Fucoxanthin inhibits tumour-related lymphangiogenesis and growth of breast cancer.

Tumour lymphangiogenesis plays an important role in promoting the growth and lymphatic metastasis of tumours. The process is associated with cell proliferation, migration and tube-like structure formation in lymphatic endothelial cells (LEC), but no antilymphangiogenic agent is currently used in clinical practice. Fucoxanthin is a material found in brown algae that holds promise in the context of drug development. Fucoxanthin is a carotenoid with variety of pharmacological functions, including antitumour and anti-inflammatory effects. The ability of fucoxanthin to inhibit lymphangiogenesis remains unclear. The results of experiments performed as part of this study show that fucoxanthin, extracted from Undaria pinnatifida (Wakame), inhibits proliferation, migration and formation of tube-like structures in human LEC (HLEC). In this study, fucoxanthin also suppressed the malignant phenotype in human breast cancer MDA-MB-231 cells and decreased tumour-induced lymphangiogenesis when used in combination with a conditional medium culture system. Fucoxanthin significantly decreased levels of vascular endothelial growth factor (VEGF)-C, VEGF receptor-3, nuclear factor kappa B, phospho-Akt and phospho-PI3K in HLEC. Fucoxanthin also decreased micro-lymphatic vascular density (micro-LVD) in a MDA-MB-231 nude mouse model of breast cancer. These findings suggest that fucoxanthin inhibits tumour-induced lymphangiogenesis in vitro and in vivo, highlighting its potential use as an antilymphangiogenic agent for antitumour metastatic comprehensive therapy in patients with breast cancer.

The potential role of Annexin 3 in diapause embryo restart of Artemia sinica and in response to stress of low temperature.

Annexins are highly conserved and ubiquitous in various somatic cell types. They are involved in membrane transport and a range of calcium-regulated activities on the cell membrane surface, including vesicular transport, membrane fusion in exocytosis, signal transduction, and formation of calcium channels. They also regulate inflammatory response, cell differentiation, and interaction between cytoskeletal proteins. In this study, for the first time, an ANX3 gene from Artemia sinica ( As-anx3) was cloned. The As-anx3 full-length complementary DNA comprises 1,024 bp and has a 948 bp open reading frame encoding a 315-amino-acid polypeptide with four ANX domains. The profiles of both As-ANX3 mRNA and protein expression exhibited peaks at the 0 hr stage and had the same significant downregulation trend throughout the post-diapause embryo development stage. The ERK1/2, the phosphorylation levels of ERK1/2, and cell cycle-related protein (CDK4) expressions were analyzed by western blot analysis. The results showed that CDK4 presented a significantly ascending trend from 0 and 40 hr, although the phosphorylation levels of ERK1/2 did not increase significantly. The transcriptional and protein expressions of As-ANX3 were highly upregulated when the temperature was lowered from 25 to 15°C, but the expressions showed a gradual downward trend when the temperature was further lowered to 5°C. These results indicated that As-ANX3 plays a crucial role in restarting diapause and low-temperature stress in A. sinica.

The Potential Roles of the Apoptosis-Related Protein PDRG1 in Diapause Embryo Restarting of Artemia sinica.

High salinity and low temperatures can induce Artemia sinica to enter the diapause stage during embryonic development. Diapause embryos stop at the gastrula stage, allowing them to resist apoptosis and regulate cell cycle activity to guarantee normal development after diapause termination. P53 and DNA damage-regulated gene 1 (pdrg1) is involved in cellular physiological activities, such as apoptosis, DNA damage repair, cell cycle regulation, and promotion of programmed cell death. However, the role of pdrg1 in diapause and diapause termination in A. sinica remains unknown. Here, the full-length A. sinica pdrg1 cDNA (As-pdrg1) was obtained and found to contain 1119 nucleotides, including a 228 bp open reading frame (ORF), a 233 bp 5'-untranslated region (UTR), and a 658-bp 3'-UTR, which encodes a 75 amino acid protein. In situ hybridization showed no tissue specific expression of As-pdrg1. Quantitative real-time PCR and western blotting analyses of As-pdrg1 gene and protein expression showed high levels at 15-20 h of embryo development and a subsequent downward trend. Low temperatures upregulated As-pdrg1 expression. RNA interference for the pdrg1 gene in Artemia embryos caused significant developmental hysteresis. Thus, PDRG1 plays an important role in diapause termination and cell cycle regulation in early embryonic development of A. sinica.

