ApoE/NOS3 Knockout Mice as a Novel Cardiovascular Disease Model of Hypertension and Atherosclerosis.

Hypertension is an independent risk factor for atherosclerosis. However, few models of hypertensive atherosclerosis have been established in medical research. In this study, we crossed the ApoE knockout (ApoE-KO; ApoE-/-) atherosclerotic mouse model with the NOS3 knockout (NOS3-KO; NOS3-/-) hypertensive mouse model to establish an ApoE/NOS3 double knockout (ApoE/NOS3-KO; ApoE/NOS3-/-) hypertensive atherosclerosis mouse model. We found that ApoE/NOS3-/- mice reproduced normally, had a blood pressure of 133.00 ± 3.85 mmHg, and developed hypertensive fundus retinopathy and hypertensive nephropathy. In addition, serum total cholesterol (TC) and low-density lipoprotein (LDL) levels in the blood were abnormally elevated, steatosis was observed in the liver cells, and atherosclerotic lesions were observed in the aortic vessels in ApoE/NOS3-/- adult mice. In conclusion, ApoE/NOS3-/- adult mice are a satisfactory model of hypertension and atherosclerosis and can be utilized for studies on cardiovascular diseases.

Involvement of Porcine ß-Defensin 129 in Sperm Capacitation and Rescue of Poor Sperm in Genital Tract Infection.

In mammals, ß-defensins have been reported to play pivotal roles in sperm protection and fertilization. However, the function and mechanism of porcine ß-defensin 129 (pBD129) in the sperm remain unclear. Here, we demonstrate that pBD129 is a glycosylated protein and broadly exists in accessory sex glands and coats the sperm surface. We inhibited the pBD129 protein on the sperm surface with an anti-pBD129 antibody and found that sperm motility was not significantly affected; however, sperm acrosome integrity and tyrosine phosphorylation levels increased significantly with time (p < 0.05) during capacitation. These changes were accompanied by an increase in sperm Ca2+ influx, resulting in a significantly reduced in vitro fertilization cleavage rate (p < 0.05). Further investigation revealed that treatment with recombinant pBD129 markedly restored the sperm motility in semen contaminated with Escherichia coli. The results suggest that pBD129 is not only associated with poor sperm motility after genital tract infection but can also protect the spermatozoa from premature capacitation, which may be beneficial for semen preservation.

Carboxypeptidase E protein regulates porcine sperm Ca2+ influx to affect capacitation and fertilization.

Mammalian spermatozoa acquire their fertilizing ability in the epididymis, which is important for sperm maturation and capacitation. Carboxypeptidase E (CPE) is a prohormone-processing enzyme and sorting receptor that functions intracellularly. Recently, CPE was identified to exist in the seminal plasma. However, little is known about the effects of CPE on reproductive function. This study focused on the effects of CPE on sperm function and fertilization. Herein, CPE was identified to be localized in the boar sperm, testis, epididymis, accessory gonad and seminal plasma, with high expression found in the bulbourethral glands and cauda epididymis. Furthermore, compared with high motility spermatozoa, a decrease in CPE abundance was observed in low motile spermatozoa by Western blot analysis. The use of specific antibody to inhibit the CPE in spermatozoa led to a decrease in sperm motility, followed by an expected decrease in acrosome exocytosis and tyrosine phosphorylation in the capacitation process. These changes were accompanied by a decrease in intracellular Ca2+ ([Ca2+]i) influx, which resulted in a significant decrease in the cleavage rate during in vitro fertilization (IVF). Based on these observations, we suggest that CPE might affect porcine sperm Ca2+ influx to participate in the regulation of sperm function during capacitation.

Glutathione S-transferase kappa 1 is positively related with sperm quality of porcine sperm.

The glutathione S-transferase (GST) superfamily members play an important role in the male reproductive tract and sperm physiology. However, the expression profiles of some members of this protein family and their effect on sperm quality remain unclear. In this study, we found that GST kappa 1 (GSTK1) encoded protein is abundant in the testes and capacitated sperm acrosome. Western blot analysis revealed that the decreased abundance of GSTK1 was observed in low motile spermatozoa; moreover, GSTK1 expression decreased in sperm stored at 17°C under a long preservation time. In vitro analyses revealed that GSTK1 had no significant effect on sperm motility, capacitation, or acrosome reaction. Notably, after capacitated sperm were incubated with 4 and 8 µg/ml anti-GSTK1 antibodies, the fertilization rate significantly decreased in vitro fertilization assay. The current study demonstrates that GSTK1 is correlated with sperm quality and is a promising marker for the assessment of sperm quality and provides a basis for understanding the potential molecular mechanism for targeting pathogenic factors in male infertility.

