Browsing by Author "Kastelic, John P."
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Item Open Access A combination of calcium hydroxide and sodium hydrosulphate controls pathogens causing environmental mastitis in recycled manure solids(2024-10-08) Praveen, Selladurai; Kataktalware, Mukund A.; Meena, Priyanka; Lavanya, Maharajan; Patoliya, Priyanka; Jeyakumar, Sakthivel; Ravindra, Menon R.; Chauhan, Mamta; Ramesha, K. P.; Devi, G. L.; Kastelic, John P.; Dhali, ArindamAbstract Recycled manure solids (RMS) are dried cow dung processed using a manure dewatering machine and subsequently sun-dried to ~ 20% moisture. Benefits of RMS include abundant availability, low cost, and eco-friendliness, but its use as bedding material for cows is hindered by a moisture content that promotes microbial growth. This in vitro study evaluated impacts of calcium hydroxide (CH; 5 and 7.5%) and sodium hydrosulphate (SHS; 6 and 8%), independently and in combinations, at various depths of RMS, on physicochemical and microbial properties. The CH-treated groups had increased pH and reduced moisture on Day 0. Incorporating 7.5% CH + 6% SHS at 15–20 cm, and 7.5% CH + 8% SHS at all depths, effectively suppressed Escherichia coli and Klebsiella spp. Furthermore, a combination of 7.5% CH + 8% SHS at 20 cm inhibited coliform growth, whereas 7.5% CH with 6% SHS inhibited Streptococcus spp. In conclusion, a combination of 7.5% CH with either 6 or 8% SHS at a depth of 15 cm in RMS was particularly effective in controlling environmental mastitis-causing pathogens, specifically E. coli and Klebsiella spp. Graphical AbstractItem Open Access Cathelicidin contributes to prompt protective inflammation against Toxoplasma gondii(2019-07-10) Tan, Yi Lin; Cobo, Eduardo R.; Kastelic, John P.; Gilleard, John Stuart; Finney, Constance A. M.Toxoplasma gondii is an intracellular parasite infecting all warm-blooded animals, including humans. Although infected immunocompetent individuals are usually asymptomatic, in immunocompromised individuals, T. gondii can affect the central nervous system and may cause congenital toxoplasmosis and death. Cathelicidins are short cationic peptides secreted by leukocytes and epithelial cells with antimicrobial and immunomodulatory activities. It has been proposed that cathelicidin might be a key defense against intracellular pathogens. For instance, human cathelicidin (LL-37), either endogenous or synthetic exogenous, reduced survival of intracellular Mycobacterium tuberculosis in macrophages. However, the role of cathelicidin in toxoplasmosis has been barely investigated. The objective was to elucidate the contributions of cathelicidin during acute systemic and long-term infection with T. gondii. In an acute generalized model of toxoplasmosis, C57BL/6 cathelicidin-deficient (Camp-/-) and wild type (Camp+/+) mice were challenged with luciferase-green fluorescence protein tagged T. gondii (high virulence Type 1 RH, 1 x 105, intra-peritoneal). Although all mice had succumbed by day 5 post infection, Camp-/- mice failed to initiate pro-inflammatory responses in vital organs (ileum, colon, liver, spleen and brain; p<0.05) at early infection (1 d). Consequently, more parasite load was detected in those vital organs (p<0.05). In long-term toxoplasmosis, we determined that Camp-/- mice were more susceptible to oral challenge with T. gondii cysts (low-virulence Type 2 ME 49); all Camp-/- infected mice died by day 12 post infection (p<0.05), whereas Camp+/+ infected and PBS treated mice survived throughout the 14 d study. These Camp-/- mice had more severe hepatitis, with evident liver necrosis and increased inflammatory cytokines, Ifn-γ and Tnf-α gene expression, than Camp+/+ mice (p<0.05). In Camp-/- infected mice, there was pronounced inflammation and Ifn-γ gene synthesis in their spleen (p<0.05) and in their ileum, more severe epithelial disruption, with fewer goblet cells. In in vitro studies, human macrophages (THP-1) infected by T. gondii (ME 49) expressed elevated endogenous gene transcriptional cathelicidin (p<0.05) together with induced gene transcriptional expression of IFN-γ and TNF-α (p<0.05). Camp-/- bone marrow derived macrophages (BMMs) challenged with T. gondii (RH or ME 49) secreted more Tnf-α (p<0.05) compared to infected Camp+/+ BMMs. The T. gondii burdens (both RH and ME 49 strains) were similar between Camp+/+ and Camp-/- BMMs. However, when THP-1 cells macrophages were pre-stimulated with recombinant human LL-37 cathelicidin (2 uM), T. gondii burden was reduced (p<0.05). In summary, this study identified the critical role of cathelicidin in initiating host immune