Protective Effects of Fucoidan on Aß25-35 and d-Gal-Induced Neurotoxicity in PC12 Cells and d-Gal-Induced Cognitive Dysfunction in Mice.

Alzheimer's disease (AD) is a chronic neurodegenerative disease which contributes to memory loss and cognitive decline in the elderly. Fucoidan, extracted from brown algae, is a complex sulfated polysaccharide and potential bioactive compound. In this study, we investigated whether fucoidan protects PC12 cells from apoptosis induced by a combination of beta-amyloid 25-35 (Aß25-35) and d-galactose (d-Gal), and improves learning and memory impairment in AD model mice. The results indicated that fucoidan could inhibit the release of cytochrome c from the mitochondria to cytosol and activation of caspases, and increase the expression of apoptosis inhibitor proteins (IAPs), including livin and X-linked IAP (XIAP) in PC12 cells damaged by Aß25-35 and d-Gal-induction. Fucoidan reversed the decreased activity of acetylcholine (ACh) and choline acetyl transferase (ChAT), as well as the increased activity of acetylcholine esterase (AChE), in AD model mice induced by infusion of d-Gal. Furthermore, fucoidan improved antioxidant activity in vitro and in vivo by activation of superoxide dismutase (SOD) and glutathione (GSH). These results suggested that fucoidan could protect PC12 cells from apoptosis and ameliorate the learning and memory impairment in AD model mice, which appeared to be due to regulating the cholinergic system, reducing oxidative stress, and inhibiting the caspase-dependent apoptosis pathway.

Identification, expression pattern and potential role of variable lymphocyte receptor Aj-VLRA from Apostichopus japonicus in response to bacterial challenge.

The variable lymphocyte receptors (VLRs) are found in jawless vertebrates (agnathans), and specifically recognize bacteria and viruses via their leucine-rich repeats (LRRs). VLRs are believed to be adaptive immune response molecules. Echinoderms do not have adaptive immune systems; however, in the present study, a VLR cDNA named Aj-VLRA was cloned and characterized from sea cucumber, Apostichopus japonicus. The complete cDNA of Aj-VLRA was 3072 bp, including a 1995 bp open reading frame encoding 664 amino acids comprising LRR domains, a predicted transmembrane helix and an N-terminal signal peptide. As determined by quantitative real-time reverse transcription polymerase chain reaction, Aj-VLRA transcripts are ubiquitously expressed in the body wall, longitudinal muscles, intestine and respiratory tree of A. japonicus. The expression level of Aj-VLRA was upregulated after challenge with four common marine bacteria. In situ hybridization showed that the expression of Aj-VLRA was widely distributed in the four tissues, particularly in the cytoplasm of epidermal cells. Recombinantly expressed Aj-VLRA (including the LRR domains) could bind to bacteria including Micrococcus lysodeikticus (Gram+) and Vibrio anguillarum (Gram-). Collectively, the results suggested that Aj-VLRA is related to an innate immune response of A. japonicus.

Fucoidan Suppresses Hypoxia-Induced Lymphangiogenesis and Lymphatic Metastasis in Mouse Hepatocarcinoma.

Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways.

Identification and expression of the elongator protein 2 (Ajelp2) gene, a novel regeneration-related gene from the sea cucumber Apostichopus japonicus.

Elongator proteins comprise six subunits (ELP1-ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5' untranslated (UTR), a 526 bp 3' UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it's the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.

Cloning and expression patterns of the brine shrimp (Artemia sinica) glycogen phosphorylase (GPase) gene during development and in response to temperature stress.

