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The aim of this study was to determine the cellular and molecular mechanisms of leptin (LEP) and the leptin receptor (LEPR) in testicular development of prepubertal ganders. In an in vivo animal experiment, active immunization against LEPR severely depressed prepubertal testicular development by significantly reducing testicular weights at 200 and 227 days of age. The number of elongated spermatids in the seminiferous tubules was also significantly decreased by immunization with LEPR at ages of 200 and 227 days. Inhibition of testicular development by LEPR immunization was associated with decreases in LHR, StAR, 3β-HSD, CYP11A1, CYP17A1, and PRLR mRNA expression levels in testicular tissue, which resulted in a significant decrease in testosterone synthesis. In the in vitro experiments, the addition of LEP combined with anti-LEPR antibodies strengthened LEPR signal transduction, and inhibited significantly testosterone production in cultured Leydig cells isolated from prepubertal gander testes. The mRNA expression of LHR, StAR, 3β-HSD, CYP11A1, CYP17A1 also decreased significantly after treatment with LEP combined with anti-LEPR antibodies in cultured Leydig cells. These results suggest that anti-LEPR antibodies strengthen LEPR signaling transduction in the presence of LEP, and immunization against LEPR inhibited testes development and testosterone secretion in prepubertal ganders.

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Magang geese exhibit a unique characteristic of follicular development, with eight largest orderly arranged pre-ovulatory follicles in the abdominal cavity. However, little is known about the mechanisms underlying this follicular development. This study aimed to compare gene expression profiles of granulosa cells (GCs) at different stages of follicular development and provide comprehensive insights into follicle selection and the mechanisms underlying the well-defined follicle hierarchy in Magang geese. GCs of large white follicles (LWFs), small yellow follicles (SYFs), F8, F4, and F1 were used for RNA-seq analysis; 374, 1117, 791, and 593 genes were differentially expressed in stages LWFs to SYFs, SYFs to F8, F8 to F4, and F4 to F1, respectively, suggesting that these genes contribute to follicle selection and development. Reliability of sequencing data was verified through qPCR analysis of 24 genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways revealed a complex mechanism that remodels the extracellular matrix and turnover of extracellular matrix components in follicular development and ovulation and involves multiple pathway, such as focal adhesion, adherens junction, and extracellular matrix-receptor interaction. Some unique characteristics were observed during the different follicular development stages. For instance, some differentially expressed genes were enriched in progesterone-mediated oocyte maturation and steroid biosynthesis from stage SYFs to F8, whereas others were enriched in actin cytoskeleton regulation and vascular smooth muscle contraction from stage F4 to F1. These findings enhance our current understanding of GC function and ovarian follicles during the key stages of follicular development.

Egg turning during incubation plays important roles in achieving high hatching performance and gosling quality. The objective of this study was to improve embryonic and muscular developments so to achieve better gosling quality by wider egg turning angles during incubation, and to unravel the associated regulatory molecular mechanisms. In each of three consecutive incubations, 1,728 goose eggs were divided into 3 groups that were set in the same type of commercial incubators with turning angles adjusted differently to 50°, 60°, and 70°, respectively. On average of the 3 tests, incubation with wider 70° turning angle reduced the post-18-day embryo mortality, promoted embryonic growth and development, improved the hatchability and gosling quality. On embryonic day of 29, gene mRNA expression levels of the hypothalamic growth hormone-releasing hormone (GHRH), pituitary growth hormone (GH), and liver insulin-like growth factor 1 (IGF-1) were higher in the 70° turning group than in the 50° or 60° groups. Wider angle turning also increased mRNA expression levels of the muscle development regulatory genes such as MYF5, MyoD, Myogenin (MyoG), and MRF4. Changes in expression of the above genes, together with the upregulation of the Pax3 and Pax7 genes in leg muscles, well explained the enhancement of the muscular growth and development when eggs were incubated by wider turning angles. These results also extended our understanding of the impacts and mechanisms of egg turning during incubation on hatching performance and gosling quality.

Ovarian angiogenesis is an extremely rapid process that occurs during the transition from follicle to corpus luteum (CL) and is crucial for reproduction. It is regulated by numerous factors including transforming growth factor-β1 (TGFB1). However, the regulatory mechanism of TGFB1 in ovarian angiogenesis is not fully understood. To address this, in this study we obtained high-throughput transcriptome analysis (RNA-seq) data from bovine luteinizing follicular cells cultured in a system mimicking angiogenesis and treated with TGFB1, and identified 455 differentially expressed genes (DEGs). Quantitative real-time PCR results confirmed the differential expression patterns of the 12 selected genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified that the MAPK and ErbB pathways, cell adhesion molecules (CAMs), and extracellular matrix (ECM)-receptor interactions may play pivotal roles in TGFB1-mediated inhibition of CL angiogenesis. TGFB1 phosphorylated ERK1/2 (MAPK1/3) and Akt, indicating that these pathways may play an important role in the regulation of angiogenesis. Several genes with specific functions in cell adhesion and ECM degradation were identified among the DEGs. In particular, TGFB1-induced upregulation of syndecan-1 (SDC1) and collagen type I alpha 1 chain (COL1A1) expression may contribute to the deposition of type I collagen in luteinizing follicular cells. These results indicate that TGFB1 inhibits cell adhesion and ECM degradation processes involving ERK1/2, ErbB, and PI3K/Akt signaling pathways, and leads to inhibition of angiogenesis during the follicular-luteal transition. Our results further reveal the molecular mechanisms underlying the actions of TGFB1 in early luteinization.

