Search Results (130)

The corpus luteum (CL) is a transient gland that can directly influence follicular dynamics and oocyte quality. The objective of this study was to evaluate the influence of the absence or presence of a small (≤3 mm), medium (4–8 mm), or large (>8 mm) CL in slaughterhouse ovaries on in vitro embryo production. Cumulus–oocyte complexes (COCs) were collected from each group of ovaries and matured in TCM-199 medium, plus hormones and fetal bovine serum. Fertilization was performed with fresh semen from a Katahdin ram of known fertility. Embryo development was carried out in commercial sequential media for 72 and 96 h, until the blastocyst stage. The number of follicles (2–6 mm in diameter) and COCs were influenced by the presence of CL, which was higher (p < 0.05) in the Large CL group (5.51 ± 0.33 and 3.62 ± 0.27) compared to the Without CL group (4.54 ± 0.19 and 2.62 ± 0.14, respectively), with no difference between the CL sizes. Likewise, the diameter and area of the COCs were higher in the Small CL group of ovaries compared to the Without CL group. In the Large CL group of ovaries, 9% more morulae (p < 0.05) were obtained compared to the Without CL group; in the Medium CL group, 13% more blastocysts were obtained compared to the Without CL group. However, in the hatching capacity and diameter of blastocysts, no statistical difference was evident (p > 0.05). In conclusion, the presence and size of the CL in the ovaries of slaughtered sheep influence the productive efficiency of embryos in vitro under the conditions in which the present study was carried out. Full article

Bovine leukemia virus (BLV) remains a major concern for cattle industries worldwide due to its persistent nature, economic impact, and challenges in control. In this study, we conducted a comprehensive nationwide survey of BLV in Kazakhstan between 2014 and 2024, utilizing serological diagnostics to assess prevalence and characterize viral genotypes (2024). A total of 433,537 serum samples were screened by agar gel immunodiffusion (AGID), revealing an overall seroprevalence of 5.87%, with the highest rates observed in the North Kazakhstan, Kostanay, and East Kazakhstan regions. In 2024, a targeted analysis of 3736 serum and 536 whole blood samples across 17 regions was performed using AGID, ELISA, real-time PCR, and nested PCR. ELISA demonstrated higher sensitivity than AGID (10.4% vs. 8.2%), confirmed by statistical correlation (r = 0.97, p < 0.001) and a Wilcoxon signed-rank test (p = 0.026). Real-time PCR detected BLV DNA in 4.7% of samples, with the highest positivity in the East Kazakhstan and Abai regions, confirming active viral circulation. Validation of a domestically developed AGID diagnostic kit showed full concordance with commercial assays (IDEXX, IDvet), supporting its use in national surveillance programs. These findings highlight the endemic status of BLV in Kazakhstan. Molecular analysis of sequenced isolates revealed the presence of genotype G-7, consistent with strains circulating in neighboring countries. Together, these results underscore the importance of integrated serological and molecular approaches for effective monitoring and control. Full article

