Orthogonal Enzyme-Substrate Design Strategy for Discovery of Human Protein Palmitoyltransferase Substrates.

Protein palmitoylation, with more than 5000 substrates, is the most prevalent form of protein lipidation. Palmitoylated proteins participate in almost all areas of cellular physiology and have been linked to several human diseases. Twenty-three zDHHC enzymes catalyze protein palmitoylation with extensive overlap among the substrates of each zDHHC member. Currently, there is no global strategy to delineate the physiological substrates of individual zDHHC enzymes without perturbing the natural cellular pool. Here, we outline a general approach to accomplish this on the basis of synthetic orthogonal substrates that are only compatible with engineered zDHHC enzymes. We demonstrate the utility of this strategy by validating known substrates and use it to identify novel substrates of two human zDHHC enzymes. Finally, we employ this method to discover and explore conserved palmitoylation in a family of host restriction factors against pathogenic viruses, including SARS-CoV-2.

Synthesis and Evaluation of Fluorine-18-Labeled L-Rhamnose Derivatives.

The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[18F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7-27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.

The p38 signaling pathway mediates the TGF-ß1-induced increase in type I collagen deposition in human granulosa cells.

Type I collagen, which is mainly composed of collagen type I alpha 1 chain (COL1A1), is the most abundant extracellular matrix (ECM) protein in the mammalian ovary; and the cyclical remodeling of the ECM plays an essential role in the regulation of corpus luteum formation. Our previous studies have demonstrated that TGF-ß1 is a potent inhibitor of luteinization in human granulosa-lutein (hGL) cells. Whether TGF-ß1 can regulate the expression of COL1A1 during the luteal phase remains to be elucidated. The aim of this study was to investigate the effect of TGF-ß1 on the regulation of COL1A1 expression and the underlying molecular mechanisms using an immortalized hGL cell line (SVOG cells) and primary hGL cells (obtained from 20 consenting patients undergoing IVF treatment). The results showed that TGF-ß1 significantly upregulated the expression of COL1A1. Using inhibition approaches, including pharmacological inhibition (a specific p38 inhibitor, SB203580, and a specific ERK1/2 inhibitor, U0126) and specific siRNA-mediated knockdown inhibition, we demonstrated that TGF-ß1 promoted the expression and production of COL1A1 in hGL cells, most likely via the ALK5-mediated p38 signaling pathway. Our findings provide insights into the molecular mechanisms by which TGF-ß1 promotes the deposition of type I collagen during the late follicular phase in humans.

Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications*.

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

Transcriptome analysis to unravel the gene expression profile of ovarian follicular development in Magang goose.

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.

Corticosterone induces growth hormone expression in pituitary somatotrophs during goose embryonic development.

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.

Time course effect of lipopolysaccharide on Toll-like receptors expression and steroidogenesis in the Chinese goose ovary.

The ovary of Chinese goose is easily infected by microorganisms because of the mating behaviour in water, which causes decreased laying performance. This study investigated the time course effect of lipopolysaccharide (LPS) on the steroidogenesis and mRNA expression of Toll-like receptors (TLRs), a class of key pattern recognition receptor, in the breeding goose ovary. The laying geese were treated intravenously with LPS for 0, 6, 12, 24 and 36 h, and all birds were slaughtered approximately 8 h after oviposition. The expression levels of TLRs in the white and yellowish follicles, and granulosa and theca layers of hierarchical follicles were examined by real-time PCR. All 10 members of avian TLR family were differentially expressed among the different follicular tissues. Moreover, at 24 and 36 h after LPS treatment, the hierarchical follicle morphological structure was altered, but the expression levels of TLRs were still higher than the control. Furthermore, during LPS treatment period, the expression pattern of TLRs 2A and 4 genes was similar to that of TLR15 in the white follicles, TLRs 1B, 5 and 15 in the yellowish follicles, TLRs 7 and 15 in the granulosa layer, and TLRs 1A, 2B, 3, 7 and 15 in the theca layer, which had a negative correlation with the kinetics of plasma P4 and E2 concentrations. In conclusion, the mechanism by which pathogen infection inhibited goose follicular growth and further decreased egg production may involve a gradually enhanced inflammatory response and reduced endocrine function. This may be due to stimulated TLRs in the ovary.

Reproductiveaxis gene regulation during photostimulation and photorefractoriness in Yangzhou goose ganders.

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.

Production of biologically active recombinant goose FSH in a single chain form with a CTP linker sequence.

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 ß subunits were amplified from goose pituitary cDNA. A chimeric gene containing α and ß 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-gFSHß-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.

Investigating right ovary degeneration in chick embryos by transcriptome sequencing.

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.

Expression and Purification of a Functional Porcine Soluble Triggering Receptor Expressed on Myeloid Cells 1.