This study was designed to investigate the effects of fermented feed diets on the growth performance and cecal microbial community in geese, and to examine associations between the gut microbiota and growth performance. A total of 720 healthy, 1-day-old male SanHua geese were used for the 55-D experiment. Geese were randomly divided into 4 groups, each with 6 replicates of 30 geese. Groups were fed a basal diet supplemented with 0.0, 2.5, 5.0, or 7.5% fermented feed. The results showed that 7.5% fermented feed had an increasing trend in the body weight and average daily gain of the geese; however, there was no significant response to increasing dietary fermented feed level with regards to ADFI and FCR. In addition, compared with the control group, there was a higher abundance of bacteria in the phylum Bacteroidetes in the cecal samples of geese in the 7.5% fermented feed group (53.18% vs. 41.77%, P0.05), whereas the abundance of Firmicutes was lower in the 7.5% fermented feed group (36.30% vs. 44.13%, P0.05). At the genus level, the abundance of Bacteroides was increased by adding fermented feed to geese diets, whereas the abundances of Desulfovibrio, Phascolarctobacterium, Lachnospiraceae_uncultured, Ruminiclostridium, and Oscillospira were decreased. These results indicate that fermented feeds have an important effect on the cecal microflora composition of geese, and may affect host growth, nutritional status, and intestinal health.

Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygen-respiring organisms, and copper/zinc SOD (Cu/ZnSOD) is one of the most important SODs. In the present study, Macrobrachium rosenbergii Cu/Zn-SOD was expressed in a yeast eukaryotic system. The open reading frame (ORF) of MrCu/ZnSOD was cloned into the plasmid vector pHAC181, and the recombinant plasmid was integrated into the downstream region of the GAL1 promoter in Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination. The resulting recombinant MrCu/ZnSOD consisted of a 3 × HA-tag at its C-terminal. Via western blot, the molecular weight of the recombinant MrCu/ZnSOD was estimated at about 30 kDa. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this recombinant MrCu/ZnSOD ranged from 0.556 to 0.840 μM, and from 0.967 to 2.015 μM, respectively. The recombinant MrCu/ZnSOD protein was able to agglutinate four Gram-negative bacterial strains, as well as two of three Gram-positive strains (except Staphylococcus aureus). This demonstrated that the recombinant protein possessed some antimicrobial activity against certain Gram-positive and Gram-negative bacteria. M. rosenbergii were fed with the recombinant yeast strain MrCu/ZnSOD for 4 weeks and then challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. This group of prawns presented lower mortality, higher enzymatic activity, and higher expression of the mRNA of immune-related genes than that in the control groups. Taken together, these results suggest that MrCu/ZnSOD is an antioxidant enzyme and antimicrobial peptide involved in the crustacean innate immune system and offers protection to the host against pathogenic bacteria.

Photoperiodic control is essential for manipulating the reproductive performance of avian species. This study was conducted to assess the neuroendocrine mechanisms that regulate reproductive functions of Yangzhou geese when there are different monochromatic light colors from light emitter diode (LED) sources. A flock of geese was divided into four groups with white, red, blue, and green light treatments being imposed. The results indicated that peak laying rates and reproductive performance were greater in geese treated with white or red as compared with blue or green light treatments. The fertilization rate of eggs and hatchability of fertilized eggs were greater with the white or red as compared with blue or green light treatments. There was a greater abundance of OPN5, Dio2, c-Fos, and GnRH-I mRNA in the hypothalamus earlier in the treatment period and abundances of these hypothalamic factors were greater with the white or red light treatments. Abundances of pituitary LH beta and FSH beta mRNA increased at a lesser rate with the blue or green light treatments and were in greater abundances with the white or red light treatments. The lighting regimen also resulted in photo-refractoriness with there being greater abundances of GnIH, VIP, and PRL mRNA with the use of white or red light treatments. The results indicate that the use of white or red monochromatic lights while imposing a long photoperiod of 11 h daily could result in sustaining functions of the reproductive system of Yangzhou geese for considerably longer times, thus, resulting in greater egg-laying performance.

Hungarian White geese are regarded as good producers of meat, eggs, and feathers, but specific lighting schedules are required to improve their egg-laying performance. This study reveals the neuroendocrine regulatory mechanisms that govern the reproductive activities and egg-laying performances of Hungarian White geese. The results indicated that increasing the daily photoperiod from a short 8 h period to either 11 h or 14 h initiated reproduction. Egg-laying rates increased faster in the 14 h group, peaking (48.2%) on day 33 as compared to the peak (52.67%) reached on day 53 in the 11 h group. Changes to the plasma estradiol and progesterone concentrations produced similar patterns in the two groups. In the hypothalamus, OPN5, Dio2, c-Fos, and GnRH-I expression levels showed similar sequential increases and decreases. Changes in GnIH and VIP expression levels were the opposite to those of GnRH-I, but the levels peaked earlier under the 14 h photoperiod conditions. Pituitary LH beta and FSH beta expression levels increased at slower rates but remained significantly higher in the 11 h group than in the 14 h group. However, pituitary PRL expression increased considerably earlier and was higher in 14 h geese than in 11 h geese, which was opposite to the observed egg-laying rate patterns. An increase from a short to a relatively long photoperiod (11 h) regulated the neuroendocrine system and led to reproductive activities being sustained for a longer period, which resulted in high egg-laying performances.