Glycogen serves as a metabolic reserve and is involved in macromolecular synthesis. Glycogen phosphorylase (GPase) is a key enzyme involved in intracellular glycogen catabolism, catalyzing the first step in glycogen degradation. In the diapause, GPase catalyzes glycogen into the closely related molecule, sorbitol. In this study, the full-length cDNA of the GPase gene (2,790 bp) was isolated from Artemia sinica for the first time by rapid amplification of cDNA ends technology. The GPase gene encoded a protein of 853 amino acids belonging to the Glycosyltransferase GTB type superfamily. The expression pattern and location of GPase were investigated at various stages during the embryonic development of A. sinica using real-time PCR and in situ hybridization. High GPase expression was detected at the 0 and 5 h stages. Subsequently, expression declined and was maintained at a low level during the stages from 10 to 40 h following by a small increase at day 3. Expression was downregulated at temperatures ranging from 25 to 20 °C and was subsequently upregulated in the range 15-5 °C. In situ hybridization assays showed wide distribution of the GPase gene during different developmental stages. From the results of this study, we conclude that the GPase gene expression is stress-related and might play an important role in Artemia development and metabolism.

Cloning and expression analysis of the gene encoding fibrinogen-like protein A, a novel regeneration-related protein from Apostichopus japonicus.

Fibrinogen-like protein A (FGLA), a member of the fibrinogen-related protein superfamily, exists in different tissues of vertebrates and invertebrates. FGLA plays crucial roles including innate immune response, blood clotting and regeneration. In this study, the fibrinogen-like protein A (fglA) was cloned from Apostichopus japonicus using rapid amplification of cDNA ends PCR techniques. The cDNA sequence of fglA is 1,524 bp with a 849 bp open reading frame encoding a polypeptide of 282 amino acids, with an N-terminal signal peptide and a conserved C-terminal domain. Bioinformatic analysis revealed that the predicted molecular weight of the whole protein is 31.9 kDa and it has an isoelectric point of 5.64. In-situ hybridization demonstrated that fglA is widely distributed in body wall, intestines, longitudinal muscles and respiratory tree. The expression levels of fglA during different regeneration stages in the body wall, intestine and respiratory trees were analyzed by real-time PCR. The expression of fglA gradually increased within 1 h in body wall, and reached a plateau before decreasing to the basal level. This indicates that fglA is associated with the regeneration of Apostichopus japonicus.

Expression and roles of As-NUPR1 protein from Artemia sinica during embryo development and in response to salinity stress.

As-NUPR1, a stress-related protein, plays an important role in post-diapause during embryonic development in the brine shrimp Artemia sinica. In the present study, successful expression of As-NUPR1 from the cDNA sequence isolated from A. sinica was demonstrated using a prokaryotic expression system. The recombinant protein consisted of 132 amino acids with a molecular weight of 15 kDa, and a predicted isoelectric point of 7.17. As-NUPR1 polyclonal antibodies were prepared by immunization of Balb/c mice with purified recombinant As-NUPR1 protein as an antigen, and immunological studies were carried out. Expression of As-NUPR1 during different developmental stages of the embryo and in response to salinity stress was analyzed in A. sinica using Western blots. The experimental results showed that the expression of As-NUPR1 is widely distributed at different developmental stages in A. sinica, and there was no tissue or organ specificity. Expression of As-NUPR1 decreased gradually during the diapause termination stage of embryo development, after which there was a general increase in expression after breaking the shell. In addition, As-NUPR1 expression was highly upregulated under conditions of high salinity. These results suggest that the As-NUPR1 protein is a stress-related protein that plays a role in protecting embryos from high salt damage in different embryonic developmental stages, especially during the post-diapause period.

Expression patterns of As-ClC gene of Artemia sinica in early development and under salinity stress.