responses, promptly during toxoplasmosis. In the absence of cathelicidin, inflammatory responses in vital organs (liver and spleen) were exaggerated at later infection (3 and 14 d) and they were detrimental to the host. Immunomodulatory roles of cathelicidin were manifested in infected macrophages, where the peptide supressed exaggerated inflammatory responses and aided macrophage killing capabilities. Cathelicidin secreted by macrophages during T. gondii infection has a vital role in host-pathogen balance, controlling parasite burden and preventing overwhelming inflammatory responses.Item Open Access Colostrum-derived extracellular vesicles: potential multifunctional nanomedicine for alleviating mastitis(2024-10-16) Xiong, Yindi; Shen, Taiyu; Lou, Peng; Yang, Jingyue; Kastelic, John P.; Liu, Jingping; Xu, Chuang; Han, Bo; Gao, JianAbstract Bovine mastitis is an infectious disease that causes substantial economic losses to the dairy industry worldwide. Current antibiotic therapy faces issues of antibiotic misuse and antimicrobial resistance, which has aroused concerns for both veterinary and human medicine. Thus, this study explored the potential of Colo EVs (bovine colostrum-derived extracellular vesicles) to address mastitis. Using LPS-induced murine mammary epithelial cells (HC11), mouse monocyte macrophages (RAW 264.7), and a murine mastitis model with BALB/C mice, we evaluated the safety and efficacy of Colo EVs, in vivo and in vitro. Colo EVs had favorable biosafety profiles, promoting cell proliferation and migration without inducing pathological changes after injection into murine mammary glands. In LPS-induced murine mastitis, Colo EVs significantly reduced inflammation, improved inflammatory scores, and preserved tight junction proteins while protecting milk production. Additionally, in vitro experiments demonstrated that Colo EVs downregulated inflammatory cytokine expression, reduced inflammatory markers, and attenuated NF-κB pathway activation. In summary, we inferred that Colo EVs have promise as a therapeutic approach for mastitis treatment, owing to their anti-inflammatory properties, potentially mediated through the NF-κB signaling pathway modulation. Graphical AbstractItem Open Access Landscape transcriptomic analysis of bovine follicular cells during key phases of ovarian follicular development(2024-10-28) Mogollón García, Henry D.; de Andrade Ferrazza, Rodrigo; Ochoa, Julian C.; de Athayde, Flávia F.; Vidigal, Pedro M. P.; Wiltbank, Milo; Kastelic, John P.; Sartori, Roberto; Ferreira, João C. P.Abstract Background There are many gaps in our understanding of the mechanisms involved in ovarian follicular development in cattle, particularly regarding follicular deviation, acquisition of ovulatory capacity, and preovulatory changes. Molecular evaluations of ovarian follicular cells during follicular development in cattle, especially serial transcriptomic analyses across key growth phases, have not been reported. This study aims to address this gap by analyzing gene expression using RNA-seq in granulosa and antral cells recovered from ovarian follicular fluid during critical phases of ovarian follicular development in Holstein cows. Results Integrated analysis of gene ontology (GO), gene set enrichment (GSEA), protein–protein interaction (PPI), and gene topology identified that differentially expressed genes (DEGs) in the largest ovarian follicles at deviation (Dev) were primarily involved in FSH-negative feedback, steroidogenesis, cell proliferation, apoptosis, and the prevention of early follicle rupture. In contrast, DEGs in the second largest follicles (DevF2) were mainly related to loss of cell viability, apoptosis, and immune cell invasion. In the dominant (PostDev) and preovulatory (PreOv) follicles, DEGs were associated with vascular changes and inflammatory responses. Conclusions The transcriptome of ovarian follicular fluid cells had a predominance of granulosa cells in the dominant follicle at deviation, with upregulation of genes involved in cell viability, steroidogenesis, and apoptosis prevention, whereas in the non-selected follicle there was upregulation of cell death-related transcripts. Immune cell transcripts increased significantly after deviation, particularly in preovulatory follicles, indicating strong intrafollicular chemotactic activity. We inferred that immune cell invasion occurred despite an intact basal lamina, contributing to follicular maturation. Graphical AbstractItem Open Access MicroRNA miR-223 modulates NLRP3 and Keap1, mitigating lipopolysaccharide-induced inflammation and oxidative stress in bovine mammary epithelial cells and