additional boron and the expression of bone morphogenetic protein-2 (BMP-2) in tibia of ostrich chicks. Therefore, forty-eight African ostrich chicks (15 days old) were supplemented with 0 mg/L, 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L, and 640 mg/L of boron in drinking water for 75 days. The paraffin sections of tibia used to measure histomorphometric parameters by hematoxylin and eosin (HE) staining, Masson’s staining, and immunohistochemistry (IHC). Enzyme-linked immunosorbent assay was performed to assess the level of BMP-2, osteocalcin (BGP), glucocorticoids (GCs), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) in serum. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) technique was performed to detect the cell apoptosis. The results indicated that low dose of supplemental boron (40 mg/L–160 mg/L) in drinking water promotes bone development by increasing the mature ossein. The expression of BMP2 on 45 days was higher than 90 days. Serum level of BMP-2, BGP, and GCs changed significantly in groups with low dosage of boron, and OPG/RANKL ratio was upregulated from 0 to 160 mg/L. Cell apoptosis was least in 40 mg/L and 160 mg/L groups. Taken together, low dose of boron supplemented in drinking water could promote osteogenesis and growth and development of tibia by regulating the expression and secretion of BMP-2 and providing a dynamically balanced environment for tibia growth, development, and reconstruction by regulating the concentrations of BGP, GCs, and OPG/RANKL ratio in serum.

Boron is a trace element which plays important roles in immune response. The relationship between boron and splenic lymphocyte proliferation, apoptosis, secretion of cytokines, and genes potentially related to immune response in ostrich chicks were investigated in the present study. Different concentrations of boron (0, 0.01, 0.1, 0.5, 1, 5, 10, 25, 50, and 100 mmol/L) were applied to splenic lymphocytes of African ostrich, respectively. The effect of boron on lymphocyte proliferation was checked by the CCK-8 method. Flow cytometry was used to detect the effect of boron on apoptosis. The secretion levels of IL-6 and IFN-α were determined by ELISA. Splenic lymphocyte gene expression profiles of ostrich chicks treated with boron (0, 0.1, 100 mmol/L) were studied using RNA-seq technology. The results showed that cell proliferation increased with 0.01–10 mmol/L boron, when it was 25–100 mmol/L, the cell proliferation gradually decreased as the boron concentration increased. Apoptosis ratio in ostrich splenic lymphocytes was closely related to boron concentrations. 0.01- and 0.1-mmol/L boron inhibited apoptosis in splenic lymphocytes, whereas 1, 10, 50, and 100-mmol/L boron promoted apoptosis. As the concentration of boron increased, the secretion of IL-6 gradually decreased; IFN-α was initially increased and then decreased with boron concentrations increased, reaching the maximum level with 1 mmol/L boron. In terms of the RNA-Seq data, there was no differentially expressed gene between the 0- and 0.1-mmol/L boron-treated samples; 21 differentially expressed genes were found between the 0- and 100-mmol/L boron-treated samples; 43 differentially expressed genes were found between the 0.1- and 100-mmol/L boron-treated samples. Functional analysis of the differentially expressed genes by Gene Ontology verified multiple functions associated with immune response. Pathway analysis showed that systemic lupus erythematosus, alcoholism, viral carcinogenesis, and necroptosis pathway were the major enriched pathways, and BIRC2-3, FTH1, and IL-1β genes showed differential expression in necroptosis pathway. These results demonstrated that low concentrations (0.01–0.1 mmol/L) of boron may promote the proliferation and the secretion of cytokines, inhibit cell apoptosis of ostrich splenic lymphocytes by enhancing the function of the cell membrane and the activity of intracellular catalytic enzymes, whereas high-concentration (25–100 mmol/L) boron had opposite effects on cells. The necroptosis pathway might play a pivotal role in regulating the immune response of boron-treated splenic lymphocytes in ostrich chicks.