The cryopreservation of ovarian tissues from fish has recently been carried out for several endangered and commercially valuable species. However, previous studies in this context have focused on the cryopreservation of immature ovaries—mainly through slow freezing and vitrification—which requires specialized freezing equipment or higher cryoprotectant concentrations to keep cell viability. Therefore, the aim of this study was to explore a convenient, rapid, efficient and less toxic method for the cryopreservation of ovaries at the stage of vitellogenesis from yellow drum (Nibea albiflora), an economically important marine fish. The ovaries at the stage of vitellogenesis were isolated and cut into blocks of approximately 1 cm³, then cryopreserved with 15% propylene glycol (PG), fetal bovine serum (FBS) and 0.2 M trehalose as cryoprotectants. Finally, the samples were treated using three different freezing procedures, including a −80 °C refrigerator, liquid nitrogen, and their combination. After 7 days, the tissues were thawed and digested, and the cell survival rates and gene expression levels were detected using cell viability assay kits and qRT-PCR, respectively. The results of the viability assay showed that the procedure of ovarian tissue storage at −80 °C in a refrigerator for 1 h, followed by transfer to liquid nitrogen, resulted in the highest cell survival rate (>90%). Furthermore, the germ cells at various phases were of normal size; presented a full, smooth surface and regular shape; and did not show any signs of cell rupture, atrophy, depression, granulation or cavitation. Furthermore, the qRT-PCR results revealed that genes related to reproductive development, such as vasa, foxl2, zp3 and gsdf, were all down-regulated under the optimal protocol, while the expression of the nanos2 gene (which is specifically distributed in oogonia) maintained a higher level, similar to that in the control group. This indicated that the viability of germ stem cells (oogonia) was not weakened after freezing and that oogonia could be isolated from the cryopreserved ovaries for germ cell transplantation. The present study successfully establishes an optimal cryopreservation protocol for ovarian tissues from Nibea albiflora, providing reference for the preservation of ovaries at the stage of vitellogenesis from other species. Full article

Majority of commercial L-asparaginase (L-ASNase) activity assays are based on coupled enzymatic reaction, which converts aspartate into pyruvate, subsequently reacting with the probe to form a stable chromophore, which can be detected spectrophotometrically. However, in complex biological samples this method can be inaccurate due to poor optical transparency or presence of compounds interfering with the coupled enzyme reaction–for this kind of cases alternative methods have been suggested. Here we suggest a strategy to rationally pick a method of choice in a variety of situations, taking into consideration the upsides and downsides of each method. A high-throughput fluorometric assay employing the substrate Asp-AMC was rigorously validated for L-ASPNase activity screening. Aassay performance is evaluated in complex biological matrices, including bovine serum, whole and diluted human blood, and finally the mouse blood and liver homogenates samples obtained from pharmacokinetic studies. This comprehensive validation process ensures the reliability and applicability of the assay for assessing L-asparaginase activity in diverse and physiologically relevant environments. Potential interfering factors and matrix effects were addressed, and assay conditions were optimized for each matrix. The optimized assay was employed to screen various L-asparaginase types (intracellular L-ASNases type I RrA, periplasmic L-ASNases type II EcA and EwA) and ASPNase formulations (conjugates with polyamines or polyelectrolyte complexes), comparing their kinetic parameters and stability. Fourier-transform infrared (FTIR) spectroscopy was further employed to investigate the fine features of molecular mechanisms of L-asparaginase catalysis. FTIR spectra of Asn during hydrolysis were analyzed in buffer solutions and in complex biological matrices, such as blood sample or liver homogenates which is crucial in the context of pharmacokinetic research. This combined fluorometric and FTIR approach provides a powerful platform for optimizing L-ASNase formulations and therapeutic strategies for ALL. Based on the results obtained we have developed a strategy to choose an approach for L-Asparaginase activity assessment for a variety of difficult situations when dealing with complex biological samples. Full article

Background: Bovine tuberculosis (bTB) is an infectious disease which causes significant damage to the farming industry and remains a disease of global significance. Although control strategies have focused on a test and cull approach primarily based around specific cell-mediated immune responses, serological assays are increasingly being used as a supplementary test alongside skin testing and interferon-gamma release (IGRA) assays. The UK is moving towards the use of the Bacillus Calmette–Guérin (BCG) vaccination of cattle as an additional targeted control tool against bTB. However, there are concerns over its potential impact on the outcomes of bTB diagnostic tests and other non-TB assays, such as serological tests for Mycobacterium avium subsp. paratuberculosis (MAP). Methods: We investigated the performance of commercially available serology tests designed to detect bTB and MAP using serum samples from BCG-vaccinated animals which were subsequently infected with Mycobacterium bovis (M. bovis). Results: BCG vaccination per se did not significantly impact the specificity of serological diagnostic tests for bTB or Johne’s disease. However, increased numbers of false-positive responses in bTB serology tests were seen in BCG-vaccinated animals 3 weeks following a tuberculin skin test, where up to 23% and 54% of animals gave a positive result in IDEXX and Enferplex tests, respectively. Furthermore, M. bovis infection gave rise to false-positive test results for Johne’s disease, irrespective of the animals’ prior BCG vaccination status. Conclusions: Caution should be taken when assessing results from serology tests for bTB if tuberculin skin testing has occurred shortly before collection of blood from BCG-vaccinated cattle. Furthermore, these results highlight the potential for misdiagnosis of MAP infection when using serology tests in bTB-infected cattle. Full article