murine mammary glands(2023-09-14) Zhou, Man; Barkema, Herman W.; Gao, Jian; Yang, Jingyue; Wang, Yue; Kastelic, John P.; Khan, Sohrab; Liu, Gang; Han, BoAbstract Bovine mastitis, the most prevalent and costly disease in dairy cows worldwide, decreases milk quality and quantity, and increases cow culling. However, involvement of microRNAs (miRNAs) in mastitis is not well characterized. The objective was to determine the role of microRNA-223 (miR-223) in regulation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and kelch like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) oxidative stress pathway in mastitis models induced by lipopolysaccharide (LPS) treatment of immortalized bovine mammary epithelial cells (bMECs) and murine mammary glands. In bMECs cultured in vitro, LPS-induced inflammation downregulated bta-miR-223; the latter interacted directly with the 3’ untranslated region (3’ UTR) of NLRP3 and Keap1. Overexpression of bta-miR-223 in bMECs decreased LPS and Adenosine 5’-triphosphate (ATP)-induced NLRP3 and its mediation of caspase 1 and IL-1β, and inhibited LPS-induced Keap1 and Nrf2 mediated oxidative stress, whereas inhibition of bta-miR-223 had opposite effects. In an in vivo murine model of LPS-induced mastitis, increased miR-223 mitigated pathology in the murine mammary gland, whereas decreased miR-223 increased inflammatory changes and oxidative stress. In conclusion, bta-miR-223 mitigated inflammation and oxidative injury by downregulating the NLRP3 inflammasome and Keap1/Nrf2 signaling pathway. This study implicated bta-miR-223 in regulation of inflammatory responses, with potential as a novel target for treating bovine mastitis and other diseases.Item Open Access Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro(2021-10-14) Liu, Yang; Deng, Zhaoju; Xu, Siyu; Liu, Gang; Lin, Yushan; Khan, Sohrab; Gao, Jian; Qu, Weijie; Kastelic, John P.; Han, BoAbstract Mycoplasma species are the smallest prokaryotes capable of self-replication. To investigate Mycoplasma induced autophagy in mammalian cells, Mycoplasma bovis (M. bovis) and bovine mammary epithelial cells (bMEC) were used in an in vitro infection model. Initially, intracellular M. bovis was enclosed within a membrane-like structure in bMEC, as viewed with transmission electron microscopy. In infected bMEC, increased LC3II was verified by Western blotting, RT-PCR and laser confocal microscopy, confirming autophagy at 1, 3 and 6 h post-infection (hpi), with a peak at 6 hpi. However, the M. bovis-induced autophagy flux was subsequently blocked. P62 degradation in infected bMEC was inhibited at 3, 6, 12 and 24 hpi, based on Western blotting and RT-PCR. Beclin1 expression decreased at 12 and 24 hpi. Furthermore, autophagosome maturation was subverted by M. bovis. Autophagosome acidification was inhibited by M. bovis infection, based on detection of mCherry-GFP-LC3 labeled autophagosomes; the decreases in protein levels of Lamp-2a indicate that the lysosomes were impaired by infection. In contrast, activation of autophagy (with rapamycin or HBSS) overcame the M. bovis-induced blockade in phagosome maturation by increasing delivery of M. bovis to the lysosome, with a concurrent decrease in intracellular M. bovis replication. In conclusion, although M. bovis infection induced autophagy in bMEC, the autophagy flux was subsequently impaired by inhibiting autophagosome maturation. Therefore, we conclude that M. bovis subverted autophagy to promote its intracellular replication in bMEC. These findings are the impetus for future studies to further characterize interactions between M. bovis and mammalian host cells.Item Open Access Nutritional Modulation of Reproduction in Bulls(2019-12-13) Johnson, Chinju Therese; Thundathil, Jacob C.; Kastelic, John P.; Chelikani, Prasanth K.; Fitzsimmons, Carolyn Jean; Lonergan, Patrick; Lee, Kee-youngNutrition is the single most important factor affecting reproductive development in most livestock species. The objective of this thesis was to investigate nutritional modulation of testis development at cellular and molecular level, and sperm function in bulls. In that regard, I investigated impacts of residual feed intake (RFI)- a measure of feed efficiency, early pre-natal diet and pre-pubertal diet on reproductive potential of bulls. In my first study, I investigated impacts of selection for RFI (based on sire and dam) on the reproductive potential of bull calves. Diets of pregnant cows were modulated during early gestation to determine the individual effects of diverse genetic background for RFI and pre-natal diet and the potential interaction between these variables on