Bolstering Buck Fertility: The Impact of Asparagus racemosus Aqueous Extract on Semen Cryopreservation and Antioxidant Defense System.

Importance of Study: Semen cryopreservation results in sperm damage due to lipid peroxidation or oxidative stress, leading to a decrease in conception rate. The sperm damage during cryopreservation can be minimized with the use of suitable antioxidant supplements in semen diluent. Some herbs have potent antioxidant potential and can be used in semen diluent to protect the spermatozoa. Objective: Hence, the investigation was planned to evaluate the effect of Asparagus racemosus (A. racemosus) aqueous extract on buck semen quality during cryopreservation. Methodology: In the current study, semen was collected from eight Sirohi bucks, and from each buck, 8 ejaculates were collected. Good-quality semen samples were pooled during each collection. Pooled semen samples were then divided into four equal parts and diluted in TRIS buffer containing different concentrations of A. racemosus aqueous extract (different groups, i.e., G I -5 mg, G II -2.5 mg, G III -1.25 mg, and G IV -0 mg of A. racemosus aqueous extract in 1 mL TRIS buffer). All the diluted semen samples were kept at equilibration temperature (5°C) for 2 hours and then cryopreserved by the manual method. Semen samples were evaluated for various sperm characteristics and antioxidant status before and after cryopreservation. Results: Asparagus racemosus aqueous extract showed significant (p < 0.05) enhancement of sperm viability, sperm motility, acrosomal integrity, and plasma membrane integrity, whereas it reduced sperm abnormality. Furthermore, in the experimental groups, the antioxidant gene expression was found to be increased compared to that of the treatment group. G III (p < 0.05) showed significantly better results in terms of sperm viability, sperm motility, acrosomal integrity, and plasma membrane integrity. Conclusion: Asparagus racemosus aqueous extract has the antioxidant potential to protect buck spermatozoa during semen cryopreservation.

Seroprevalence and risk factor analysis of brucellosis among dairy farmers in Aligarh region, North India: creating awareness of a neglected disease.

Introduction. Brucellosis, a globally distributed zoonotic disease, is caused by the Gram-negative bacteria known as Brucella. Humans acquire infection through direct contact with the blood, urine and placenta of animals, inhalation of dust or aerosols at infected animal farms, and raw milk and meat intake. This study aimed to assess the prevalence of brucellosis in dairy farmers in and around the Aligarh region of North India, to document various clinical signs and symptoms in Brucella-positive individuals, and to create awareness in dairy farmers concerning brucellosis and ways to prevent it. Methods. This was an observational study that included 125 dairy farmers in and around the Aligarh region. Serum samples were taken from this high-risk group after obtaining informed consent. Further, a pre-designed proforma was used to collect information about their knowledge, attitude and practices (KAP) concerning brucellosis and assess the risk factors for the disease. The Rose Bengal test (RBT), serum agglutination test (SAT) and enzyme-linked immunosorbent assay (ELISA) were performed to detect the seroprevalence of brucellosis. Result.Brucella infection was diagnosed in 64 (51.20 %) cases by indirect ELISA (IgM+IgG), 41 (32.8 %) by RBT and 4 (3.2 %) by SAT. Significant clustering of patients was seen in the 20-55 years age group. The most common symptoms in ELISA IgM-positive patients were joint pain (16.07 %), fatigue (14.28 %), anorexia (12.50 %), weight loss (8.92 %), malaise (5.35 %), undulant fever (3.57 %), night sweats (3.57 %) and headache (1.78 %). The findings of this study indicate that ELISA (IgM+IgG) exhibits great sensitivity as compared to SAT and RBT. KAP was very poor among dairy farmers. Conclusion. In India, Brucella is a frequent but severely underreported illness. ELISA is the most sensitive serological test for diagnosing brucellosis. No potential vaccine has yet been introduced for humans against brucellosis. Thus, it is necessary to impart awareness and sensitize high-risk groups concerning brucellosis.

Metal-based nanomaterials and nanocomposites as promising frontier in cancer chemotherapy.