Triggering receptor expressed on myeloid cells 1 (TREM-1) plays a vital role in the pathogen-triggered amplification loop required for proinflammatory responses. Blockade of TREM-1 signaling may inhibit expansion of sepsis and prolong survival of animals. In the present study, the gene of porcine soluble TREM-1 was cloned and expressed in E. coli. After purification, the bioactivity of recombinant porcine soluble TREM-1 was tested in vitro on porcine alveolar macrophages. The results showed that supplementation with the recombinant porcine sTREM-1 protein rapidly and dose-dependently attenuated the upregulation of cytokines (IL-1ß, IL-2, IL-4, IL-8, IL-10, IL-12, IL-16, IL-18, and TNF-α) caused by LPS stimulation in the cultured porcine alveolar macrophages. These results indicate that the recombinant porcine sTREM-1 protein can prevent TREM-1-mediated hyperinflammatory responses after exposure to LPS.

Molecular mechanisms of enhancing porcine granulosa cell proliferation and function by treatment in vitro with anti-inhibin alpha subunit antibody.

BACKGROUND: This study was conducted to clarify the effect of the inhibiting action of inhibin on porcine granulosa cell proliferation and function, and to investigate the underlying intracellular regulatory molecular mechanisms. METHODS: Porcine granulosa cells were cultured in vitro, and were treated with an anti-inhibin alpha subunit antibody, with or without co-treatment of follicle-stimulating hormone (FSH) in the culture medium. RESULTS: Treatment with anti-inhibin alpha subunit antibody led to a significant increase in estradiol (E2) secretion and cell proliferation. Anti-inhibin alpha subunit antibody worked synergistically with FSH at low concentrations (25 microg/mL) to stimulate E2 secretion, but attenuated FSH action at high concentrations (50 microg/mL). Immunoneutralization of inhibin bioactivity increased FOXL2, Smad3, and PKA phosphorylation, and mRNA expression of the transcription factors CEBP and c-FOS. The expression of genes encoding gonadotropin receptors, FSHR and LHR, and of those involved in steroidogenesis, as well as IGFs and IGFBPs, the cell cycle progression factors cyclinD1 and cyclinD2, and the anti-apoptosis and anti-atresia factors Bcl2, TIMP, and ADAMTS were upregulated following anti-inhibin alpha-subunit treatment. Treatment with anti-inhibin alpha subunit down regulated expression of the pro-apoptotic gene encoding caspase3. Although expression of the pro-angiogenesis genes FN1, FGF2, and VEGF was upregulated, expression of the angiogenesis-inhibiting factor THBS1 was downregulated following anti-inhibin alpha subunit treatment. CONCLUSIONS: These results suggest that immunoneutralization of inhibin bioactivity, through augmentation of the activin and gonadotropin receptor signaling pathways and regulation of gene expression, permits the development of healthy and viable granulosa cells. These molecular mechanisms help to explain the enhanced ovarian follicular development observed following inhibin immunization in animal models.

[Accumulation of antibiotics, heavy metals and antimicrobial-resistant bacteria in duck bio-bed].

OBJECTIVE: The objective of this study is to understand the accumulation of antibiotics, metals in the litter, and evolution features of bacterial antimicrobial-resistance during the fermentation of duck bio-bed. METHODS: The experiment was conducted in meat duck bio-bed farm of Jiangsu province from November 2011 to July 2013. The new litter and spent litter from 4th, 8th meat duck flock were studied for the accumulation of consumed antimicrobials and metals. Bacterial resistance levels to consumed antimicrobials were measured in the collected litter. RESULTS: The residues of doxycycline in the litter increased significantly after successive flocks of meat ducks, but ofloxacin had not been detected in all litter samples. The litter for the 8th meat duck flock had the highest level of resistant cultivable bacteria in the three sorts of medium amended with 16 microg/mL, 100 microg/mL doxycycline. Meanwhile, the level of resistant cultivable bacteria in the same sort of medium amended with 8 microg/mL, 50 microg/mL ofloxacin differed insignificantly between different flock litter sample. Different flock litter differed insignificantly in the accumulation content of As, Pb and Hg, and was low in Cd. The 4th, 8th flock litter increased significantly in Zn and Mn than new litter, and increased slowly in Cu and Cr. CONCLUSION: With the application of doxycycline in each flock of meat ducks, the content of doxycycline and doxycycline resistance in enterobacteriaceae bacteria in the litter increased significantly, Zn and Mn had an increasing trend overall.

Improving duckling hatchability and quality by optimization of egg turning angle during incubation.