Pattern recognition proteins (PRPs) activate the innate immune system in invertebrates, and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) is an important PRP with various biological functions. Here, the open reading frame (ORF) of Macrobrachium rosenbergii LGBP (MrLGBP) was cloned into plasmid vector pHAC181, then integrated into downstream of the GAL1 promoter of Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination, followed by its expression in the yeast eukaryotic system. The resulting recombinant LGBP contained a 3 × HA-tag at its C terminus and had a molecular weight of about 45 kDa, as evaluated by western blot analysis. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were ranged from 0.340 to 0.802 and 1.189–1.810 μM, respectively. The recombinant MrLGBP protein agglutinated almost all tested bacteria except Bacillus thuringiensis and Staphylococcus aureus. These results revealed that this recombinant protein exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. M. rosenbergii prawns were fed with the recombinant yeast strain MrLGBP for 1 month and challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. These prawns showed lower mortality and higher enzymatic activity and expression levels of immunity genes than did the control groups. All these results suggest that MrLGBP may play important roles in the innate immunity of crustaceans, and recombinant strain S. cerevisiae MrLGBP may be useful for the development of an effective immune feed additive in the future.

Growth hormone (GH) is required for normal postnatal development in poultry; however, no immunoassay exists to assess its levels in geese plasma, hindering the study of endocrine regulation in this species. We developed a sandwich ELISA to determine the GH concentrations in the plasma of geese. Recombinant goose GH was produced using a eukaryotic expression system and purified for use as the reference standard in ELISA and the antigen for producing the polyclonal antibodies in rabbits. Rabbit anti-goose GH polyclonal antibody was used to coat the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and catalyze the chromogenic reaction of 3,3,5,5-tetramethylbenzidine and H2O2. A sigmoidal curve was fitted to the optical density and the log of the standard GH concentration using the four-parameter logistic model. The sensitivity of the assay was less than 0.156 ng/mL. The intra- and interassay coefficients of variation were less than 9 and 13%, respectively. The response curve of the serially diluted plasma samples from geese exhibited a good parallel relationship with that observed for the reference standards. The assay effectively detected differences in GH concentrations in plasma samples from geese at various physiological stages; thus, it will be useful for future study of their growth and metabolism.

Lipopolysaccharide (LPS) from gram-negative bacteria was found to be involved in the decrease in laying performance in goose flocks with high stocking density during summer months. LPS injection delayed the increase in the laying rate and altered hierarchical follicle morphology. While there is evidence that LPS exerts suppressive effects on goose reproduction, the time course effects of LPS on the hypothalamus-pituitary-ovary (HPG) axis remain elusive. In this study, we investigated the expression of genes in the HPG axis and the plasma gonadotrophin hormone concentrations in breeding geese at 0, 6, 12, 24, and 36 h after intravenous injection with LPS. The results showed that LPS treatment enhanced and suppressed expression of hypothalamic gonadotropin-inhibiting hormone (GnIH) and gonadotrophin-releasing hormone (GnRH) mRNA, respectively, and similar effects were observed on the mRNA expression of their receptors, GnIHR and GnRHR, in the pituitary. LPS treatment transiently increased follicle FSH&beta, mRNA expression at 12 h and exerted no significant effect on LH&beta, mRNA expression in the pituitary. Regardless of the expression of FSH&beta, and LH&beta, plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were significantly increased during 24&ndash, 36 h after LPS treatment. In the ovary, StAR and Cyp11a1 were mainly expressed in the granulosa layer (GL) of hierarchical follicles, while Cyp17a1 and Cyp19a1 were mainly expressed in white follicles (WFs) and yellowish follicles (YFs), and to a lesser extent in the theca layer (TL). After LPS treatment, the mRNA levels of Cyp11a1 in the GLs, Cyp17a1 in the WFs and TL, and Cyp19a1 in the WFs, YFs, and TL were significantly decreased. However, LPS treatment transiently upregulated StAR expression at 12 h. These results indicate that the exposure of laying geese to LPS may impair the HPG axis and disturb ovarian steroidogenesis. Our research provides new insights into reproductive dysfunction caused by LPS and the immune challenge in birds.

Treatment of fetal rat and embryonic chicken with exogenous glucocorticoids induces premature differentiation of growth hormone (GH) secreting cells. The effect of corticosterone (CORT) on somatotroph differentiation was mostly studied in pituitary cells in vitro. Currently, there is no evidence for glucocorticoid-mediated induction of somatotroph differentiation during pituitary development in bird species other than chicken. In this study, we sought to find out if in ovo injection of corticosterone into developing goose embryos could induce premature increase of GH in somatotrophs. On embryonic day (e) 15, the albumen of fertile goose eggs was injected with 300 μl of 0.9% saline, 300 μl 5 × 10-8M CORT, or 300 μl 5 × 10-6 M CORT. Embryos were allowed to develop until e20 and e28 and isolated pituitaries were subjected to quantitative real-time PCR and immunocytochemistry to detect GH mRNA and protein, respectively. At e20 and e28, blood from chorioallantoic vessels was subjected to radioimmunoassay for analysis of plasma GH protein. In ovo administration of exogenous corticosterone brought about a 2.5-fold increase in the expression of GH mRNA and increased the in situ expression of GH protein in goose pituitary cells, and enhanced plasma GH levels compared to that of the respective controls at e20. These findings prove that administration of glucocorticoid could stimulate the expression of GH in somatotrophs during goose embryonic development. Our results suggest the probable involvement of membrane glucocorticoid receptor in the corticosterone mediated expression of GH. Together with previously published data, our results suggest that corticosterone mediated induction of GH expression during embryonic development is relatively conserved among different vertebrates.