As-ClC (chloride channels protein from Artemia sinica), a member from the chloride channels protein family, is a α-helical membrane protein predicted to traverse the cell membrane 11 times. It is important for several physiological functions such as cell volume regulation, cell proliferation, growth and differentiation. In this paper, the complete cDNA sequence of As-CIC was cloned from A. sinica for the first time using RACE technology. The expression pattern and location of the As-CIC gene was investigated in different stages of the embryonic development by means of quantitative real-time PCR and in situ hybridization (ISH) assay. As-CLC was distributed throughout the whole body in cells of different embryonic development of A. sinica as shown by ISH. There was a low expression level of the As-ClC gene after 0 h and a higher expression level after 15 and 40 h when the embryo entered the next growth period and the environmental salinity changed. At adult stage, the As-ClC maintained a high expression level. The results of the real-time PCR assay showed an increasing trend of As-ClC transcripts with increasing salinity. The expression of As-ClC was higher in the control group (28) than in the experimental group except at a salinity of 200 PSU. It indicated that As-ClC functions as salinity-stress-related gene, probably participated in cell volume regulation and osmotic regulation during the early embryonic development of A. sinica.

Fucoidan derived from Undaria pinnatifida induces apoptosis in human hepatocellular carcinoma SMMC-7721 cells via the ROS-mediated mitochondrial pathway.

Fucoidans, fucose-enriched sulfated polysaccharides isolated from brown algae and marine invertebrates, have been shown to exert anticancer activity in several types of human cancer, including leukemia and breast cancer and in lung adenocarcinoma cells. In the present study, the anticancer activity of the fucoidan extracted from the brown seaweed Undaria pinnatifida was investigated in human hepatocellular carcinoma SMMC-7721 cells, and the underlying mechanisms of action were investigated. SMMC-7721 cells exposed to fucoidan displayed growth inhibition and several typical features of apoptotic cells, such as chromatin condensation and marginalization, a decrease in the number of mitochondria, and in mitochondrial swelling and vacuolation. Fucoidan-induced cell death was associated with depletion of reduced glutathione (GSH), accumulation of high intracellular levels of reactive oxygen species (ROS), and accompanied by damage to the mitochondrial ultrastructure, depolarization of the mitochondrial membrane potential (MMP, Δψm) and caspase activation. Moreover, fucoidan led to altered expression of factors related to apoptosis, including downregulating Livin and XIAP mRNA, which are members of the inhibitor of apoptotic protein (IAP) family, and increased the Bax-to-Bcl-2 ratio. These findings suggest that fucoidan isolated from U. pinnatifida induced apoptosis in SMMC-7721 cells via the ROS-mediated mitochondrial pathway.

Standardized ileal digestibility of amino acids in extruded full-fat soybean fed to non-gestating, gestating, and lactating sows.

This study was conducted to determine and compare the apparent ileal digestibility and standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AA) in extruded full-fat soybean (EFSB) fed to nongestating, midgestating, late-gestating, and lactating sows. Six EFSB samples were collected from different sources. Fourteen nongestating sows (Landrace × Yorkshire; parity 3 to 5) were fitted with a T-cannula at the distal ileum. After recovery, sows were assigned to a replicated 7 × 3 incomplete Latin square design. The diets included a nitrogen-free (NF) diet and six experimental diets (EFSB 1 to 6). Eight midgestating sows (Landrace × Yorkshire; parity 3; day 48 of gestation), eight late-gestating sows (Landrace × Yorkshire; parity 3; day 90 of gestation), and eight lactating sows (Landrace × Yorkshire; parity 3; day 6 of lactation) were all assigned to four dietary treatments in a repeated 4 × 3 incomplete Latin square design. The diets included a NF diet and three experimental diets (EFSB 4 to 6). Results showed that there were significant differences in the AID and SID of CP and other AA in nongestating sows (P < 0.05), the AID and SID values of EFSB 1 to 3 were higher than those of EFSB 4 to 6, and the value of EFSB 5 was the lowest. For midgestating sows, there were differences in the AID of methionine (EFSB 5 had a lower value than EFSB 4 and 6) (P < 0.01). For late-gestating sows, only the AID of methionine (EFSB 5 had a lower value than EFSB 4 and 6), tryptophan (EFSB 5 had a higher value than EFSB 4 and 6), and proline (EFSB 5 had a higher value than EFSB 4) was different (P < 0.05), and the SID of methionine (EFSB 4 had a higher value than EFSB 5) and tryptophan (EFSB 5 had a higher value than EFSB 4 and 6) was different (P < 0.05). The SID of histidine and valine was greater in lactation than in nongestation (P = 0.045 and P = 0.02, respectively). The SID of isoleucine was greater in lactation than in nongestation and gestation (P < 0.01). The SID of methionine in nongestation was lower than in gestation and lactation (P < 0.01). The SID of cysteine was the lowest in midgestation (P = 0.045), and the SID of proline was greater in midgestation than in lactation and nongestation (P < 0.01). In conclusion, the AA ileal digestibility of six EFSB samples from different sources was different, and the ileal digestibility of CP and most AA was not affected by the physiological stage of sows.