Egg turning in incubation is crucial to the development of embryos and hatching performance. We aimed to develop a high performance duck egg incubation technique by enlarging and changing egg turning angles. Increasing turning angle from 45 to 75° did not affect the embryo early mortality during the first 15 d of incubation, which ranged from 3.5 to 4.0%, but accelerated chorioallantoic membrane (CAM) development by 17 h, and significantly (P < 0.01) reduced the late mortality from 9.4 ± 0.98% to 5.31 ± 0.63%. As the result, fertile egg hatchability increased from 91.03 ± 0.97% to 94.64 ± 0.61% (P < 0.05), so was healthy duckling rate from 87.24 ± 1.17% to 92.08 ± 0.55% (P < 0.05), and duckling live weight from 60.74 ± 0.63 g to 63.15 ± 0.35 g (P < 0.05). Changing turning angle from 75°to 60°during incubation d 15 to 25 further reduced late embryo mortality to 3.88 ± 0.47 and increased hatchability to 96.58 ± 0.68%. This changing angle turning hatched ducklings exhibited the highest growth performance during rearing than those hatched by 45 and 75° egg turning. The enhanced growth rate was paralleled by upregulations of somatotropic axis genes mRNA expression levels of the hypothalamus GHRH, liver GHR and IGF-1 during embryo incubation and duckling rearing. In conclusion, a changing angle egg turning incubation technique, 75°in the first 15 d and 60°thereafter, can enhance CAM development, upregulate somatotropic axis genes expressions, and can maximally improve embryo livability, duckling hatchability and growth performance.

Effects of ambient temperature on growth performance, slaughter traits, meat quality and serum antioxidant function in Pekin duck.

The present study investigated the effects of temperature on growth performance, slaughtering traits, meat quality and antioxidant function of Pekin ducks from 21-42 d of age. Single factor analysis of variance was used in this experiment, 144 21 d-old Pekin ducks were randomly allotted to 4 environmentally controlled chambers: T20 (20°C), T23 (23°C), T26 (26°C) and T29 (29°C), with 3 replicates in each group (12 ducks in each replicate), the relative humidity of all groups is 74%. During the 21-day trial period, feed and water were freely available. At 42 d, the BW (body weight) and ADG (average daily gain) of T26 were significantly lower than T20 (p < 0.05), and the T29 was significantly lower than T20 and T23 (p < 0.05). The ADFI (average daily feed intake) of T26 and T29 were significantly lower than T20 and T23 (p < 0.05). Compared to the T29, the T20 showed a significant increase oblique body length and chest width, and both the keel length and thigh muscle weight significantly increased in both the T20 and T23, while the pectoral muscle weight increased significantly in other groups (p < 0.05). The cooking loss of the T29 was the lowest (p < 0.05). The T-AOC (total antioxidant capacity) of T29 was significantly higher than the other groups (p < 0.05), the SOD (superoxide dismutase) in the T29 was significantly higher than the T23 and T26 (p < 0.05). In conditions of 74% relative humidity, the BW and ADFI of Pekin ducks significantly decrease when the environmental temperature exceeds 26°C, and the development of body size and muscle weight follows this pattern. The growth development and serum redox state of Pekin ducks are more ideal and stable at temperatures of 20°C and 23°C.

Development and preclinical validation of 2-deoxy 2-[18F]fluorocellobiose as an Aspergillus-specific PET tracer.

The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity. Aspergillus fumigatus is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an Aspergillus-specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by Aspergillus species, and synthesized 2-deoxy-2-[18F]fluorocellobiose ([18F]FCB) by enzymatic conversion of 2-deoxy-2-[18F]fluoroglucose ([18F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [18F]FCB injection in A. fumigatus pneumonia as well as A. fumigatus, bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live A. fumigatus infection. In vitro testing confirmed production of ß-glucosidase enzyme by A. fumigatus and not by bacteria, resulting in hydrolysis of [18F]FCB into glucose and [18F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at A. fumigatus infectious sites. We conclude that [18F]FCB is a promising and clinically translatable Aspergillus-specific PET tracer.

The characteristics of oviposition and hormonal and gene regulation of ovarian follicle development in Magang geese.