为了研究硼对小鼠脾淋巴细胞增殖及细胞因子的影响,分别采用CCK-8、ELISA及qPCR技术检测不同浓度硼协同刀豆凝集素(concanavalin A,ConA)或脂多糖(lipopolysaccharide,LPS)对小鼠脾淋巴细胞增殖、细胞因子分泌及其mRNA表达的影响.结果 显示,当硼协同ConA作用于脾淋巴细胞时,随着硼浓度的增加,细胞存活率呈先上升后下降的趋势;IL-2的分泌量逐渐下降,IL-6的分泌量没有显著变化;IL-2、IFN-γ及IL-6 mRNA的相对表达量有所增加,IL-10则表现为逐渐下降的趋势.当硼协同LPS作用于脾淋巴细胞时,随着硼浓度的增加,细胞存活率也呈先上升后下降的趋势;显著促进了IL-2、IL-6的分泌(P＜0.05);硼浓度为1,10,50 mmol/L时,IL-2及IL-10 mRNA的相对表达量均显著低于LPS单独刺激组(P＜0.05),IL-6 mRNA的相对表达量先下降后上升,IFN-γ则表现为逐渐下降.结果 说明低浓度的硼对脾淋巴细胞的增殖及细胞因子的分泌起促进作用,高浓度的硼则对细胞产生毒性作用且在一定程度上抑制相关细胞因子的分泌,且硼对脾淋巴细胞的细胞因子表达也起到了一定的调控作用.

The nuclear-related factor 2 (Nrf2) pathway is the most important mechanism in antioxidant capacity, which regulates the cell’s redox homeostasis. In addition, Nrf2 pathway also can inhibit cell apoptosis. The mechanism of boron actions on various organs is well documented. But, it is not known whether boron can also regulate the Nrf2 pathway in the kidneys. Therefore, in this research, the actions of boron on the kidneys of ostrich chicks, especially the antioxidant effects, have been studied. The ostrich chicks were divided into six groups and supplemented with boric acid (BA) (source of boron) in the drinking water (0, 40, 80, 160, 320, 640 mg respectively) to examine apoptotic, antioxidant, biochemical, and histochemical alterations induced by boron administration in the ostrich chick’s kidney. The cellular apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. The relative antioxidant enzymes (T-AOC, MDA, GSH-Px, SOD, GR, CAT) and biochemical indices (ALT, AST, ALP, CK, LDH, BUN, CREA, UA) in the kidney were determined by spectrophotometric method. The expression of three important genes in the antioxidant pathway (Nrf2, HO-1, GCLc) was measured by quantitative real-time PCR (qPCR), and the localization of key regulator Nrf2 was examined by immunohistochemistry (IHC) method. Western blotting was also performed to further validate our results. Our results revealed that low doses of boron (up to 160 mg) had positive effect, while high doses (especially 640 mg) caused negative effect on the development of the kidney. The cellular apoptosis was in a biphasic manner by altering the boron quantities. The low doses regulate the oxidative and enzyme activity in the kidney. The IHC and western blot showed maximum localization of Nrf2 in 80 mg/L BA dose group. Furthermore, supplementation of boron at low doses upregulated the expression of genes involved in the antioxidant pathway. Taken together, the study demonstrated that low levels of boron (up to 160 mg) inhibited the cell apoptosis, regulate the enzyme activity, and improved the antioxidant system, thus may encourage the development of the ostrich chick’s kidney, while a high amount of boron especially 640 mg/L promoted cell apoptosis and reduced the antioxidant capacity, thus caused negative effect to the ostrich chick’s kidney.

为了探讨犀牛心脏的组织学结构,取非洲白犀牛心壁,采用石蜡包埋切片方法,HE染色,明视野显微镜观察,显微摄影,对非洲白犀牛心脏的组织学结构特点进行观察,并用图像分析系统进行分析.结果显示:非洲白犀牛的心壁由内到外分为心内膜、心肌膜、心外膜3层.心内膜平均厚度约为260.48 μm;心肌膜很厚,左心室最厚,平均厚度可达32.52 mm;心外膜的平均厚度为225.13 μm.心内膜下的浦肯野纤维成群排列为2～3行,在房室交界处则形成一个特殊的结节.浦肯野纤维直径约51.98 μm,多为不规则四边形,或呈长条状,最大长径可达286 μm.细胞核多为圆形,位于细胞的边缘.心室肌纤维直径约10.19 μm,心肌横纹不明显,闰盘不明显.肌纤维排列较为紧密,心肌纤维间血管丰富.本研究为犀牛生理学和兽医临床等相关学科提供了可靠的形态学依据.