Spiroplasma eriocheiris is a novel pathogen similar to the Spiroplasma mirum and also had an ability to infect the newborn mice and caused cataract. Our study was designed to study how S. eriocheiris infects mouse 3T6-Swiss albino cells and to elucidate the cellular molecular pathogenesis of Spiroplasma. FCM analysis and MTT analysis clearly shown that S. eriocheiris could induce 3T6 cell apoptosis and cause cell viability decreased seriously. Immunofluorescence experiments and TEM analysis shown that S. eriocheiris can invade 3T6 cells and form typical inclusion bodies and exhibit vacuolization in vitro. S. eriocheiris-oxytetracycline protection assay show that the infective bacteria already were detected at 1h post infection, and sharply increased at 12h after the bacteria infection. To further study the infection mechanism of S. eriocheiris, global mRNA and microRNA (miRNA) expression profiling were analyzed after the cells infected with the bacteria. A total of 619 non-redundant annotated transcripts (183 up-regulated and 436 down-regulated) and 22 miRNAs (8 up-regulated and 14 down-regulated) were differential expression after 6h S. eriocheiris infection compared to control group. Integrated analysis shown that homologous genes from differential expression miRNA targets and the differential expression genes of the mRNA microarray were major focused on two important pathways focal adhesion and MAPK signaling pathway. To validate the results of microarray, eight focal adhesion (β-Catenin, Parvin, Grb2 and ERK) and MAPK signaling pathway (FGFR, Grb2, ERK, MKK3, p38 and JNK) genes and the housekeeping gene GAPDH were assayed by qPCR and Western blot to confirm the results. Eight miRNAs (miR-143-3p, miR-214-5p, miR-322-3p, miR-328-5p, miR-351-5p, miR-466h-5p, miR-503-5p and miR-30c-1-3p) and the housekeeping gene U6 miRNA were assayed by qPCR to confirm the results of microarray. All the results help us better understand the infection mechanism of S. eriocheiris.

Triggering receptor expressed on myeloid cells (TREM) is a cell-surface receptor primarily expressed on macrophages. Here, two novel TREM genes, AcTREM1 and AcTREM2, were identified from Anas cygnoides. AcTREM1 cDNA contained a putative signal peptide, two IG domains, and a transmembrane domain. The deduced AcTREM2 sequence also contained a signal peptide, an IG domain, and a transmembrane domain. qRT-PCR, fluorescence in situ hybridization, and immunofluorescence experiments showed that AcTREM1 and AcTREM2 were mainly expressed in the liver and spleen, and both genes and proteins were mainly distributed in cytoplasm. AcTREM1 expression in the liver and spleen was significantly upregulated following lipopolysaccharide (LPS) challenge at an early stage of infection and then decreased at a later stage. Changes in AcTREM2 expression were reciprocal to those of AcTREM1 in the liver and spleen after LPS challenge. Our results indicate that AcTREM1 and AcTREM2 participate in the antibacterial immunity of A. cygnoides.

In asymmetric chick gonads, the left and right female gonads undergo distinct programs during development, generating a functional ovary on the left side only. Despite some progress being made in recent years, the mechanisms of molecular regulation remain incompletely understood, and little genomic information is available regarding the degeneration of the right ovary in the chick embryo testis. In this study, we performed transcriptome sequencing to investigate differentially expressed genes in the left and right ovaries and gene functions at two critical time points; embryonic days 6 (E6) and 10 (E10). Using high-throughput RNA-sequencing technologies, 539 and 1046 genes were identified as being significantly differentially expressed between 6R-VS-6L and 10R-VS-10L. Gene ontology analysis of the differentially expressed genes revealed enrichment in functional pathways. Among these, candidate genes associated with degeneration of the right ovary in the chick embryo were identified. Identification of a pathway involved in ovarian degeneration provides an important resource for the further study of its molecular mechanisms and functions.

FSH is a glycoprotein hormone secreted by the pituitary gland that is essential for gonadal development and reproductive function. In avian reproduction study, especially in avian reproduction hormone study, it is hindered by the lack of biologically active FSH. In order to overcome this shortcoming, we prepared recombinant goose FSH as a single chain molecule and tested its biological activities in the present study. Coding sequences for mature peptides of goose FSH α and β subunits were amplified from goose pituitary cDNA. A chimeric gene containing α and β subunit sequences linked by the hCG carboxyl terminal peptide coding sequence was constructed. The recombinant gene was inserted into the pcDNA3.1-Fc eukaryotic expression vector to form pcDNA-Fc-gFSHβ-CTP-α and then transfected into 293-F cells. A recombinant, single chain goose FSH was expressed and verified by SDS-PAGE and western blot analysis, and was purified using Protein A agarose affinity and gel filtration chromatography. Biological activity analysis results showed that the recombinant, chimeric goose FSH possesses the function of stimulating estradiol secretion and cell proliferation, in cultured chicken granulosa cells. These results indicated that bioactive, recombinant goose FSH has been successfully prepared in vitro. The recombinant goose FSH will have the potential of being used as a research tool for studying avian reproductive activities, and as a standard for developing avian FSH bioassays.

Lipopolysaccharide (LPS) is an inhibitory factor that causes hormonal imbalance and subsequently affects ovarian function and fertility in mammals. Previous studies have shown that the exposure of granulosa cells (GC) to LPS leads to steroidogenesis dysfunction. However, the effects of LPS on the viability of GC remain largely unclear. In the present study, we aimed to address this question and unveil the underlying molecular mechanisms using cultured porcine GC. Results showed that GC proliferation and tumor necrosis factor α (TNFα) secretion were significantly increased after exposure to LPS, and these effects were completely reversed by blocking the TNFα sheddase, ADAM17. Moreover, GC proliferation induced by LPS was mimicked by treatment with recombinant TNFα. In addition, SerpinE1 and SerpinB2 expression levels increased in GC after treatment with LPS or recombinant TNFα, whereas blocking the Erk1/2 pathway completely abolished these effects and also inhibited GC proliferation. Further, consistent with the effects of blocking the Erk1/2 pathway, cell proliferation was completely inhibited by knocking down SerpinE1 or SerpinB2 in the presence of LPS or recombinant TNFα. Mitochondrial membrane potential (MMP) polarization in GC was increased by LPS or recombinant TNFα treatment, and these changes were completely negated by Erk1/2 inhibition, but not by SerpinE1 or SerpinB2 knockdown. Taken together, these results suggested that the TNFα-mediated upregulation of SerpinE1 and SerpinB2, through activation of the Erk1/2 pathway plays a crucial role in LPS-stimulated GC proliferation, and the increase in GC MMP may synergistically influence this process.