reproductive potential of bull calves born to them. Selection for RFI negatively impacted reproductive development of bull calves. However, there was no interaction between pre-natal diet and RFI on the reproductive development (age at puberty, SC, sperm production potential and sperm function) of bull calves. On investigating the testes of bulls with varying RFI, I found a possible role of IGF-I signaling in modulating the differences induced by RFI (age at puberty, SC and motility). To further elucidate IGF-I signaling, puberty and its associated testicular change in bulls, I utilized tissues from a differential pre-pubertal feeding study where the pre-pubertal bulls had exhibited elevated IGF-I levels and early puberty when fed a high nutrition diet (energy and protein) from 2-32 wk of life. On evaluating the testes of pre-pubertal bulls fed high vs low diet, I detected enhanced testicular cholesterol biosynthesis and Sertoli cell maturation in the high diet bulls at 16 and 24 wk respectively, with an indirect interaction between the cholesterol biosynthesis genes and the IGF-I receptor. These results suggested that IGF-I signaling is involved in Sertoli cell function. Using a cell culture model, I successfully tested this theory and was able to prove its role in promoting cholesterol biosynthesis. When evaluating the post-pubertal testes of bulls fed differential pre-pubertal nutrition, I detected upregulated mitochondrial function both in the testes and sperm of high diet fed bulls and proposed this as a mechanism to support their greater sperm motility, sperm production potential and enhanced cholesterol biosynthesis. Considering impacts of our dietary modulation on mitochondrial function and its potential involvement in epigenetic regulation, I evaluated the sperm epigenome and detected differential methylation mainly in genes implicated in spermatogenesis, sperm function and early embryo development. To conclude, the results of my thesis offer further confirmation to the AI industry about the benefits of allocating higher nutrition to bulls during their early post-natal life. Nutritional modulation has also been validated as an effective model to study male reproductive development.Item Open Access On-farm control of digital dermatitis in dairy cows(2019-07-30) Jacobs, Casey Elizabeth; Barkema, Herman W.; Orsel, Karin; Kastelic, John P.; Pajor, Edmond Anthony; Mason, SteveDigital dermatitis (DD) is an infectious foot lesion causing painful skin erosions which can lead to large economic and welfare implications. Currently, it is the most common foot lesion affecting dairy cattle in much of the world. Control of DD includes detection, prevention, and treatment of lesions to minimize their impact. This thesis aims to elucidate epidemiology of DD by investigating on-farm methods of detection, prevention, and treatment to generate new knowledge regarding DD control. The detection methods studied included pen walks for young stock and infrared thermography. Aspects of prevention included an evaluation of a novel footbath product and a systematic review and network meta-analyses to determine the most appropriate footbath protocol for prevention of DD. The treatment component investigated the routine treatment of DD lesions with commercially available products compared to negative and positive controls. Pen walks were able to detect DD in young stock and identified DD on 39% of farms with a mean prevalence of 1.4%. Increasing age and a high lactating DD prevalence were associated with increased DD presence in young stock. Infrared thermography was adequate to identify ulcerative, erosive lesions; however, image analysis is cumbersome and should be standardized and automated for use as a detection device. The novel footbath product evaluated was inferior to both copper sulfate and non-interference protocols and is not recommended for prevention of DD. The systematic review and network meta-analysis of footbath protocols available in the literature identified ≥ 5% copper sulfate used ≥ 4 times/wk as superior to no treatment and water for the treatment of DD lesions; however, no footbath protocol was identified as superior to any other for the prevention of DD. Routine treatment of DD lesions with commercially available topical applications was no more effective than saline (negative control) in clinically curing active lesions to nonactive stages over the 8-wk study. Routine detection, combined with consistent prevention practices and prompt treatment of DD lesions should be included in comprehensive DD control programs to maximize DD control and limit the negative consequences