Serum beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) are biomarkers of situations of negative energetic balance in bovine. However, knowledge about their possible measurement and use in saliva is limited. In this report, two commercially available methods for the measurement of BHB and NEFAs were validated for use in bovine saliva. Both methods showed good precision and accuracy. The BHB concentrations were correlated between the saliva and the serum, but not the NEFA concentrations. The cows with hyperketonemia (n = 17) had increased salivary BHB compared to the cows with no clinical signs and no hyperketonemia (n = 34) and those with clinical signs of metritis (n = 17). The salivary NEFA concentration increased in newborn calves (n = 10) on days 1 and 2 of life compared to the day of birth before colostrum intake. The calves with symptomatic bovine respiratory disease complex (BRD, n = 7) showed higher salivary NEFA concentrations than those without clinical symptoms (n = 6). Thus, BHB and NEFAs can be reliably measured in bovine saliva using easily automatable colorimetric methods. Salivary BHB increased in hyperketonemia and could be a potential biomarker of this condition. Further studies should be undertaken to clarify the mechanism and possible use of salivary NEFAs as biomarkers. Full article

Achieving protein stability is one of the main objectives before bottling wine. Traditionally, this is accomplished via bentonite fining, but the application has drawbacks and is not the most sustainable practice. A promising alternative was previously identified in modified cellulose, which is approved for tartrate stabilization but, as a side activity, could also help remove protein from wine. This study was designed to evaluate powdered carboxymethyl cellulose (CMC) and a liquid formulation in model wine using bovine serum albumin (BSA) and egg white as model proteins. The solubility of BSA proved to be insufficient, so experiments in wine were conducted with egg white protein only. Low-addition levels of liquid CMC showed the highest protein removal rates in real wine, so final trials were conducted with 13 commercial wines to evaluate the performance in different wine styles. The protein removal rate ranged from 12% to 84%, with an overall average of 57%. While these results do not reach the efficiency of bentonite, CMC is showing promise as an additional stabilization tool for a wide variety of wines. It can stabilize over the entire pH range of wine between 2.9 and 4.1, which is a unique feature of this method. Full article

There are few studies on how dairy × beef crossbred calves perform during the pre-weaning phase compared to dairy calves. This observational study evaluated birth weight, average daily gain (ADG), and disease occurrence in Angus × Holstein (Ang × Hol) crossbred and Holstein calves reared under the same conditions on a commercial dairy farm. Retrospective data from 379 calves (290 Holstein females; 89 Ang × Hol crossbreds: 46 males, 43 females) born between January 2022 and August 2023 were analyzed. Variables included dam parity, calving type, birth weight, colostrum Brix levels, serum total protein (STP), mortality, disease occurrence, ADG, and weaning weight. Statistical analysis used linear and logistic regression models. Ang × Hol male calves had higher odds of assisted calving. Male and female Ang × Hol calves had greater birth weights than Holstein calves, with males being the heaviest. No differences in STP were observed. Ang × Hol calves (both sexes) showed higher ADG than Holsteins but did not differ from each other. Holstein calves had higher odds of diarrhea (OR: 2.95, 95% CI: 1.63–5.35), while Bovine Respiratory Disease (BRD) incidence was similar across groups. Overall, Ang × Hol crossbred calves demonstrated superior growth and lower diarrhea risk under the same management conditions. Full article