The present study was designed to investigate the potential role of immunization against inhibin on testicular development, plasma testosterone concentration and expression of relevant genes in hypothalamus, pituitary, Leydig and Sertoli cells in Yangzhou ganders. For this purpose, Yangzhou ganders, n = 30 were divided into groups A and B. Group B ganders were actively immunized against inhibin α-subunit, while group A ganders were immunized with bovine serum albumin (BSA), which served as control. Immunization against inhibin elevated testes weights. In addition, immunization against inhibin elevated GnRH, StAR, CYP11A1 and AMH mRNA transcription expressions as depicted by qRT-PCR. Furthermore, hypothalamic GnRH-I mRNA expressions were up regulated, while GnIH mRNA transcription expression showed reciprocal expression on day 227. LH-β mRNA transcription expression remained unaffected. In conclusion, our findings suggest that active immunization against inhibin affect spermatogenesis and testicular development through regulations of hypothalamic, pituitary and testicular genes expressions.

The aim of this study was to determine the cellular and molecular mechanisms of prolactin (PRL) in testicular development of prepubertal cockerels. In an in vivo animal experiment, active immunization against PRL severely depressed prepubertal testicular development by significantly reducing testicular weights at both 122 and 164 d of age. The number of elongated spermatids in the seminiferous tubules was also significantly decreased by immunization with 199-residue chicken PRL (cPRL) at age 122 d. Inhibition of testicular development by cPRL immunization was associated with decreases in LH receptor (LHR), FSH receptor (FSHR), Stat5b, P450scc, steroidogenic acute regulatory (StAR) protein, and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression levels in testicular tissue. In in vitro experiments, testosterone production by cultured Leydig cells isolated from prepubertal cockerel testes was dose-dependently enhanced by treatment with bioactive recombinant PRL, but a lesser response was seen with high concentrations of PRL. The distinct changes in testosterone production in response to high and low concentrations of added PRL were paralleled by similar patterns of change in the mRNA levels of Stat5b, LHR, P450scc, StAR, 3β-HSD, and CYP17A1 in cultured Leydig cells, as well as protein amounts of phosphorylated Jak2 and Stat5a/b. In conclusion, low to medium doses of PRL potentiate testis development in prepubertal cockerels by enhancing testosterone secretion from Leydig cells via activation of PRLR/Stat5b signal transduction, which upregulates mRNA expression of LHR and testosterone synthesizing enzymes. However, this positive regulation was weaker in response to a high dose of PRL, which reduced PRLR/Stat5b signal transduction and the expression of genes involved in LH signaling and testosterone synthesis.

The present study was conducted to establish a sandwich ELISA for the determination of prolactin (PRL) concentrations in the plasma of domestic fowls. The assay uses a recombinant goose PRL as the reference standard, expressed in a eukaryotic system, and as the antigen for raising a polyclonal antibody in rabbit. This rabbit anti-goose PRL polyclonal antibody was used for coating the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and to catalyze the chromogenic reaction using 3,3',5,5'-tetramethylbenzidine as the substrate. The assay showed a linear relationship between the optical density and concentration of the standard PRL in the 0 to 12.5 ng/mL range, and the assay was sensitive to a concentration as low as 0.39 ng/mL. The intra- and inter-assay CVs were7% and 11%, respectively. The response curves of the serially diluted plasma samples from goose, duck, and chicken exhibited similar parallel relationships to that observed for the reference standards. Consistent with previous findings, the assay effectively detected differences in PRL concentration in plasma samples from chicken, duck, and goose at various reproductive stages.

The toll-like receptor/NF-κB signaling pathways play an important role in the innate immune system. In the present study, one Pelle gene (named EsPelle) was identified for the first time from the Chinese mitten crab Eriocheir sinensis. The full-length cDNA of EsPelle is 3797 bp with a 3156 bp-long open reading frame that encodes a 1051 amino acid protein. EsPelle protein contains a death domain at the N-terminal and a serine/threonine kinase domain at the C-terminal. A neighbor joining phylogenetic tree showed that the EsPelle protein, which is closest to those of Scylla paramamosain Pelle and Litopenaeus vannamei Pelle, was clustered to a group of crustacean Pelle proteins. EsPelle was expressed in all tested tissues of normal crabs, and its expression was regulated in hemocytes and hepatopancreas of crabs challenged with lipopolysaccharide, peptidoglycan, Staphyloccocus aureus, Vibrio parahaemolyticus, and Aeromonas hydrophila. Overexpression of EsPelle in Drosophila Schneider 2 cells could upregulate the expression of Drosophila antimicrobial peptides, namely, metchnikowin (Mtk), attacinA (Atta), drosomycin (Drs), and cecropinA (CecA). Moreover, EsPelle silencing by siRNA reduced the transcription of anti-lipopolysaccharide factor 1 and 2, crustin 2, and lysozyme in crabs challenged with V. parahaemolyticus. From the results, we speculated that EsPelle was involved in innate immune defense against V. parahaemolyticus in E. sinensis.