Epigallocatechin-3-Gallate Promotes the in vitro Maturation and Embryo Development Following IVF of Porcine Oocytes.

PURPOSE: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and exerts protective effects because of its strong antioxidant properties. As far as we know, there is still a lack of systematic research on the effects of EGCG on the in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes. The present study aimed to determine the effects of EGCG on the IVM and IVF of porcine oocytes. METHODS: Porcine oocytes were treated with different concentrations of EGCG (5, 10 and 20 µM), and the cumulus cell expansion, oocyte maturation rate, reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) levels, total antioxidant capacity were determined. The mRNA expression levels of oxidative stress- and apoptosis-associated genes were determined by quantitative real-time PCR. The cleavage rate and blastocyst rate of oocytes after 10 µM EGCG treatment during IVM and IVF were also evaluated. RESULTS: EGCG at 5, 10 and 20 µM significantly promoted cumulus cell expansion, and EGCG at 10 µM increased the oocyte maturation rate. EGCG (10 µM) treatment reduced the ROS and MDA levels, while increased the antioxidant capacity and GSH concentrations in the mature oocytes. The qRT-PCR results showed that EGCG treatment up-regulated the mRNA expression of catalase, glutathione peroxidase and superoxide dismutase in the mature oocytes. In addition, EGCG treatment also decreased the mRNA expression levels of Bax and caspase-3 and increased the Bcl-2 mRNA expression level in the mature oocytes. In addition, the cleavage rate and blastocyst rate of oocytes treated with 10 µM EGCG during IVM and IVF were significantly higher than those of the control group. CONCLUSION: Our results suggest that EGCG promotes the in vitro maturation and embryo development following IVF of porcine oocytes. The protective effects of EGCG on the oocytes may be associated with its antioxidant and anti-apoptosis properties.

Analysis of differentially abundant proteins related to boar fertility in seminal plasma using iTRAQ-based quantitative proteomics.

Animal fertility is one of the most important characteristics for the livestock breeding industry. Conventional semen analysis provides basic information on sperm quality, but the predictive value of such analysis with regard to fertility remains questionable. Therefore, it is important to determine and predict male fertility more accurately in the clinic. To identify seminal plasma proteins involved in fertility, isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography with tandem mass spectrometry (quantitative proteomic analysis) were used to identify differentially abundant proteins (DAPs) in seminal plasma between high- and low-reproductive-efficiency Landrace boars. A total of 141 DAPs were identified, of which 125 upregulated and 16 downregulated proteins were subjected to bioinformatics analysis. These DAPs were found to be mainly involved in proteolysis, ATP binding, and energy metabolism. We investigated the relevance of three DAPs-ceruloplasmin, carboxypeptidase E (CPE), and serpin family A member 12 (SERPINA12)-in an in vitro fertility assay. This assay revealed that the inhibition of these proteins with antibodies can reduce or increase the fertilization rate. These results indicate possible biomarkers for the selection of high-fertility boars and provide a theoretical basis for the use of protein biomarkers in the livestock breeding industry. SIGNIFICANCE: Our study identified differentially abundant proteins in the seminal plasma of high-reproductive-efficiency and low-reproductive-efficiency Landrace boars. These proteins may be used as biomarkers to screen out high-fertility boars. The study can provide not only a new method for improving the effects of artificial insemination and reproductive efficiency of boars but also an important reference for boar breeding. Meanwhile, because pigs and humans have similar physiological parameters and organ sizes, our findings can also serve as a reference for human reproduction research.

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus.

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 102 copies/µL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.

Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.

Rapid and sensitive real-time recombinase polymerase amplification for detection of Marek's disease virus.

Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/µL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

Development of a real-time loop-mediated isothermal amplification assay for detection of porcine circovirus 3.

BACKGROUND: Porcine circovirus type 3 (PCV3) is an emerging circovirus species, that has been reported in major pig-raising countries including the United States, China, South Korea, Brazil, Spain, and Poland. RESULTS: A real-time loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of porcine circovirus 3 (PCV3). The method had a detection limit of 1 × 101 copies/µL with no cross-reactions with classical swine fever virus (CSFV) C strain, foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV2) LG vaccine strain, porcine epidemic diarrhoea virus (PEDV), porcine respiratory and reproductive syndrome virus (PRRSV), or pseudorabies virus (PRV). The PCV3 positive detection rate of 203 clinical samples for the real-time LAMP assay was 89.66% (182/203). CONCLUSIONS: The real-time LAMP assay is highly sensitive, and specific for use in epidemiological investigations of PCV3.