BACKGROUND: Egg laying in Magang geese is characterized by extended interruption between clutches and lowing laying rate. Both the ovarian follicular development and ovulation characteristics, and the associated endocrine and molecular regulatory mechanisms involved are poorly understood, but could be important for guiding development of molecule aided selection of egg laying performances in geese. This study, therefore, recorded egg-laying characteristics of Magang geese, and the endocrine and molecular regulatory mechanisms of ovarian follicular development, maturation, and ovulation in Magang geese. METHODS: Oviposition, ovarian follicle development, and reproductive hormone and gene expression profiles were observed in a small flock of Magang geese. RESULTS: Greater than 73% of eggs were laid during the day. The average oviposition interval was 46.8 h (36-55 h). It took approximately 18 days for large white follicles to develop into mature F1 follicles; follicular growth was exponential. LHR expression levels increased from the small to the large mature follicles, but FSHR expression decreased in the granulosa and thecal layers. As the follicles matured, inhibin alpha and inhibin betaA expression increased in the granulosa layer. Activin IR, activin IIRA, activin IIRB, and beta-glycan expressions also increased as the follicles increased in size, but were more abundantly expressed in the thecal than in the granulosa layers. During the oviposition cycle, plasma concentrations of gonadal hormones decreased rapidly, whereas the level of PGFM peaked around ovulation. The profiles of activin, inhibin, follistatin, estradiol, and progesterone leading to ovulation were characterized. CONCLUSIONS: The molecular and endocrine mechanisms that regulate follicular development in Magang geese are similar to those in chickens. Moreover, gonadotropin regulation and interaction between activin, inhibin, and follistatin secretion may govern 3-stage maturation in the final preovulatory follicles in Magang geese. The rapid rebound of post-ovulatory secretions of inhibin and follistatin may inhibit recruitment of new SYF recruitment once a sequence of eggs is started, and may limit the egg clutch size to no more than the number of LYFs present before the first sequence egg.

Bacterial lipopolysaccharide with different administration routes affects intestinal mucosal morphological, immunological, and microbial barrier functions in goslings.

The current study was conducted to evaluate the effects of different administration routes of bacterial lipopolysaccharide (LPS) on intestinal mucosal morphological, immunological, and microbial barrier functions in goslings. First, we compared intestinal villi morphology of goslings under intraperitoneal or oral LPS treatment through hematoxylin and eosin staining. Then, we determined the signatures of the microbiome in the ileum mucosa of goslings subjected to oral LPS treatment at 0, 2, 4, and 8 mg/kg BW by 16S sequencing, and analyzed the changes in intestinal barrier functions and permeability, levels of LPS in the ileum mucosa, plasma, and liver tissue, and the induced inflammatory response of Toll-like receptor 4 (TLR4). As a result, intraperitoneal LPS injection resulted in a thicker intestinal wall in the ileum within a short time, whereas villus height was less affected; in contrast, oral LPS treatment exerted a stronger influence on villus height but not on intestinal wall thickness. We also found that oral LPS treatment affected the structure of the intestinal microbiome, reflected by changes in the clustering of intestinal microbiota. The average abundance of Muribaculaceae showed an increasing trend with increasing LPS levels, and that of the genus Bacteroides decreased, compared with the control group. In addition, oral LPS treatment with 8 mg/kg BW affected the intestinal epithelial morphology, damage the mucosal immune barrier, downregulated the expression of tight junction proteins, increased circulating D-lactate levels, and stimulated the secretion of various inflammatory mediators and activation of the TLR4/MyD88/NFκB pathway. This study presented the injuries of intestinal mucosal barrier function induced by LPS challenges in goslings and provided a scientific model for searching the novel strategies to attenuate the immunological stress and gut injury caused by LPS.

Impact of Accessory Corpus Luteum Induced by Gonadotropin-Releasing Hormone or Human Chorionic Gonadotropin on Pregnancy Rates of Dairy Cattle following Embryo Transfer: A META-Analysis.

The circulation of progesterone (P4) concentrations of recipients has positive correlations with embryo survival and pregnancy success of embryo transfer (ET) in dairy cows. One strategy to improve P4 concentration is the administration of gonadotropin-releasing hormone (GnRH) or human chorionic gonadotropin (hCG), thereby inducing the formation of accessory corpus luteum (CL). This study aimed at determining the efficacy of GnRH or hCG treatment regarding embryo transfer (ET) and providing a better clinical veterinary practice guidance. A meta-analysis was conducted on the data from 2048 treated recipient cows and 1546 untreated cows. By inducing the formation of accessory CL with GnRH (100 µg), GnRH analogue Buserelin (8-10 µg), or hCG (≥1500 IU) 5-11 days after synchronized ovulation, hCG alone achieved an improvement (RR = 1.39, p < 0.05), while GnRH and GnRH analogue did not result in significant changes (RR = 1.04, p = 0.26). Treatment with GnRH or hCG 5-7 days after synchronized ovulation was associated with increased chances of pregnancy compared with later treatment (11-14 days). Owing to the treatment, the pregnancy rate of cows with very poor fertility (<40%) was improved, while that of cows with good fertility (≥40%) was not affected. Treatment with GnRH or hCG greatly improved pregnancy rates of parous lactating cows (RR = 1.32, p < 0.05) compared with heifers (RR = 1.02, p > 0.05). Additionally, as indicated by pregnancy loss analysis, the treatment had no benefit on late embryo/early fetus survival at days 28-81. In conclusion, the induction of accessory CL with GnRH or hCG may benefit fertility and have important implications for the management of reproductive performance in the dairy industry.