Background The Yangzhou goose is a long-day breeding bird that has been increasingly produced in China. Artificial lighting programs are used for controlling its reproductive activities. This study investigated the regulations of photostimulation and photorefractoriness that govern the onset and cessation of the breeding period. Results Increasing the daily photoperiod from 8 to 12 h rapidly stimulated testis development and increased plasma testosterone concentrations, with peak levels being reached 2 months after the photoperiod increase. Subsequently, testicular activities, testicular weight, spermatogenesis, and plasma testosterone concentrations declined steadily and reached to the nadir at 5 months after the 12-hour photoperiod. Throughout the experiment, plasma concentrations of triiodothyronine and thyroxine changed in reciprocal fashions to that of testosterone. The stimulation of reproductive activities caused by the increasing photoperiod was associated with increases in gonadotropin-releasing hormone (GnRH), but decreases in gonadotropin-inhibitory hormone (GnIH) and vasoactive intestinal peptide (VIP) gene messenger RNA (mRNA) levels in the hypothalamus. In the pituitary gland, the levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mRNA abruptly increased during the longer 12-hour photoperiod. The occurrence of photorefractoriness was associated with increased GnIH gene transcription by over 250-fold, together with increased VIP mRNA levels in the hypothalamus, and then prolactin and thyroid-stimulating hormone in the pituitary gland. FSH receptor, LH receptor, and StAR mRNA levels in the testis changed in ways paralleling those of testicular weight and testosterone concentrations. Conclusions The seasonal reproductive activities in Yangzhou geese were directly stimulated by a long photoperiod via upregulation of GnRH gene transcription, downregulation of GnIH, VIP gene transcription, and stimulation of gonadotrophin. Development of photorefractoriness was characterized by hyper-regulation of GnIH gene transcription in the hypothalamus, in addition of upregulation of VIP and TRH gene transcription, and that of their receptors, in the pituitary gland.

The red swamp crayfish, Procambarus clarkii (Girard, 1852) is one of the most commonly-farmed freshwater species in inland China, but the yield is very low. The aim of this study was to assess whether the gender has effect on growth performance of P. clarkii. Male and female juvenile crayfish were individually reared in net cages for 75 days (trial 1). In addition, juvenile crayfish and adult crayfish of different gender were group-reared for 60 days and 30 days respectively (trials 2 and 3). The weight growth rate (WGR, %), length growth rate (LGR, %) and survival rate (SR, %) were monitored. Results showed that the length-weight regressions differed significant between males and females juvenile crayfish (P females > males) was noted among monosex and mixed-sex groups. No significant difference was noted, in terms of LGR, between females and males (P>0.05). However, the LGR of each of these was significantly lower than that of the mixed-sex groups (P

Spiroplasma eriocheiris is known to cause tremor disease in the Chinese mitten crab Eriocheir sinensis; however, the molecular characterization of this pathogen is still unclear. S. eriocheiris has the ability to invade and survive within mouse 3T6 cells. The invasion process may require causing damage to the host cell membrane by chemical, physical or enzymatic means. In this study, we systematically characterized a novel lysophospholipase (lysoPL) of S. eriocheiris TDA-040725-5T. The gene that encodes lysoPL in S. eriocheiris (SE-LysoPL) was cloned, sequenced and expressed in Escherichia coli BL21 (DE3). Enzymatic assays revealed that the purified recombinant SE-LysoPL hydrolysed long-chain acyl esterases at pH 7 and 30 °C. SE-LysoPL was detected in the membrane and cytoplasmic protein fractions using the SE-LysoPL antibody in Western blot. The virulence ability of S. eriocheiris was effectively reduced at the early stage of infection (m.o.i.=100) by the SE-LysoPL antibody neutralization test. To the best of our knowledge, this is the first study to identify and characterize a gene from S. eriocheiris encoding a protein exhibiting lysoPL and esterase activities. Our findings indicate that SE-LysoPL plays important roles in the pathogenicity of S. eriocheiris.

A growth experiment was conducted to determine the optimal dietary carbohydrate-to-lipid (CHO : L) ratio for the Australian redclaw crayfish, Cherax quadricarinatus (von Martens). Specimens were divided into five treatment groups and fed twice a day to apparent satiation with isonitrogenous and isoenergetic diets with dietary CHO : L ratios of 10.75:1, 5.99:1, 3.60:1, 2.18:1, or 1.33:1. Over the 8-wk growth trial, redclaw crayfish fed the 2.81:1 and 3.60:1 diets exhibited better weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) than specimens fed the other diets (P 0.05). On the basis of the analysis of growth, FCR, and costs of culturing redclaw crayfish, the optimal growth and costs occurred when the dietary carbohydrate and lipid levels were 290.10 g/kg and 80.70 g/kg, respectively, corresponding to a CHO : L ratio of 3.60:1. These results indicate that a CHO : L ratio of 3.60:1 is optimal for rearing redclaw crayfish and that this species uses carbohydrate more efficiently than lipid.