Cancer is a disease associated with complex pathology and one of the most prevalent and leading reasons for mortality in the world. Current chemotherapy has challenges with cytotoxicity, selectivity, multidrug resistance, and the formation of stemlike cells. Nanomaterials (NMs) have unique properties that make them useful for various diagnostic and therapeutic purposes in cancer research. NMs can be engineered to target cancer cells for early detection and can deliver drugs directly to cancer cells, reducing side effects and improving treatment efficacy. Several of NMs can also be used for photothermal therapy to destroy cancer cells or enhance immune response to cancer by delivering immune-stimulating molecules to immune cells or modulating the tumor microenvironment. NMs are being modified to overcome issues, such as toxicity, lack of selectivity, increase drug capacity, and bioavailability, for a wide spectrum of cancer therapies. To improve targeted drug delivery using nano-carriers, noteworthy research is required. Several metal-based NMs have been studied with the expectation of finding a cure for cancer treatment. In this review, the current development and the potential of plant and metal-based NMs with their effects on size and shape have been discussed along with their more effective usage in cancer diagnosis and treatment.

Development of duplex real-time PCR for quick detection of cryptosporidiosis in goats.

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy and antigen trap-ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA-based duplex TaqMan® probe PCR was developed to target the Cryptosporidium oocyst wall protein gene and 18SSU rRNA gene in a single tube that can detect metabolically active cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and they will help decipher the active stages of its lifecycle in a host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 1004 and 5.95 × 1005 , respectively. During active infections, the oocyst wall protein will be active and so its COWP gene transcripts will act as a marker for active infection. While transcripts for 18SSU rRNA are constitutively expressed in cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among healthy animals.

Multiplex qPCR for differentiation of Mycobacterium avium subspecies paratuberculosis in active and passive infection of goats.

Mycobacterium avium paratuberculosis (MAP) is causative agent of Johne's disease (JD) in domestic animals and has broad host range. JD infected animals shed viable MAP in their milk, feces, blood, and tissues which get transmitted to human beings directly or indirectly by consumption of animal products, through contact, animal handling and through contaminated environment, aerosols. In this current study, we developed hydrolysis probe based TaqMan® real-time PCR assay where samples were investigated by targeting IS900 mRNA and ModD gene to differentiate live MAP shedders from inactive/dead MAP bacilli shedding animals. The IS900 mRNA and ModD gene primers were designed using discontiguous unique conserved sequences of IS900 more towards the 3' end and fibronectin attachment protein (FAP) genes, respectively. Two different reporter dyes Cy5 and TexasRed, with compatible quenchers BHQ-1 and BHQ-2, respectively, were used for probe designing of IS900 and ModD genes. Triplex PCR assay was developed by using serially diluted positive MAP culture in log10 dilution and probe and template titration. TaqMan® probe real-time PCR targeting IS900 mRNA and ModD gene detects the MAP infection at early stage with high sensitivity and specificity. The specificity of developed TaqMan probe real-time PCR was found to be high while validated by using Escherichia coli and Staphylococcus aureus in addition to the MAP culture as there is no non-specific signal from other microbes. The sensitivity of developed TaqMan® probe real-time PCR was computed based on copy numbers ranged from 4.14 × 1011 to 4.14 × 104 for IS900 (FAM), 1.27 × 1011 to 1.27 × 104 for IS900 mRNA (Cy5), and 3.68 × 1010 to 3.68 × 104 for ModD (TexasRed), and lowest limit to detect MAP was 4.14 × 104, 1.27 × 104, and 3.68 × 104 copies for respective genes. This assay would be of great aid to contain the MAP infection in the large herd, where silent shedders spread active infection can be differentiated from passive shedding by non-infected animals. This test would also be equivalent to culture test in terms of specificity and hence can be able to be undertaken in molecular epidemiological studies to represent the actual disease prevalence in the future. KEY POINTS: • Multiplex mRNA-based qPCR was developed to identify the actively infective MAP bacilli from passive ones. • ModD and IS900 used as targets to assess active MAP bacilli in fecal samples of suspected animals. • The LOD was computed using copy numbers with 4.14 × 104 and 3.68 × 104 copies for IS900 and ModD, respectively.