Boron is an essential mineral that plays an important role in several biological processes. Boron is required for growth of plants, animals, and humans. There are increasing evidences of this nutrient showing a variety of pleiotropic effects, ranging from anti-inflammatory and antioxidant effects to the modulation of different body systems. In the past few years, the trials showed disease-related polymorphisms of boron in different species, which has drawn attention of scientists to the significance of boron to health. Low boron profile has been related with poor immune function, increased risk of mortality, osteoporosis, and cognitive deterioration. High boron status revealed injury to cell and toxicity in different animals and humans. Some studies have shown some benefits of higher boron status, but findings have been generally mixed, which perhaps accentuates the fact that dietary intake will benefit only if supplemental amount is appropriate. The health benefits of boron are numerous in animals and humans; for instance, it affects the growth at safe intake. Central nervous system shows improvement and immune organs exhibit enhanced immunity with boron supplementation. Hepatic metabolism also shows positive changes in response to dietary boron intake. Furthermore, animals and human fed diets supplemented with boron reveal improved bone density and other benefits including embryonic development, wound healing, and cancer therapy. It has also been reported that boron affects the metabolism of several enzymes and minerals. In the background of these health benefits, low or high boron status is giving cause for concern. Additionally, researches are needed to further elucidate the mechanisms of boron effects, and determine the requirements in different species.

Increased synthesis of heat shock protein 70 (Hsp70) occurs in prokaryotes and eukaryotes in response to physiological, environmental, and chemical exposures, thus allowing the cell survival from fatal conditions. Hsp70 cytoprotective properties may be clarified by its anti-apoptotic function. Boron has been reported to play an essential role in various organ developments and metabolisms. However, it is not known if boron is also able to modulate the Hsp70. In the present study, the actions of boron on ostrich spleen and expression level of Hsp70 were investigated. Thirty healthy ostrich chicks were randomly assigned to six groups: groups I, II, III, IV, V, and VI and fed the basal diet spiked with 0-, 40-, 80-, 160-, 320-, and 640-mg boric acid (BA)/L, respectively, in drinking water. The histomorphological examination in the spleen was done by hematoxylin and eosin (HE) staining. The expression level of Hsp70 was analyzed by immunohistochemistry (IHC) and western blotting, and mRNA expression of Hsp70 was investigated by quantitative real-time PCR (qPCR). In order to investigate apoptosis, TUNEL assay reaction in all treatment groups was analyzed. Our results showed that the histological structure of spleen up to 160 mg/L BA supplementation groups well developed. The Hsp70 expression level first induced at low-dose groups (up to group IV) and then inhibited dramatically in high-dose groups (V and VI) while comparing with the group I (0 mg BA). The TUNEL assay reaction revealed that the cell apoptosis amount was decreased in group IV, but in group V and especially in group VI, it was significantly increased (P < 0.01). Taken altogether, proper dietary boron treatment might stimulate ostrich chick spleen development by promoting the Hsp70 expression level and inhibiting apoptosis, while a high amount of boron supplementation would impair the ostrich spleen structure by inhibiting Hsp70 expression level and promoting cell apoptosis.

The objective of this study is to construct a digital gene expression tag profile to identify genes potentially related to immune response in the ostrich. Exposure to boron leads to an immune response in the ostrich, although the underlying mechanism remains obscure. Thus, a dire need of biological resource in the form of transcriptomic data for ostriches arises to key out genes and to gain insights into the function of boron on the immune response of thymus. For this purpose, RNA-Seq analysis was performed using the Illumina technique to investigate differentially expressed genes in ostrich thymuses treated with different boric acid concentrations (0, 80, and 640 mg/L). Compared with the control group, we identified 309 upregulated and 593 downregulated genes in the 80 mg/L treated sample and 228 upregulated and 1816 downregulated genes in 640 mg/L treated sample, respectively. Trend analysis of these differentially expressed genes uncovers three statistically significant trends. Functional annotation analysis of the differentially expressed genes verifies multiple functions associated with immune response. When ostrich thymuses were treated with boron, expression changes were observed in genes predominantly associated with MAPK and calcium signaling pathways. The results of this study provide all-inclusive information on gene expression at the transcriptional level that further enhances our apprehension for the molecular mechanisms of boron on the ostrich immune system. The calcium and MAPK signaling pathways might play a pivotal role in regulating the immune response of boron-treated ostriches.

B cell activating factor (BAFF) belongs to the tumor necrosis factor (TNF) family,which plays a critical role in B cell survival,proliferation,maturation and immunoglobulin secretion.In this study,two primers Os-F and Os-R contained EcoR I and Hind Ⅲ restriction sites were designed to amplify the soluble part of OsBAFF based on the complete OsBAFF sequence in African ostrich in our previous research.The results of SDS-PAGE and western blot analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in E.coli Rosset (DE3)In vitro,purified OsBAFF was not only able to promote the survival rate of African ostrich bursal lymphocytes,but also able to co-stimulate proliferation of mouse splenic B cells.These findings indicated that OsBAFF played an important role in survival and proliferation of African ostrich bursal lymphocytes,which could provide valuable information for research on the immune system of African ostrich and OsBAFF could serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and other birds.