This study was carried out to induce out-of-season breeding, in the summer, and to achieve high reproductive performance using artificial photoperiod manipulation in the long-day breeding Yangzhou goose. Young geese were subject to a two-phase short-to-long (group A) or a three-phase (long-short-long; group B) photoperiod program February through October. Egg-laying was induced to start similarly in both groups in May, increased to a peak level in July, and then decreased gradually through to October. The peak and post-peak laying rates were higher with the three-phase than with the two-phase program. Plasma progesterone concentrations changed similarly in the two groups, increasing from low levels during the pre-lay periods until the peak laying stage, then decreasing with decline in the egg-laying rate. Plasma T3 concentrations increased from the beginning of the experiment to form the first peak under a short photoperiod, declined to a trough at peak lay and then progressively increased to high levels towards the end of the experiment. Plasma T4 concentrations increased throughout the experiment, showing little response to changes in photoperiod. GnIH mRNA expression level in the hypothalamus steadily decreased from high levels under the short photoperiod to a nadir at peak of lay, but was abruptly up-regulated by over a thousand-fold thereafter. This mRNA expression pattern was also shared by GnIHR, VIPR, TRHR, TSH, and PRL genes in the pituitary gland, and to lesser extent, by GnRH, VIP, and TRH genes in the hypothalamus. Pituitary GnRHR mRNA expression levels changed in a similar manner to that of reproductive activities of geese in both groups. FSH beta subunits mRNA expression levels increased to high levels after day 11 of the long photoperiod, and were higher in group B than in group A at peak laying. LH beta gene expression level was similarly upregulated by photoperiod and was higher in group B than in group A when used the multivariable and two-way analyses of variance. Taken together, photoperiod, through regulation of expression of an array of genes in the hypothalamus and pituitary gland, synchronized stimulation and refractoriness of the reproductive system in Yangzhou geese. The higher out-of-season egg laying performance following the three-phase photo-program treatment was mediated by higher FSH beta and LH beta subunit mRNA expression levels.

Flow cytometry provides rapid and reproducible methods for analyzing crustacean cellular immune responses to pathogens. We used this method to investigate the hemocytes sub-populations of freshwater prawn Macrobrachium rosenbergii and their immune responses to a novel pathogen spiroplasma MR-1008. M. rosenbergii inoculated with 100 μl spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for total hemocytes count (THC) and changes in differential involvement of hemocytes sub-populations during 1-28 d after inoculation. The results showed that THC was dramatically lowered 1 d after inoculation, and it obviously increased at the 5 d after inoculation; thereafter, a high level of THC was maintained to 15 d. Three morphologically distinct hemocytes sub-populations including granular cells (GC), semigranular cells (SGC) and hyaline cells (HC) could be identified by flow cytometry, and the proportions of the 3 kinds of cell categories varied obviously during the infection of spiroplasma suggesting differential involvement according to the pathogen. The flow cytometry used in this study confirmed that the semigranular cells were the main hemocytes involved in the cellular defense against spiroplasma in the M. rosenbergii.

Integrins belong to a superfamily of conserved α β heterodimeric cell surface receptors that have critical function in cell migration, differentiation, and survival. In this study, an integrin called EsIntegrin was identified from Chinese mitten crab Eriocheir sinensis. EsIntegrin cDNA is 4415 bp long with a 2457 bp open reading frame that encodes an 818 amino acid protein. EsIntegrin contains a signal peptide, an integrin beta subunit (N-terminal portion of extracellular region) INB domain, an epidermal growth factor (hEGF) domain, an integrin B tail domain, a transmembrane region, and an integrin b cyt domain. EsIntegrin was mainly expressed in hemocytes and the heart, with a relatively lower expression level in gills, nerves, intestine, hepatopancreas, muscles, and eyestalk. When healthy crabs were challenged with LPS, PGN, Staphyloccocus aureus, or Vibrio parahaemolyticus, EsIntegrin expression level was upregulated significantly. Recombinant EsIntegrin has agglutination activity to Gram-positive (e.g., S. aureus and Bacillus subtilis) and Gram-negative bacteria (e.g., V. parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. Furthermore, rEsIntegrin could not only bind to various bacteria such as S. aureus, Micrococcus luteus, B. subtilis, Bacillus megaterium, Bacillus thuringiensis, V. parahaemolyticus, Vibrio anguillarum, A. hydrophila, Vibrio natriegens, and Escherichia coli, but this compound also helped crabs in clearing virulent Gram-negative bacterium, V. parahaemolyticus, in vivo. These data suggested that EsIntegrin might function as cellular receptor that is involved in anti-bacterial immunity from E. sinensis.

The 70-kDa family of heat-shock proteins (Hsp70) plays an important role in the host immunity, which is widely expressed in eukaryotic cells as a major chaperone protein. In the present study, the full-length complementary DNA (cDNA) of a second cognate cytosolic Hsp70 family member (MnHsc70-2) was cloned and characterized from Macrobrachium nipponense, which is an economically and nutritionally important crustacean. The cDNA was 2,717 bp, containing an open reading frame (ORF) of 1,950 bp, which encodes a protein of 649 amino acids with a theoretical molecular weight of 71.1 kDa and an isoelectric point of 5.27. Sequence alignment showed that the MnHsc70-2 shared 75–97 % identity with other heat-shock proteins. Compared to the previously identified cognate Hsp70 (MnHsc70-1) in M. nipponense, MnHsc70-2 showed quite different expression profiles under unstressed conditions in all tested tissues, including the hemocytes, heart, hepatopancreas, gill, intestine, nerve, and muscle. The phylogenetic analysis demonstrated that MnHsc70-2 showed the closest relationship with MnHsc70-1. Heat-inducibility assays showed that two isolated messenger RNAs (mRNAs) displayed different expression profiles in both the hepatopancreas and gill tissues. MnHsc70-1 mRNA expression level decreased at first and then increased to the normal level, whereas MnHsc70-2 mRNA level increased at first and then decreased. The expressions of two MnHsc70s showed substantial obvious heat-inducible regulation in both the hepatopancreas and gill. Under bacterial challenge by Aeromonas hydrophila, both MnHsc70-1 and MnHsc70-2 mRNA level was up-regulated moderately. The results suggested that two cognate Hsc70s may play essential functions in mediating responses to heat-shock and bacterial challenge.