Enterocolitis in goats associated with enterotoxaemia in the perspective of two toxins: Epsilon toxin and beta-2 toxin - An immunohistochemical and molecular study.

Caprine intestinal diseases associated with clostridia are generally caused by Cpa and Etx encoded alpha (α) and epsilon (ε) toxinotypes of Clostridium perfringens type D respectively. A recent study on goat enterocolitis, demonstrated that the incidence of Clostridium perfringens type-D toxinotype and beta 2 toxins is high, suggesting its role in enterocolitis and many other diseases of goats affecting intestinal tract. Considering this scenario, the present prevalence study was planned to screen the goat intestinal tissues for the presence of the epsilon toxin and beta 2 toxin. Tissue sections from enterotoxaemia suspected cases in 189 goats were collected and epsilon-toxin was demonstrated by immuno-histochemically and toxinotyping multiplex polymerase reaction in 19 animals and beta 2 toxin in 19 animals by multiplex polymerase reaction. Immuno-reactivity to epsilon toxin was detected maximum in distal ileum of goat intestine and this toxin was produced by Clostridium perfringens type D. It suggests that immunohistochemistry is a confirmatory tool for detection of bacterial toxin especially epsilon toxin where isolation and characterisation of bacteria is not possible. Here, we have reported a strong association between ε-toxin (epsilon) and beta-2 toxin in causing disorders of intestine in goats. In addition, we have explored the possible role of cpb2 positive isolates of C. perfringens and their pathogenic effects in causing enterotoxaemia. These determinants help in the understanding of the pathogenesis of enterotoxaemia in goats which needs to be further investigated.

Bacteriophages: A possible solution to combat enteropathogenic Escherichia coli infections in neonatal goats.

Due to awareness and benefits of goat rearing in developing economies, goats' significance is increasing. Unfortunately, these ruminants are threatened via multiple bacterial pathogens such as enteropathogenic Escherichia coli (EPEC). In goat kids and lambs, EPEC causes gastrointestinal disease leading to substantial economic losses for farmers and may also pose a threat to public health via the spread of zoonotic diseases. Management of infection is primarily based on antibiotics, but the need for new therapeutic measures as an alternative to antibiotics is becoming vital because of the advent of antimicrobial resistance (AMR). The prevalence of EPEC was established using bfpA gene, uspA gene and Stx1 gene, followed by phylogenetic analysis using Stx1 gene. The lytic activity of the isolated putative coliphages was tested on multi-drug resistant strains of EPEC. It was observed that a PCR based approach is more effective and rapid as compared to phenotypic tests of Escherichia coli virulence. It was also established that the isolated bacteriophages exhibited potent antibacterial efficacy in vitro, with some of the isolates (16%) detected as T4 and T4-like phages based on gp23 gene. Hence, bacteriophages as therapeutic agents may be explored as an alternative to antibiotics in managing public, livestock and environmental health in this era of AMR.

In vivo kinetics of peripheral cellular immune responses in Mycobacterium avium subspecies paratuberculosis (MAP) infected and vaccinated goats.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (ParaTB) also known as Johne's disease (JD) in ruminants, which is characterized by chronic intestinal inflammation. A similar counterpart has been observed in the form of Crohn's disease in humans. The present study is the first trail in goats to understand the peripheral cellular immune responses following experimental MAP infection and vaccination. Fifteen apparently healthy male kids (3-6 months old) of Barbari breed were included in this study. In the experimental study, 5 kids were infected with 'S 5' strain of MAP ("Indian Bison Type"), 5 were vaccinated (Indigenous Vaccine) against MAP infection (Singh et al., 2007) and the remaining 5 kids were uninfected and non-vaccinated controls. Kids were observed for a period of 180 days post exposure (infection and vaccination) and were tested for development of infection. Cellular immune responses (in blood) were recorded post-exposure by three assays. We measured the frequencies of CD4 and CD8T cells, estimated plasma IFNγ and TNα and in the third assay, in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) from vaccinated, infected and controls were examined in response to polyclonal stimulation. The frequencies of peripheral CD4 and CD8T cells were comparable in control, infected and vaccinated animals except around day 49 post-infection where MAP infected animals showed a trend towards significantly reduced frequencies of CD4 T cells compared to apparently healthy controls. Significantly reduced plasma TNFα levels were also observed in infected animals compared to vaccinated animals,during the course of infection. Diminished levels (although non significant) of TNFα were observed in the supernatants from polyclonally stimulated PBMCs at around day 49 post infection. It is conceivable that the diminished cellular immune responses may coincide with an impairment (immune exhaustion) of perhaps antigen-specific CD4T cells that might, in the course of infection, contribute to the progressive nature of caprine paratuberculosis.