Extruded full-fat soybean (EFSB) is produced by treating soybean with high temperature and pressure. It is rich in oil, protein, and energy. The standardized ileal digestibility (SID) of amino acids in EFSB has been studied in growing or finishing pigs, but not in sows. The ileal digestibility of amino acids in EFSB may be different between sows and growing pigs and even between sows at different physiological stages. Therefore, the SID of amino acids of six EFSB samples from different sources was evaluated in nongestating, midgestating, late-gestating, and lactating sows. Results indicate that the amino acid ileal digestibility of six EFSB samples from different sources was different, and the ileal digestibility of crude protein and most amino acids was not affected by the physiological stage of sows.

Nutritional values of cottonseed meal from different sources fed to gestating and non-pregnant sows.

This study set out to determine the apparent total tract digestibility (ATTD) of the nutrients and energy in six cottonseed meal (CSM) feedstuffs fed to pregnant and non-pregnant sows. The six types of CSM were: two expelled CSMs with crude protein (CP) levels of 40.67% and 44.64%, and four solvent-extracted CSMs with CP levels of 45.18%, 51.16%, 56.44%, and 59.63%. Fourteen gestating sows (at the fourth parity with body weights of 220.6 ± 18.4 kg at days 30 of gestation) and 14 non-pregnant sows (after the third parity with body weights of 219 ± 14.6 kg) were assigned to a replicated 7 × 3 Youden square design with seven diets and three periods. The seven diets included an entirely corn-based diet and six diets each containing 20.0% of the six CSMs tested. Each period included a 5-d acclimation to the experimental diets, followed by a 5-d period during which urine and feces were collected. Significant differences were found among the six CSM diets, regardless of reproductive stage, regarding 1) the ATTD of neutral detergent fiber (NDF) (P < 0.05) and 2) the ATTD of dry matter (DM), organic matter (OM), and CP and the gross energy (GE) (P < 0.01). Non-pregnant sows had a greater ATTD of OM and CP (P < 0.01) compared with gestating sows. The digestible energy (DE) and metabolizable energy (ME) of the six CSM samples ranged from 12.48 to 17.15 MJ/kg and 11.35 to 15.88 MJ/kg, respectively, for non-pregnant sows, and from 12.86 to 16.41 MJ/kg and 12.43 to 14.72 MJ/kg, respectively, for gestating sows. However, the DE, ME, and ME:DE ratios of each CSM were similar between gestating and non-pregnant sows. DE and ME were negatively correlated with NDF and ADF, respectively, but were positively corrected with CP level (P < 0.01). Collectively, the DE, ME, and nutrient digestibility of CSM varied greatly according to the chemical compositions, and CSMs with higher protein and lower fiber levels had greater DE and ME levels.