of DD.Item Open Access Pathogenesis of Heat-Induced Infertility in Male Mammals(2020-09-04) Rizzoto, Guilherme; Kastelic, John P.; Caulkett, Nigel; Klein, Claudia; Thundathil, Jacob C.; McGowan, Michael R.; Easton, Paul A.Testicular temperature must be 3-5 ºC below body temperature for physiological spermatogenesis and testicular function. Therefore, increased testicular temperatures, either the entire body or just the testes, reduce sperm quality and fertility. Our understanding regarding the pathophysiology of testicular heat stress is unclear. There is a long-standing dogma that as testicular temperature increases, there is no change in blood flow, and the testes, which are regarded as physiologically functioning on the brink of hypoxia, undergo frank hypoxia. However, recent data challenged this dogma, indicating that temperature itself was the major pathological agent. Therefore, this thesis was developed to further investigate the subject. In a series of five studies, the overall aim was to investigate changes in testicular blood flow in response to testicular heat stress and its pathophysiology on testes and testicular function. In the first two studies, we investigated how heat stress and hypoxia affect testicular blood flow and metabolism in rams; both treatments increased testicular blood flow which supported metabolic needs, with no indications of hypoxia. The third study was a comparison of responses between Bos indicus and Bos taurus bulls to increased testicular temperature. Once again, testicular blood flow significantly increased, supporting metabolic needs, with no indications of hypoxia. These three studies provided new knowledge to debunk the previous dogma and to support the new understanding that temperature itself was the main pathological factor of testicular heat stress. In the last two studies, we investigated how heat stress modulates gene expression in bull and mouse testes. Heat stress caused modulation of gene P53 and components of the P53-dependent apoptotic pathway, also upregulation of genes associated with the antioxidant (GPX1) and chaperone systems (Hsp70) and downregulation of the StAR gene and reduced testosterone concentrations (impaired steroidogenesis). Collectively, these studies provided novel information regarding testicular vascular physiology under local heat stress and described several factors associated with its pathophysiology in the testes. Lastly, it is expected that these findings will serve as a strong base for new studies in this area, to elucidate in more detail, how heat stress affects reproduction in male mammals.Item Open Access Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures(2021-12-11) Zhao, Wenpeng; He, Fumeng; Barkema, Herman W.; Xu, Siyu; Gao, Jian; Liu, Gang; Deng, Zhaoju; Shahid, Muhammad; Shi, Yuxiang; Kastelic, John P.; Han, BoAbstract Emergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1β and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1β, and IL-1β proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.Item Open Access Rapid and reliable detection of Leishmania antibodies in canine serum with double-antigen sandwich homogeneous chemical luminescence(2024-07-30) Zhao, Xiangjun; Ma, Licai; Jin, Yipeng; Barkema, Herman W.; Kastelic, John P.; Wang, Lu; Wen, Kai; Liu, GangAbstract Background Leishmaniasis, caused by Leishmania spp. parasites, is an important zoonotic disease globally, posing severe threats to humans and animals. In the absence of effective vaccines, reliable serological diagnostic methods are critical for disease control. However, the enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay have limitations due to complexity, time required and/or sensitivity. Therefore, our objective was to develop an accurate, rapid and user-friendly detection method of canine leishmania antibody based on double-antigen sandwich homogeneous chemical luminescence. Methods Homogeneous chemiluminescent technology was employed, and expressed recombinant fusion proteins containing full-length K9, K39 and K26 repeat sequences were used as diagnostic antigens. To establish a dual-antigen sandwich serological assay capable of detecting various antibody types, a factorial design was used to optimize concentrations of diagnostic antigen-receptor microspheres and of biotinylated diagnostic antigens, as well as of reaction solution composition and reaction duration. To evaluate and validate this newly developed method, we collected 41 Leishmania-positive serum samples, 30 Leishmania-negative control serum samples and 78 clinical serum samples for which no diagnostic information was available. Comparative