This study evaluated the effects of serum (egpS) and follicular fluid (egpFF) from estrus guinea pigs as in vitro maturation (IVM) supplements for natural cycle guinea pig oocytes. Two experiments were conducted: the first assessed three IVM media, a commercial medium (CMOM) and two homemade media (HMOM-P and HMOM-S), for oocyte in vitro maturation using 615 bovine oocytes, while the second evaluated the incorporation of 5%, 10%, and 20% egpFF or egpS into IVM media for 1744 guinea pig oocytes. Initially, we optimized the IVM base medium using bovine oocytes to determine the most suitable culture conditions. The results obtained from these experiments served as a critical foundation for subsequent supplementation trials conducted with guinea pig oocytes. The oocytes were cultured in 70 μL drops under controlled atmospheric conditions, and maturation rates were assessed based on cumulus cell expansion and nuclear progression. The HMOM-S medium significantly enhanced cumulus cell expansion (72.5 ± 2.88%) compared with the CMOM medium (57.7 ± 5.33%; p < 0.05). Supplementation with egpFF at 5% (68.8 ± 6.22%), 10% (76.3 ± 5.39%), and 20% (80.9 ± 6.22%) significantly improved both cumulus cell expansion and nuclear progression in high-quality oocytes (types A and B), compared to the control group (43.3 ± 4.82%). Conversely, supplementation with egpS did not have a significant effect on cumulus cell expansion (p > 0.05); however, it notably improved nuclear maturation in low-quality oocytes (type C) at concentrations of 10% and 20% (p < 0.05). This resulted in an overall improvement in maturation outcomes, particularly for oocytes with compromised initial quality. These findings demonstrated that the IVM of guinea pig oocytes using HMOM-S medium was significantly enhanced by the presence of egpFF, whereas egpS supplementation exhibited a less pronounced effect on IVM outcomes. Full article

Sulfonylureas (SUs)—a class of drugs primarily used to treat type 2 diabetes—have recently attracted interest for their potential anticancer properties. While some studies have explored the chemical modification or design of new SU derivatives, our work instead centers on biological evaluations of all commercially available SUs in combination with doxorubicin (DOXO). These antidiabetic agents act by stimulating insulin secretion via K_ATP channel inhibition, and because K_ATP channels share structural features with ATP-binding cassette (ABC) transporters involved in multidrug resistance (e.g., P-glycoprotein, MRP1, and MRP2), SUs may also reduce cancer cell drug efflux. In this study, we systematically examined each commercially available SU for potential synergy with DOXO in a panel of human cancer cell lines. Notably, combining DOXO with glimepiride (GLIM), the newest SU, results in a 4.4-fold increase in cytotoxicity against MCF-7 breast cancer cells relative to DOXO alone. Mechanistic studies suggest that the observed synergy may arise from increased intracellular accumulation of DOXO. Preliminary in vivo experiments support these findings, showing that DOXO (5 mg/kg, i.v.) plus GLIM (4 mg/kg, i.p.) is more effective at inhibiting 4T1 tumor growth in mice than DOXO alone. Additionally, we show that adding a small amount of the surfactant Tween-80 to culture media affects SU binding to bovine serum albumin (BSA), potentially unmasking anticancer effects of SUs that strongly bind to proteins. Overall, these results underscore the potential of repurposing existing SUs to enhance standard chemotherapy regimens. Full article