Cottonseed meal (CSM) is a high-protein feedstuff produced as a by-product of cottonseed processing. Concerns regarding the negative effects of antinutritional factors such as gossypol toxicity have limited the use of CSM as sow feed. As genetic breeding and feed processing technology improve, the potential for CSM as a supplementary protein in sow diets has increased. However, precise evaluation of the nutritive value of CSM for sows is key to accurate diet formulation. We evaluated the nutritional values of six CSMs prepared using different processing methods with different chemical compositions, fed to pregnant and non-pregnant sows, and investigated the relationships between their chemical composition and digestible (DE) and metabolizable energy (ME). We found no differences in DE and ME between gestating and non-pregnant sows for the six CSMs tested. However, there were large variations in DE and ME among the six CSMs tested, and most DEs and MEs were higher than the values published by the NRC (2012), which were measured from pigs in the growth and finishing stages. These findings demonstrate the different DE and ME values of CSM with different chemical compositions and provide a basis for precise diet formulation for sows.

Standardized ileal digestibility of amino acids in cottonseed meal fed to pregnant and non-pregnant sows.

This study determined the apparent ileal digestibility (AID) and standard ileal digestibility (SID) of crude protein (CP) and amino acids (AA) of six cottonseed meal (CSM) samples in pregnant and non-pregnant sows. Two CSM samples were processed by expelling with a CP level of 40.67% (ECSM41) and 44.64% (ECSM45), and four samples were processed by solvent-extracted which contained graded CP levels of 45% (SECSM45), 51.16% (SECSM51), 56.44% (SECSM56), and 59.63% (SECSM60). Landrace ×Yorkshire third parity sows, 7 at gestation and 14 non-pregnant, were fitted with T-cannula in the distal ileum. Pregnant sows were allotted to a 7 × 6 Latin square design with a cornstarch-based nitrogen-free (NF) diet and the six CSM diets, and non-pregnant sows were allotted to a replicated 7 × 3 Latin square design with seven diets and three periods, respectively, resulting in a total of six replicates per treatment. All experimental sows were fed 3.0 kg/d of the experimental diets. The AID of CP in ECSM41 (75.58%) was lower than in SECSM51 (80.42%), SECSM56 (80.50%), and SECSM60 (82.44%) diets for pregnant sows (P < 0.05). The AID of CP in ECSM41 (77.88%) was significantly lower than in SECSM60 (81.87%) diets for non-pregnant sows (P < 0.05). The physiological phase did not affect the AID of CP (P > 0.05). The SID of CP was affected by diets for both pregnant (P < 0.01) and non-pregnant sows (P = 0.06). The physiological phase also affected the SID of CP (P < 0.01). The AID of histidine, leucine, methionine, threonine, and tryptophan significantly differed between different CSM samples in both pregnant (P < 0.05) and non-pregnant sows (P < 0.05). The AID of dispensable AA aspartic acid, cysteine, glutamic acid, serine, and tyrosine differed between different CSM samples of both pregnant (P < 0.05) and non-pregnant sows (P < 0.05). For pregnant sows, the indispensable AA cysteine, glycine, proline, and tyrosine had significantly different SID between different groups (P < 0.05). For non-pregnant sows, the SID of arginine, lysine, methionine, threonine, aspartic acid, cysteine, and serine had different values among different diets (P < 0.05). In conclusion, the current study presented that the ileal AA digestibility of CSM fed to pregnant and non-pregnant sows increased with the decreased of fiber content, and the current findings can contribute to a precise formulation of diets for sows using CSM.

As a protein-rich cottonseed byproduct, cottonseed meal (CSM) is considered a vegetable protein source that can substitute soybean meal in the feed of livestock animals. However, the presence of free gossypol and high fiber levels in CSM have been limiting factors for its use in growing and finishing pigs, yet its nutritive value is still uncertain for sows. There is a lack of standard ileal digestibility (SID) of amino acids (AA) for plant proteins because fitting a T-cannula in the distal ileum is difficult. Therefore, this study evaluated the apparent ileal digestibility and SID of 18 AA of CSM in sows at two physiological stages (gestation and non-pregnancy). We found that CSM with different chemical compositions impacted the SID of AA when fed to pregnant and non-pregnant sows. Additionally, the physiological stage of the sow has a substantial impact on the SID of some AA. The current findings of this study provided a basis for the precise formulation of sow diets with CSM.