analyses were performed using parasitological testing and an indirect ELISA as reference methods, focusing on diagnostic sensitivity and specificity. Results Sodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed the purification of the diagnostic antigens, which exhibited clear bands without impurities. Based on results from the 41 Leishmania-positive samples and 30 Leishmania-negative samples, there was sufficient sensitivity to detect samples diluted up to 256-fold, with analytical specificity of 100%. Overall diagnostic sensitivity was 100% and diagnostic specificity was 93.3%. Diagnostic performance was highly consistent between the newly developed method and the indirect ELISA (Kappa = 0.82, P < 0.01). Testing could be completed within 35 min with the new method Conclusions We have developed a novel double-antigen sandwich homogeneous chemical luminescence method to detect canine Leishmania antibodies, with high sensitively and specificity, a short incubation interval and a simple protocol. This streamlined approach not only offers a sensitive and efficient method for clinical diagnosis but also has great potential for use in automated testing. Graphical AbstractItem Open Access Role of GnIH and GnRH in Paracrine Control of Gametogenesis in Zebrafish (Danio rerio)(2020-03) Pourmohammadi Fallah, Hamideh; Habibi, Hamid R.; Habibi, Hamid R.; Syme, Douglas A.; Thundathil, Jacob C,; Kastelic, John P.; Wiseman, Steve B.Control of gonadal function is multifactorial and involves a number of hypothalamic, hypophyseal, and peripheral hormones. It is well established that hypothalamic GnRH (Gonadotropin-Releasing Hormone) and GnIH (Gonadotropin-Inhibitory Hormone) are key peptides involved in the neuroendocrine control of gonadal function. However, the role of GnRH and GnIH as paracrine regulators of testicular and ovarian function has not been fully investigated. Variants of GnIH and GnRH peptides have been discovered in different vertebrates, including teleost. In addition, the presence of GnRH and GnIH receptors in extra-pituitary organs, including gonads, has been demonstrated in various species. The existence of GnRH and GnIH peptides and their receptors in the testis and ovary suggest a potential for paracrine/autocrine roles of these peptides in the control of reproduction. However, information on the physiological significance of locally produced GnRH and GnIH is rather limited, and much less is known regarding the extra-pituitary actions of these peptides. The main goal of my thesis was to study the role of zebrafish GnIH (zGnIH) and GnRH peptides, which are locally produced in the gonads, in the regulation of basal and gonadotropin hormone (GTH)-induced spermatogenesis and final oocyte maturation in zebrafish, in vitro. The findings highlight the presence of GnIH and GnRH gonadal peptides and their receptors in zebrafish (Danio rerio) and provide strong evidence for direct actions of these hormones at the level of gonads. Treatment of testicular tissue with zGnIH at the lower concentrations tested, inhibited gonadotropin-induced spermatids/spermatozoa (SPD/SPZ) production ex vivo. However, at the highest concentration, zGnIH increased the basal number of SPD/SPZ and showed a paradoxical effect. The effect of zGnIH on testosterone and haploid cell production was blocked in the presence of flutamide (FLU) androgen receptor antagonist. A number of transcripts were also measured to better understand zGnIH mechanisms of action on zebrafish spermatogenesis. Further analysis of paracrine actions of zGnIH on earlier stages of spermatogenesis demonstrated that a lower concentration of zGnIH significantly decreases caspase-3 activity and changes the proliferative activity of spermatogonia cells. It was also found that zGnIH at all concentrations tested inhibits both human chorionic gonadotropin hormone (hCG) and follicle-stimulating hormone (FSH)-induced spermatogenesis. With respect to GnRH peptides, both native isoforms (GnRH2 & GnRH3) stimulated basal spermatogenesis by increasing numbers of type A undifferentiated spermatogonia, spermatozoa, and testosterone release, and GnRH2 exerted higher relative activity than GnRH3. Also, two GnRH isoforms were found to have different effects on Fsh and hCG-induced response depending on stages of spermatogenesis and concentration of peptides. In studies regarding final oocyte maturation, treatment of the late-vitellogenic ovarian follicles with GnRH and GnIH in isolation stimulated germinal vesicle break down (GVBD) in vitro. GnRH treatment increased caspase-3 activity, while GnIH treatment showed a protective effect on