Point-of-care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method.

Porcine deltacoronavirus (PDCoV) has emerged and spread throughout the porcine industry in many countries over the last 6 years. PDCoV caused watery diarrhoea, vomiting and dehydration in newborn piglets. A sensitive diagnostic method would be beneficial to the prevention and control of PDCoV infection. Recombinase polymerase amplification (RPA) is an isothermal amplification method which has been widely used for virus detection. A probe-based reverse transcription RPA (RT-RPA) assay was developed for real-time detection of PDCoV. The amplification can be finished in 20 min and fluorescence monitoring was performed by a portable device. The lowest detection limit of the PDCoV RT-RPA assay was 100 copies of RNA molecules per reaction; moreover, the RT-RPA assay had no cross-reaction with other common swine viruses. The clinical performance of the RT-RPA assay was evaluated using 108 clinical samples (54 intestine specimens and 54 faecal swab specimens). The coincidence rate of the detection results for clinical samples between RT-RPA and RT-qPCR was 97.2%. In summary, the real-time RT-RPA assay offers a promising alternative to RT-qPCR for point-of-care detection of PDCoV.

Development of a SYBR green-based real-time RT-PCR assay for rapid detection of the emerging swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus which was associated with severe diarrhea disease in pigs. SADS-CoV was first detected and identified as the causative agent of a devastating swine disease outbreak in southern China in 2017. Routine monitoring and early detection of the source of infection is therefore integral to the prevention and control of SADS-CoV infection. In this study, a SYBR green-based real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique was established for rapid detection and monitoring of this emerging virus. Specific primers were designed based on the conserved region within the M gene of the viral genome. The lowest detection limit of the RT-qPCR assay was 10 copies/µL. This assay was specific and had no cross-reaction with other 11 swine viruses. The positive rate of 84 clinical samples for the SYBR green-based RT-qPCR and the conventional RT-PCR was 73.81% (62/84) and 53.57% (45/84), respectively. These results demonstrated that the SYBR green-based RT-qPCR technique was an effectively diagnostic method with higher sensitivity than probe-based RT-qPCR and gel-based RT-PCR for detection and epidemiological investigations of SADS-CoV.

Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay.

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus.

BACKGROUND: Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV. RESULTS: The detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA. CONCLUSIONS: A broadly reactive RT-RPA assay was successfully established for MNV detection.

Development of a real time loop-mediated isothermal amplification method for detection of Senecavirus A.

Senecavirus A (SVA), formerly known as Seneca Valley Virus (SVV), is one of causative agents of vesicular diseases in swine. Recently, the outbreaks associated with vesicular disease caused by SVA infection in pig herds have been reported in Brazil, USA, China, Thailand and Canada. Several molecular detection methods have been established to determine the infection of SVA, including real time reverse transcription PCR assay, nested PCR, a TaqMan-based qRT-PCR assay and RNA-based in situ hybridization method. In our study, an assay for the identification of SVA in pig herds using real time reverse transcription loop-mediated isothermal amplification (real time RT-LAMP) was developed. The limit of detection for the assay was 1 TCID50/ml. One hundred and eighteen field samples from pigs were used to validate the assay for clinical application. Our result demonstrated that real time RT-LAMP assay is a cost-effective and highly specific and sensitive alternative for the rapid detection of SVA in clinical samples.

Development of a real time reverse transcription loop-mediated isothermal amplification method (RT-LAMP) for detection of a novel swine acute diarrhea syndrome coronavirus (SADS-CoV).

A novel swine acute diarrhea syndrome Coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in southern China in 2017. A simple and rapid detection test was developed for this virus using real-time RT-LAMP based on the conserved N gene of the virus. The method had a detection limit of 1.0 × 101 copies/µL with no cross-reactions with classical swine fever virus, porcine and respiratory syndrome virus NA, porcine and respiratory syndrome virus EU, transmissible gastroenteritis coronavirus, foot and mouth disease virus, porcine epidemic diarrhea virus (S-INDEL and non-S-INDEL), swine influenza virus subtype H1N1, porcine circovirus type 2, seneca valley virus, porcine parvovirus, porcine deltacoronavirus and rotavirus. This method was also reproducible. Twenty of 24 clinical samples were identified as SADS-CoV RNA-positive by the real-time RT-LAMP and the results were consistent with that of the real time RT-PCR method. This new method for detecting SADS-CoV is specific and sensitive for the detection of SADS-CoV.