Chemotactic factor inducing Interleukin-8 (IL8) gene is transcriptionally elevated in experimental enterotoxaemia in goats caused by Clostridium perfringens type D.

The current study was designed to analyse the effects of experimental induction of enterotoxaemia through intra-duodenal inoculation of C. perfringens type D culture isolated from spontaneous outbreaks in goats. Twenty goats (6-9 month age) were divided into four groups and C. perfringens type D culture was inoculated intra-duodenally as per following: Group-I (whole cultures-WC), group-II (culture supernatant-CS), group-III (washed cells-WS), and group-IV (uninfected control-C). The treated animals were sacrificed after 72 h post infection (hpi), and necropsy showed gross changes including haemorrhages and congestion in the ileal and colon mucosa, pulmonary congestion and edema in lung. Kidney, brain and spleen exhibited severe to moderate congestion. Microscopic changes like haemorrhages, degenerative and necrotic changes in the mucosal epithelium of intestine and haemorrhages in kidney parenchyma were observed in the H&E stained sections. Lung alveolar sacs were filled with proteinaceous fluid. Immunohistochemistry revealed positive immunolabelling for etx (epsilon toxin) in the mucosa of intestine in WC and CS group. Control animals did not exhibit any significant gross or microscopic changes. PCR amplification of DNA extracted from intestinal tissues of WC and CS groups showed positive for etx gene demonstrating the production of epsilon toxin. Transcriptional responses in experimental groups were assessed by quantitative reverse transcription real time PCR (qRT-PCR). Genes including IL-1ß and IL2 showed up-regulation in all the experimental groups (WC, CS&WS). Specifically the toxin-based experimental groups (WC&CS) showed up-regulation of the gene responsible for chemotaxis viz. IL-8, while the washed cells group (WS) showed higher transcriptional response to Cathepsin-L (Cat-L) gene denoting the acute inflammatory response due to neutrophil elastase activity. These results take a cue on the evolving nature of the enterotoxaemia in goats due to various strains circulating in the field. The host response and its modulation due to the novel enterotoxaemia strains throws light on the current challenges in efficient control of the disease in goats.

Status of Beta Defensin-1 and its Effect on Post-thaw Semen Fertility Gene Expression in Indian Goat Breed.

BACKGROUND: Defensins are antimicrobial peptides and uniformly spans the entire sperm surface and is not exclusive to a specific domain. Goat ß-defensin-1 helps in initiation of motility and capacitation of sperm. OBJECTIVE: To know the status of ß-defensin-1 in blood, semen and its effect on post thaw fertility gene expression in Indian goat breeds. MATERIALS AND METHODS: Semen was extended and divided for estimation of ß-defensin-1 and cryopreserved having different concentrations of ß-defensin-1. RESULTS: Bet defensin-1 concentration (pg/mL) in neat semen, sperm pellet and seminal plasma was significantly higher (P< 0.05) in goat breed Barbari followed by Jamunapari and Jakhrana. ß-defensing-1 was also high in Jakhrana blood followed by Barbari and Jamunapari. The post thaw motility, live sperm, acrosome intactness and hypo osmotic swelled sperms were significantly higher (P< 0.05) with 10 ng/mL ß-defensin in the semen dilutor. CONCLUSION: Beta defensin (10 ng/mL) in semen dilutor may be used as immuno-modulator to get better post thaw quality suitable for artificial insemination.