We used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from hemocytes of Procambarus clarkii under normal and infection conditions. The high-throughput sequencing approach resulted in approximately 12,801,827 and 8,410,455 raw reads corresponding to 10,949,754 and 6,648,161 high-quality mappable reads for the normal and infected hemocyte samples, respectively. Bioinformatic analyses identified 195 unique miRNAs, including 30 that are conserved in crustaceans, 48 that are novel to crayfish but are present in other arthropods (PN-type), and 117 that are completely new (PC-type). Thirty-three miRNAs displayed significant differential expressions between the two hemocyte samples (p < 0.0001). Of these, 15 (45.5%) were significantly up-regulated and 18 (54.5%) were significantly down-regulated upon challenge with Spiroplasma eriocheiris. Integrating comparative genomic and bibliomic analysis, of the 33 significant miRNAs identified, 19 were conserved and immune-related in P. clarkii and Eriocheir sinensis infected with S. eriocheiris infection; 24 were conserved and immune-related in P. clarkii and Marsupenaeus japonicus immune response to S. eriocheiris or white spot syndrome virus (WSSV) infection. Function annotation of target genes revealed a broad range of biological processes and signal transduction pathways that regulated by crayfish miRNAs. Thereinto, pcl-miR-34, pcl-miR-7, PN-pcl-let-7, pcl-miR-1, and pcl-miR-2b are highly conserved in vertebrates and invertebrates and function in the similar pathways. To our knowledge, this is the first report of comprehensive identification of P. clarkii miRNAs and of expression analysis of P. clarkii miRNAs after exposure to S. eriocheiris in crayfish, and many miRNAs were differentially regulated under normal and infection conditions. Our results should help develop new control strategies for efficient immune protection against S. eriocheiris infections in crustaceans.

Lectins play important roles in crustacean innate immunity through recognition of foreign pathogens. In this study, 20 lectins including C-type lectins [dual-carbohydrate recognition domain (CRD) type and single-CRD type], L-type lectin, and lectin with low-density lipoprotein class A (LDLa) domain were identified from the freshwater prawn Macrobrachium rosenbergii. The tissue distribution and expression patterns of these lectins under spiroplasma strain MR-1008 challenge were investigated. Most of the lectins were found to be mainly distributed in the hepatopancreas. Lectin5, Lectin14, Lectin17, and Lectin18 exhibited the highest expression level in the hemocytes, nerve, intestine, and heart, respectively. MrLec1 to MrLec6 (dual-CRD lectins) in the hepatopancreas were up-regulated by spiroplasma challenge. Single-CRD lectins reached the highest level at 72 h after spiroplasma challenge. Lectin9 and Lectin15 both belong to L-type lectins. At post-spiroplasma challenge, Lectin9 expression was up-regulated, whereas Lectin15 expression was down-regulated. Lectin11 with LDLa domain showed the highest level after 12 h Lectin18 and Lectin20, namely, CD209, were also up-regulated by spiroplasma challenge. Lectin14, a C-type lectin, quickly reached the highest level after 2 h Lectin16 showed the highest level after 72 h Lectin5 reached the highest level in cultured hemocytes after 6 h Lectin17 in the intestine and Lectin14 in the nerve were slightly up-regulated after 6 and 2 h, respectively. Our research results indicate that lectins may play important roles in early or late immune responses against spiroplasma challenge.

Freshwater prawn Macrobrachium rosenbergii inoculated with 100 μl novel pathogen spiroplasma strain MR-1008 in logarithmic phase (10 8 spiroplasmas ml −1 ) were examined for alkaline phosphatase (AKP) activity, acid phosphatase (ACP) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, as well as expressions of 7 immune related genes in hepatopancreas after 1–28 d. Hematoxylin-eosin (HE) staining showed obvious pathological features in hepatopancreas connective and epithelial tissue. Enzyme activity analyze showed that hepatopancreas AKP and ACP activity increased markedly ( P ) when inoculated with spiroplasma MR-1008 after 5 d and 10 d, respectively. SOD enzyme activity changed less obviously and slightly increased at 1 day post-inoculation, but CAT activity decreased significantly after 5 d inoculation. The expression levels of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglobulin (α2M), AKP, ACP, CAT, and copper/zinc SOD (Cu, Zn-SOD) genes in the hepatopancreas were examined by Real-Time PCR (qRT-PCR) and the results demonstrated that these immune related genes were induced by challenge with spiroplasma MR-1008. The results suggested that the prawn immune responses could be activated or inhibited by spiroplasma MR-1008, and that the hepatopancreas also plays key roles in innate immunity for defense against the pathogen.

Superoxide dismutase (SOD) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species. We cloned cDNA encoding SOD activated with manganese (Mn-SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of Mn-SOD was 1,016 bp and had a 669 bp open reading frame encoding 222 amino acids. The deduced amino acid sequence of B. calyciflorus Mn-SOD showed 89.1, 71.3, and 62.1 % similarity with the Mn-SOD of the marine rotifer Brachionus plicatilis, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, respectively. The phylogenetic tree constructed based on the amino acid sequences of Mn-SODs from B. calyciflorus and other organisms revealed that this rotifer is closely related to nematodes. Analysis of the mRNA expression of Mn-SOD under different conditions revealed that expression was enhanced 5.6-fold (p0.001) at 30 °C after 2 h, however, low temperature (15 °C) promoted Mn SOD temporarily (2.5-fold, p0.001) and then decreased to normal level (p0.05). Moderate starvation promoted Mn-SOD mRNA expression (p 120.01, p 360.05), which reached a maximum value (15.3 times higher than control, p 240.01) at 24 h. SOD and CAT activities also elevated at the 12 h-starved group. These results indicate that induction of Mn-SOD expression by stressors likely plays an important role in aging of B. calyciflorus.