In order to explore the building conditions of acute lung injury,healthy Kunming mice were selected,by intraperitoneal injection of cascade dose of LPS(0,2,4,6,8,10 mg/kg)to observe the mice respiratory frequency,mortality,lung W/D value and the changes of tissue structure in different conditions,and detect the expression changes of inflammation cytokine,IL-10,TNF-α,IL-1β and IFN-γ.The results showed that compared with control group,lung W/D value in 4 mg/kg LPS group significantly increased(P<0.05)and lung W/D values in 6,8 and 10 mg/kg LPS groups extremely significantly increased(P<0.01).Pathological analysis showed that 6 mg/kg LPS group appeared lung congestion,alveolar luminal and pulmonary interstitial exudation,alveolar septal thickening,and a small amount of neutrophil infiltration.8 and 10 mg/kg LPS groups appeared pulmonary congestion,alveolar and pulmonary interstitial exudation,alveolar septal thickening and neutrophil infiltration.Compared with control group,collagen fibers of the lung tissue in 6,8,10 mg/kg LPS group increased,and more appeared in the lung around the trachea,10 mg/kg LPS group was the most obvious.The inflammatory factors in 2,4,6,8 and 10 mg/kg LPS groups had extremely significantly increased compared with control group(P<0.01).Pathological biopsy and related indicators showed that the model of mice could be successfully constructed by intraperitoneal injection of 8 mg/kg LPS for 12 h.

Lipopolysaccharide (LPS) induces acute liver injury and the complex mechanisms include the activation of toll like receptor 4 (TLR4) signaling pathway in many species. However, immuno-pathological changes during TLR4 signaling under LPS stress in acute liver injury is poorly understood in avian species. The present investigation was therefore carried out to evaluate these alterations in TLR4 signaling pathway during acute liver injury in young chickens.

Boron is an essential trace element which plays an important role in process of metabolism and the function of the tissues. However, the effects of boron on the intestinal cells in African ostrich chicks are poorly reported. Therefore, this study was designed to investigate the role of boron on proliferation and apoptosis of the intestinal cells. A total of 36, ten day-old ostrich chicks were randomly divided into six groups and fed on the same basal diet supplemented with 0, 40, 80, 160, 320 and 640 mg/L boric acid in drinking water for 80 days. Proliferating cell nuclear antigen (PCNA) was used to test the proliferation index of intestine in different group by immunohistochemical staining (IHC). Apoptotic cells of intestine were detected by Dutp-biotin nick end labeling (TUNEL) reaction and evaluated by integral optical density (IOD). Results showed that proliferation of intestinal cells significantly increased in groups of 80, 160, 320 and 640 mg/L. TUNEL reaction showed that apoptosis significantly decreased in 80 mg/L groups, while significantly increased in high dose of boron groups (320 and 640 mg/L), especially in epithelium. In conclusion, low dose of boron-supplemented water could promote cell proliferation and depress apoptosis, while high dose of boron could cause intestinal apoptosis and thus we found increased proliferation of intestine cell as a compensatory adaption. These findings may support optimal dosage of boron that could protect the development of ostrich intestine, while high dosage of boron could suppress it, or even has toxic effects on it.

Visfatin is an adipocytokine displaying multiple functional properties, which plays a role in the regulation of cell apoptosis and inflammation by an as yet unidentified mechanism. The aim of the present study was to determine if visfatin is involved in apoptosis pathway induced by LPS in rat Mesenteric lymph nodes (MLNs). Experimental rats were divided into four groups and MLNs samples were collected from each group. The morphological changes of the MLNs were examined by histological imaging. CD68 and ENPP1 were detected with immunohistochemistry and Western Blot. Apoptosis was evaluated with TUNEL and Flow Cytometry, the mRNA levels of the apoptosis-related genes were detected by qRT-PCR, and the protein levels of the apoptotic-related factors were detected by western blot. The main results showed that visfatin could significantly increase the macrophages in MLNs and prevent cell apoptosis from LPS-induced mesenteric lymph nodes, activate apoptotic signaling pathways and regulate the mRNA levels of the apoptosis-related genes. Visfatin had a pro-apoptotic effect on normal MLNs, whereas it exerted an anti-apoptotic effect during LPS-induced cell apoptosis in rat MLNs. In short, visfatin plays a dual role in the apoptosis in rat MLNs, which is mediated by both the mitochondrial apoptotic pathway and the death-receptor apoptotic pathway.