Cloning and characterization of ß-catenin gene in early embryonic developmental stage of Artemia sinica.

ß-Catenin plays a crucial role in embryonic development and responds to the activation of several signal transduction pathways. In this paper, in order to understand the functions of ß-catenin gene in early embryonic development of Artemia sinica, the complete cDNA sequence was cloned for the first time using RACE technology, then the sequence was analyzed by some bioinformatic methods. The expression of the ß-catenin gene was investigated at various stages during the embryonic development using quantitative real-time PCR and immunohistochemistry assay. Through the investigation, the result of real-time PCR illustrated that ß-catenin gene might relate to the response of A. sinica's immune system and osmotic pressure system in early embryonic developmental stage. Meanwhile, Immunohistochemistry assay demonstrated that during embryonic development, ß-catenin was mainly expressed in the cephalothorax. Besides, we discovered that ß-catenin might not be a maternal gene in A. sinica, and this new phenomenon may explain a constitutive and regional expression during the early embryonic development of A. sinica.

Cloning and the expression pattern of Spätzle gene during embryonic development and bacterial challenge in Artemia sinica.

Spätzle gene codes for a NGF-like protein, it involves in the embryonic development and innate immune response of insects and other invertebrate. In dorsal ventral axis differentiation, proSpätzle is activated by serine endoproteases Easter and then binds to the Toll receptor in ventral axis of oocyte which initiates the ventral axis development. Besides, it could also be activated by another protease named Spätzle-processing enzyme (SPZ) to mediate Toll pathway which involves in innate immune response in fungal and Gram-positive bacterial infection of invertebrate. In this paper, a full-length cDNA of Spätzle was firstly isolated from Artemia sinica which belonged to Spätzle-4 family. The expression of Spätzle was investigated at various stages during the embryonic development of A. sinica using real-time PCR and immunohistochemistry assays. The result showed that the high expression level of Spätzle appeared at 7 and 10 days of the embryo. A gradual increased level of Spätzle transcript occurred after being challenged with Gram-positive bacteria. Immunohistochemistry assay showed that Spätzle was mainly expressed in the cephalothorax and on the alimentary canal surface during embryonic development. This new Spätzle member showed a constitutive and regional expression during the embryonic development of A. sinica. It may play an important role in dorsal-ventral differentiation at the early development stages and in immune response pathway at the pseudoadult and adult stage, as well as during infection.

Molecular cloning and its expression of trachealess gene (As-trh) during development in brine shrimp, Artemia sinica.

Basic helix-loop-helix-PAS (bHLH-PAS) family transcription factors are implicated in multiple developmental and physiological regulatory processes. Herein, a full-length cDNA encoding a bHLH-PAS domain transcription factor trachealess gene (designated as As-trh) was cloned and characterized from brine shrimp (Artemia sinica) for the first time. The full-length cDNA of As-trh was 2,698 bp with a 2,319 bp open reading frame encoding a deduced protein of 772 amino acid polypeptide with a calculated molecular mass of 86.02 kDa and an isoelectric point of 5.87. Sequence alignment revealed that As-trh had high homology with other species trh gene, including the D-trh gene in Drosophila melanogaster and Bm-trh in Bombyx mori. The early and persistent expression of As-trh in the naupliar stages by whole-mount embryonic in situ hybridization and immunohistochemistry suggest that As-trh functions very early in the salt gland and may be required continuously in this tissue. Later in development, expression of As-trh begins to decrease and disappear in salt gland of the older nauplius and appears in the thoracic epipods of the sub-adult Artemia. These results indicated that As-trh might play an important role in osmoregulatiory organ development from the larvae stages through adult stages.

Molecular analysis and its expression of a pou homeobox protein gene during development and in response to salinity stress from brine shrimp, Artemia sinica.