the late-vitellogenic follicles. GnRH and GnIH treatments were found to have similar effects on the hCG-induced resumption of meiosis while showed different effects on caspase-3 activity. Further, the interaction of GnRH and GnIH peptides in the absence and presence of hCG provides strong support for the hypothesis that gonadal GnRH and GnIH, in addition to gonadotropins are key components involved in final oocyte maturation in zebrafish. The findings in this thesis indicate that locally produced zGnIH and GnRH are important components of the complex multifactorial system that regulates gametogenesis in zebrafish in a paracrine manner.Item Open Access Streptococcus lutetiensis Induces Autophagy via Oxidative Stress in Bovine Mammary Epithelial Cells(2022-02-07) Chen, Peng; Yang, Jingyue; Wu, Naiwen; Han, Bo; Kastelic, John P.; Gao, JianStreptococcus lutetiensis, an emerging pathogen causing bovine mastitis, has not been well characterized. We reported that S. lutetiensis was pathogenic both in vivo and in vitro and caused inflammatory reactions in the mammary gland. However, roles of autophagy and oxidative stress in the pathogenesis of S. lutetiensis-induced mastitis are unclear. In this study, an autophagy model of S. lutetiensis-infected bovine mammary epithelial cells (bMECs) was used to assess oxidative stress and autophagy flux. Expressions of Beclin1, light chain 3II, and Sequestosome 1/p62 were elevated in bMECs after S. lutetiensis infection. In addition, autophagosome and lysosome formation confirmed autophagy occurred. Based on LysoTracker Red and acridine orange, lysosome degradation was blocked, and lower expressions of lysosomal-associated membrane protein 2, cathepsins D, and cathepsins L confirmed lysosomal damage. Concurrently, the nuclear factor erythroid 2-related factor 2 (Nrf2), kelch-like ECH-associated protein 1 (Keap1), heme oxygenase 1 (HO1), and NAD (P)H: quinone oxidoreductase 1 (NQO1), and basilic proteins associated with the Nrf2/Keap1 signaling pathway, were detected. Decreased keap1 and increased Nrf2, HO1, NQO1, and reactive oxygen species (ROS) indicated increased oxidative stress. Treatment with N-Acetyl-L-cysteine (NAC), an ROS inhibitor, decreased both oxidative stress and autophagy. Therefore, we concluded that S. lutetiensis caused intracellular oxidative stress and autophagy in bMECs. In addition, crosstalk between autophagy and oxidative stress affected the autophagic flux and blocked downstream autophagy. The Nrf2-keap1-p62 pathway participated in this process, with ROS acting upstream of these effects, interfering with normal cell functions.Item Open Access Wound inflammation post-orchiectomy affects the social dynamic of Nelore bulls(2023-07-15) Marcelino, Caique M.; Trindade, Pedro H. E.; García, Henry D. M.; Pupulim, Antonio G. R.; Martins, Cyntia L.; Rizzoto, Guilherme; Teixeira-Neto, Francisco; Macitelli, Fernanda; Kastelic, John P.; Ferreira, João C. P.Abstract Background Confinement of cattle imposes spatial restrictions and predisposes to aversive social encounters that can lead to contusions, wounds, pain, stress, fright, and reduced productivity. Although endogenous testosterone concentrations are linked to agonistic dominance behaviors in males, it is unknown whether decreased blood testosterone concentrations after castration alter social hierarchy rank in Nelore bulls. Therefore, in this study, we investigated the impact of the surgical would inflammation post-orchiectomy on social dynamics in a group of Nelore bulls (Bos indicus). Fourteen Nelore (Bos indicus) bulls were castrated and assessed pre- and post-surgically. Parameters evaluated were agonistic (mounting, headbutting, and fighting) and affiliative (head-play) behavior, plasma testosterone concentrations, average daily weight gain (ADG), and a score for severity of post-surgical infection. Exploratory statistics included social network analysis (SNA), hierarchy rank delta (Δ), and principal component analysis (PCA). Furthermore, statistical inferences included the Wilcoxon test, multiple logistic regression models, and Spearman's correlation. Results The social dynamic of Nelore bulls was modified after castration based on the findings of the SNA and the PCA. The moderate correlation between the postoperative inflammation level with the Δ, and the significant effect of this level in the logistic model post-castration were partially attributed to effects of pain on social relations. Conclusions Our findings suggest the severity of post-surgical inflammation, which has an association with pain intensity, was closely associated with changes in the social hierarchy.