Morphological and Transcriptomic Analysis of the Supplemental Boron in the Liver of Ostrich Chicks.

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.

Boron Supplementation Promotes Osteogenesis of Tibia by Regulating the Bone Morphogenetic Protein-2 Expression 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.

Transcriptional Study Revealed That Boron Supplementation May Alter the Immune-Related Genes Through MAPK Signaling in Ostrich Chick Thymus.

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.

Effects of Boron Supplementation on Expression of Hsp70 in the Spleen of African Ostrich.

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.

Effects of supplemental boron on intestinal proliferation and apoptosis in African ostrich chicks / Efectos del boro suplementario sobre la proliferación intestinal y apoptosis en polluelos de avestruz africana

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. Proliferatingcell nuclearantigen (PCNA) wasused to test the proliferation indexof intestine in different group byimmunohistochemicalstaining (IHC). Apoptoticcellsofintestinewere detectedbyDutp-biotin nick end labeling (TUNEL) reaction and evaluated by integral optical density (IOD). Results showed that proliferationof 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 inepithelium. 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.

El boro es un elemento esencial que desempeña un importante rol en el proceso del metabolismo y en la función de los tejidos. Sin embargo, existe poca información de los efectos del boro en las células intestinales de polluelos de avestruz Africana. Por lo tanto, este estudio fue diseñado para investigar el papel del boro sobre la proliferación y la apoptosis de las células intestinales. Un total de 36 polluelos de avestruz de diez días se dividieron, aleatoriamente, en seis grupos y se alimentaron con una misma dieta basal suplementada con 0, 40, 80, 160, 320 y 640 mg/L de ácido bórico en agua potable durante 80 días. Se utilizó el antígeno nuclear celular de células en proliferación (PCNA) para probar el índice de proliferación de intestino en diferentes grupos por tinción inmunohistoquímica. Las células apoptóticas del intestino fueron detectadas por dUTP-biotina nick etiquetado para reacción (TUNEL) y evaluadas por la densidad óptica integrada (DOI). Los resultados mostraron que la proliferación de las células intestinales aumentó significativamente en los grupos de 80, 160, 320 y 640 mg /L. La reacción TUNEL mostró que la apoptosis se redujo significativamente en los grupos de 80 mg/L, mientras que el aumento fue significativo en grupos tratados con dosis alta de boro (320 y 640 mg/L), especialmente en el epitelio. En conclusión, la baja dosis de boro en agua suplementada podría promover la proliferación celular y deprimir la apoptosis, mientras que altas dosis de boro podrían provocar apoptosis intestinal y, por lo tanto, se halló una mayor proliferación de las células del intestino como una adaptación compensatoria. Estos hallazgos indican que una dosis óptima de boro podría proteger el desarrollo del intestino del avestruz, mientras que altas dosis de boro podrían suprimirla, o incluso tener efectos tóxicos sobre ella.

Effect of Boric Acid Supplementation on the Expression of BDNF in African Ostrich Chick Brain.

The degree of brain development can be expressed by the levels of brain brain-derived neurotrophic factor (BDNF). BDNF plays an irreplaceable role in the process of neuronal development, protection, and restoration. The aim of the present study was to evaluate the effects of boric acid supplementation in water on the ostrich chick neuronal development. One-day-old healthy animals were supplemented with boron in drinking water at various concentrations, and the potential effects of boric acid on brain development were tested by a series of experiments. The histological changes in brain were observed by hematoxylin and eosin (HE) staining and Nissl staining. Expression of BDNF was analyzed by immunohistochemistry, quantitative real-time PCR (QRT-PCR), and enzyme linked immunosorbent assay (ELISA). Apoptosis was evaluated with Dutp-biotin nick end labeling (TUNEL) reaction, and caspase-3 was detected with QRT-PCR. The results were as follows: (1) under the light microscope, the neuron structure was well developed with abundance of neurites and intact cell morphology when animals were fed with less than 160 mg/L of boric acid (groups II, III, IV). Adversely, when boric acid doses were higher than 320 mg/L(groups V, VI), the high-dose boric acid neuron structure was damaged with less neurites, particularly at 640 mg/L; (2) the quantity of BDNF expression in groups II, III, and IV was increased while it was decreased in groups V and VI when compared with that in group I; (3) TUNEL reaction and the caspase-3 mRNA level showed that the amount of cell apoptosis in group II, group III, and group IV were decreased, but increased in group V and group VI significantly. These results indicated that appropriate supplementation of boric acid, especially at 160 mg/L, could promote ostrich chicks' brain development by promoting the BDNF expression and reducing cell apoptosis. Conversely, high dose of boric acid particularly in 640 mg/L would damage the neuron structure of ostrich chick brain by inhibiting the BDNF expression and increasing cell apoptosis. Taken together, the 160 mg/L boric acid supplementation may be the optimal dose for the brain development of ostrich chicks.

Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich.

Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory response to pathogens under boron stress.

Effect of boric acid supplementation of ostrich water on the expression of Foxn1 in thymus.

Foxn1 is essential for thymus development. The relationship between boric acid and thymus development, optimal dose of boric acid in ostrich diets, and the effects of boric acid on the expression of Foxn1 were investigated in the present study. Thirty healthy ostriches were randomly divided into six groups: Group I, II, III, IV, V, VI, and supplemented with boric acid at the concentration of 0 mg/L, 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L, 640 mg/L, respectively. The histological changes in thymus were observed by HE staining, and the expression of Foxn1 analyzed by immunohistochemistry and western blot. TUNEL method was used to label the apoptotic cells. Ostrich Foxn1 was sequenced by Race method. The results were as following: Apoptosis in ostrich thymus was closely related with boric acid concentrations. Low boric acid concentration inhibited apoptosis in thymus, but high boric acid concentration promoted apoptosis. Foxn1-positive cells were mainly distributed in thymic medulla and rarely in cortex. Foxn1 is closely related to thymus growth and development. The nucleotide sequence and the encoded protein of Foxn1 were 2736 bases and 654 amino acids in length. It is highly conserved as compared with other species. These results demonstrated that the appropriate boric acid supplementation in water would produce positive effects on the growth development of ostrich thymus by promoting Foxn1 expression, especially at 80 mg/L, and the microstructure of the thymus of ostrich fed 80 mg/L boric acid was well developed. The supplementation of high dose boron (>320 mg/L) damaged the microstructure of thymus and inhibited the immune function by inhibiting Foxn1 expression, particularly at 640 mg/L. The optimal dose of boric acid supplementation in ostrich diets is 80 mg/L boric acid. The genomic full-length of African ostrich Foxn1 was cloned for the first time in the study.

Effects of supplemental boron on growth performance and meat quality in African ostrich chicks.

To investigate the effects of boron on growth performance and meat quality, 10-day-old Africa ostrich chicks were randomly divided into 6 groups with 6 replicates in each group. For 80 days, birds in the treatments were fed the same basal diet but given different concentrations of boron-supplemented water. The highest final BW (33.4 ± 0.30 kg), ADFI (376 ± 1.83 g), and ADG (224 ± 1.01 g) appeared in the group receiving 160 mg/L boron (group 4). 160 mg/L boron also decreased drip loss (2.20 ± 0.59), cooking loss (35.3 ± 1.14), and elevated pH value (6.13 ± 0.28) of meat (P < 0.05). Ostrich chicks in the 640 mg/L treatment group (group 6) had the lowest final BW (30.8 ± 1.05 kg) and ADG (208 ± 0.74 g) (P < 0.05). The highest ash (1.35 ± 0.01%) and pH (6.18 ± 0.03) and the lowest protein (20.4 ± 1.74%), drip loss (2.10 ± 0.76%), cooking loss (35.0 ± 0.41%), C18:1 (28.2 ± 0.65%), and C18:3ω3 (2.60 ± 0.51%) appeared in group 6 (P < 0.05) as well. Overall, the optimum concentration of 160 mg/L supplemental boron improved ostrich growth performance and meat quality; however, high concentrations of boron decreased both performance and meat quality.

Effect of additional boron on tibias of African ostrich chicks.

The aim of the present study was to find out the effects of boron on ostrich chicks fed with 0 mg/l, 100 mg/l, 200 mg/l, and 400 mg/l of additional boron in water. We measured bone mineral density (BMD), perimeter, length, weight, ash content of ostrich tibias, thickness of cortical bone, and diameter of the marrow cavity. We also analyzed the apoptosis status of paraffin sections using a TUNEL kit and examined serum levels of leptin and estradiol (E(2)). The results were dramatic. Compared with the control group, group C had a very high BMD. The serum levels of leptin in groups C and D were significantly higher than control values, and the levels of E(2) fluctuated. The perimeter, length, weight, and ash content of ostrich tibias all increased significantly with increasing dosage of boron. The cross-section analysis revealed that the bone marrow cavity shifted closer to one side in group D, which was observed on a macro-scale. This shift may be related to the toxicity of excessive boron, as indicated by the apoptosis status. According to the present data, additional boron was helpful for ostrich chick bone development, and 200 mg/l supplement boron in the drinking water appeared to be the most beneficial.