A primary cell culture derived from the gill tissues of largemouth bass (Micropterus salmoides) was successfully established and characterized, providing a physiologically relevant model for virological research. Gill tissues were enzymatically dissociated, and their cells were cultured in M199 supplemented with 20% fetal bovine serum at 25 °C, yielding optimal growth. Viral replication within these primary cells was confirmed by transmission electron microscopy, and further qRT-PCR demonstrated the upregulation of antiviral genes (IFN1, Mx1, ISG15, and Viperin). These primary gill cells of spindle-like morphology exhibited significantly higher susceptibility to Micropterus salmoides rhabdovirus (MSRV) compared to established cell lines, as evidenced by higher viral titers, thus establishing their suitability for studying host–virus interactions. Furthermore, these cells were amenable to genetic manipulation, with the successful transfection of an mCherry reporter gene using commercially available reagents. These findings highlight the utility of the largemouth bass gill-derived primary cell culture as an alternative in vitro system for investigating MSRV pathogenesis and host immune responses, which serves as a stepping stone for improved antiviral strategies in largemouth bass aquaculture. Full article

Cultured meat technology is a form of cellular agriculture where meat is produced from animal cells grown in a lab, instead of raising and slaughtering animals. This technology relies heavily on fetal bovine serum (FBS) in cell media; hence, production is costly and contributes significantly to ammonia and greenhouse gas emissions. Achieving the successful commercialization of cell-cultured food requires the critical resolution of manufacturing cost and safety concerns. Hence, our research efforts are focused on identifying commercially viable and ecologically sustainable alternatives to FBS. In this study, we evaluated the potential of twenty-six water-based algal and cyanobacterial extracts to stimulate cell growth for meat cultivation under 90% reduced serum conditions. The extracts were compared in viability, proliferation, and Trypan blue exclusion assays. In the first screening phase, the extracts were evaluated in a ZEM2S (zebrafish) cell culture in a 1% FBS regimen. Based on their ability to exhibit protein tolerance or promote cell proliferation, ten extracts were selected and further assayed in a QM7 cell culture. The QM7 cell line (myoblasts from Japanese quail) is highly relevant for meat cultivation because of its ability to differentiate into muscle fibers. Extracts derived from two microalgae species, Arthrospira platensis (Spirulina) and Dunaliella tertiolecta, demonstrated the highest tolerance in cell culture, above 10 μg/mL (expressed as total protein concentration). Tolerance at a 100 μg/mL concentration was demonstrated exclusively using an extract of blue spirulina (commercially purified Spirulina), which supported cell growth through multiple passages. Full article

Background: Lumpy skin disease virus (Poxviridae family—Capripoxvirus genus) is the aetiological agent of LSD, a disease primarily transmitted by hematophagous biting, affecting principally cattle. Currently, only live attenuated vaccines are commercially available, but their use is limited to endemic areas. There is a need for safer vaccines, especially in LSD-free countries. This research aims to develop and test a safe and efficacious inactivated vaccine. Moreover, in this study, we used keyhole limpet hemocyanin (KLH) as a positive marker to distinguish infected from vaccinated animals (DIVA). Methods: Lumpy skin disease virus was propagated on primary lamb testis cells and Madin–Darby bovine kidney cells (PLT and MDBK, respectively), and four inactivated vaccines were produced. The vaccines differed from each other with the addition or not of KLH and in cells used for virus propagation. To evaluate the safety and immunogenicity, the vaccines and two placebos were administered to six groups comprising six male calves each, and antibody response was investigated using both an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization (SN) test. In addition, the LSD/γ-interferon test and KLH (IgM-IgG) ELISA were performed on the collected samples. Furthermore, the use of KLH allowed us to distinguish vaccinated animals in the ELISA results, without any interference on the strength of the immune response against the LSDV. Finally, the efficacy of one of four vaccines was investigated through a challenge, in which one group of vaccinated animals and one animal control group were infected with a live field strain of LSDV. Results: Four out of the six control animals showed severe clinical signs suggestive of LSD, and, therefore, were euthanized for overcoming the predetermined limit of clinical score. By contrast, the vaccinated animals showed only mild symptoms, suggesting a reduction in severe disease notwithstanding the incapability of the vaccine in reducing the virus shedding. Conclusion: The vaccines produced were safe and able to elicit both a humoral and a cellular immune response, characteristics that, together with the demonstrated efficacy, make our vaccine a good candidate for countering the LSD spread in disease-free countries, thus also facilitating disease containment throughout the application of a DIVA strategy. Full article