Brine shrimps of the genus Artemia are aquatic species of economic importance because of their important significance to aquaculture and are used as a model species in physiology and developmental biology. Research on Artemia POU homeobox gene function will enhance our understanding of the physiological and developmental processes of POU homeobox gene in animals. Herein, a full-length cDNA encoding an Artemia POU homeobox protein gene 1 (APH-1) from Artemia sinica (designated as As-APH-1) was cloned and characterized by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. The As-APH-1 gene encoded a protein of 388 amino acid polypeptide with a calculated molecular mass of 42.85kDa and an isoelectric point of 6.90 and the protein belongs to the POU III family. Multiple sequence alignments revealed that A. sinica As-APH-1 protein sequence shared a conserved POU homeobox domain with other species. The early and persistent expression of As-APH-1 in the naupliar stages by semi-quantitative RT-PCR and whole-mount embryonic immunohistochemistry suggest that As-APH-1 functions very early in the salt gland and may be required continuously in this organ. Later in development, expression of As-APH-1 begins to dramatically decrease and disappear in salt gland of the sub-adult Artemia. In addition, we also discovered that As-APH-1 increased obviously as the salinity increased, indicating that As-APH-1 might be used as a good indicator of salinity stress. In summary, we are the first to identify the As-APH-1 gene and to determine its gene expression patterns in early embryogenesis stages and in different salinity stress in brine shrimp, A. sinica. The result of expression of As-APH-1 affected by salinity changes will provide us further understanding of the underlying mechanisms of osmoregulation in Artemia early embryonic development.

The nutritive value of soybean meal from different sources for sows during mid- and late gestation.

A precise understanding of the nutritive value of soybean meal (SBM) for pregnant sow is required for accurate feeding. Hence, we evaluated the nutritive value of 11 SBM samples from different sources for sows during mid and late gestation. In total, 24 mid-gestating sows (parity three; 230.3 ± 12.0 kg on day 37 of gestation) and 24 late-gestating sows (parity three; 238.8 ± 20.9 kg on day 72 of gestation) were assigned to a replicated 12 × 3 Youden square design with 12 diets and 3 periods. The 12 diets included a corn-based diet and 11 diets containing 25.50% SBMs from different sources. After 5-d adaptation, urine and feces were collected for 5 d. Although the chemical characteristics of SBM varied between samples, no differences were observed in digestible energy (DE), metabolizable energy (ME), apparent total tract digestibility (ATTD) of dry matter, gross energy, crude fiber, and neutral detergent fiber values in SBMs fed to both animal groups. However, de-hulled SBM 4 from Brazil displayed greater ATTD for nitrogen (N) in late-gestating sows (P < 0.05); animals displayed significantly (P < 0.01) greater ME, ME:DE ratio, and N net utilization values when compared with mid-gestating sows. The chemical composition of SBMs can be used to predict DE and ME values. In conclusion, ME, ME:DE ratio, and N net utilization SBM values for late-gestating sows were greater than in mid-gestating sows. Therefore, we should consider differences in ME values for SBMs when formulating diets for sows in mid and late gestation periods.

Soybean meal (SBM) is the most commonly used protein source in swine diets, with high available energy. Sows have crucial roles in the pig industry, therefore precise knowledge of actual SBM nutritive values at different gestation stages is vital for efficient livestock production and management. In our study, we evaluated the nutritive value of SBMs from different sources in mid- and late-gestating sows, and generated prediction equations for digestible energy (DE) and metabolizable energy (ME) values. We identified no differences in DE and ME values in SBMs from different sources when fed to sows during mid- and late gestation. However, regardless of the pregnancy stage, DE and ME SBM values for sows, identified in this study, were greater than values published by NRC (2012). Also, ME, ME:DE ratio, and nitrogen (N) net utilization SBM values for late-gestating sows were greater than in mid-gestating sows. The chemical composition of SBMs can be used to predict DE and ME values. Our study data can be used to accurately formulate diets for pregnant sows.