The fouling of protein on the surface of reverse osmosis (RO) membranes is a surface phenomenon strongly dependent on the physical and chemical characteristics of both the membrane surface and the foulant molecule. Much of the focus on fouling mitigation is on the synthesis of more hydrophilic membrane materials. However, hydrophilicity is only one of several factors affecting foulant attachment. A more systematic and rationalized methodology is needed to screen the membrane materials for the synthesis of fouling-resistant materials, which will ensure the prevention of the accumulation of foulants on the membrane surfaces, avoiding the trial and error methodology used in most membrane synthesis in the literature. If a clear correlation is found between various membrane surface properties, in combination or singly, and the amount of fouling, this will facilitate the establishment of a systematic strategy of screening materials and enhance the selection of membrane materials and therefore will reflect on the efficiency of the membrane process. In this work, eight commercial reverse osmosis membranes were tested for bovine serum albumin (BSA) protein fouling. The work here focused on three surface membrane properties: the surface roughness, the water contact angle (hydrophilicity), and finally the Hansen solubility parameter (HSP) distance between the foulant understudy (BSA protein) and the membrane surface. The HSP distance was investigated as it represented the affinities of materials to each other, and therefore, it was believed to have an important contribution to the tendency of foulant to stick to the surface of the membrane. The results showed that the surface roughness and the HSP distance contributed to membrane fouling more than the hydrophilicity. We recommend taking into account the HSP distance between the membrane material and foulants when selecting membrane materials. Full article

Bovine brucellosis and neosporosis are reported as potential abortifacient infections in cattle worldwide. Brucellosis is additionally a zoonotic bacterial infection caused by numerous Brucella species. Meanwhile, neosporosis is a protozoan parasitic disease that is implicated in causing high economic losses in the cattle industry. Herein, we attempted to investigate the seroprevalence of specific antibodies to Brucella spp. and Neospora caninum using commercially available ELISAs. In addition, we conducted risk factor analysis and estimated the correlation of seropositivity of both pathogens with the recorded abortions in the tested herds. Serum samples from cattle (n = 460) collected from various governorates in the Delta region, northern Egypt, were targeted in this study. Overall, a seroprevalence of 5.4%, 33.3%, and 1.3% was revealed for Brucella spp., N. caninum, and mixed seropositivity, respectively. The location (Kafr El Sheikh vs. Dakahlia vs. Al-Qalyubiya vs. Damietta governorates) and a history of abortion (yes vs. no vs. unknown) were analyzed as risk factors of infection. Kafr El Sheikh governorate (57.7%, p = < 0.0001) and a history of abortion (54.1%, p = < 0.0001) were considered risk factors for Brucella spp. seropositivity compared to the reference factors Al-Qalyubiya (1.1%) and unknown abortion history (0.6%). In the case of N. caninum, the location was also considered a risk factor because the seropositive rates were significantly higher in Damietta (51%, p = 0.001) and Dakahlia (33.4%, p = 0.026) compared to Kafr El Sheikh (11.3%, set as a reference). Conversely to Brucella, animals without a history of abortion exhibited a higher seropositive rate for N. caninum (47.6%, p = 0.009) compared to those with a history of abortion (21.6%, set as reference). For further investigations into the association between abortion and the obtained seropositive rates, we also analyzed the reactivity by comparing samples of animals with, without, and unknown history of abortion. We detected high seroreactivity for Brucella spp. in samples collected from animals with a history of abortion, as demonstrated in the recorded antibody levels and correlation coefficient (Pearson r = 0.919). Based on our data, despite the higher seroprevalence of N. caninum compared to that of Brucella species, Brucella spp. might be the primary cause of abortion in our tested cattle population. Full article