Scanning Electron and Light Microscopic Characterization of the Endometrium Exposed to Ozonated Sunflower Oil at High Peroxide Index Value in Postpartum Primiparous Cows.

Scanning Electron and Light Microscopic Characterization of the Endometrium Exposed to Ozonated Sunflower Oil at High Peroxide Index Value in Postpartum Primiparous Cows. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Scanning Electron and Light Microscopic Characterization of the Endometrium Exposed to Ozonated Sunflower Oil at High Peroxide Index Value in Postpartum Primiparous Cows. Jair Camargo Ferreira, Matheus dos Santos Oliveira, Cristiane Marisa Piacitelli Prado Ferreira, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7520703/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The goals of the present study were to characterize the uterine response to the infusion of ozonized sunflower oil in first-calf heifers and to determine the in vitro antimicrobial action of the therapy. A total of 18 cows were submitted to a single treatment with ozonized or non-ozonized oil (O 3 and non-O 3 groups, respectively; n=9 cows/group) between days 10 and 12 after calving. Scanning electron and light microscopy were performed immediately before and 15 days after treatment (D0 and D15, respectively) to assess morphometric and morphological parameters of the endometrium. Additionally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the treatments were done against uterine bacteria ( Escherichia coli , Staphylococcus ssp., Streptococcus ssp. and Arcanobacterium pyogenes ) isolated at D10. On D15, the number of endometrial glands was greater ( P <0.01) in the O 3 group (32.6±2.5) than in non-O 3 group (11.0±1.0). Endometrial glands from O 3 group had reduced ( P <0.01) number of cells (34.1±2.2), diameter (111.9±10.8 µm) and luminal area (2376±830 µm 2 ) after treatment, when compared to the non-O 3 group (57.7±2.9, 2013.5±20.9 µm and 15008±1757 µm 2 , respectively). Scanning electron microscopy indicated the absence of surface structure abnormalities in the endometrium of the O 3 group. Ozonized sunflower oil inactivated (MIC and MBC ≤0.18 µg mL -1 ) all isolated uterine bacteria. In conclusion, the infusion of ozonized oil at high peroxide index value enhanced the endometrial gland proliferation and the uterine involution through the first weeks after calving in cows. In addition, the ozonized oil had a broad-spectrum germicidal action against uterine bacteria. Cattle endometrial gland integrative medicine ozone puerperium uterus Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights Ozonated sunflower oil infusion enhanced postpartum uterine involution in cows. Ozone therapy intensified the endometrial gland proliferation after calving. The surface microstructure of the bovine uterus is not compromised by the infusion of ozonized oil. Ozonized oil has germicidal action against uterine bacteria isolated from post-partum cows. Introduction Endometritis ranks among the most prevalent disorders observed during the postpartum period in cattle (Foldi et al. 2006). In addition to localized inflammation, approximately 90% of cows experience uterine infection within the initial days following calving (Sheldon et al. 2008 ). Clinical and subclinical endometritis are also detected in 20 and 30% of the cows, respectively, beyond three weeks postpartum (Cheong et al. 2011 ; Gilbert et al. 2005 ). Chronic endometritis exerts a profoundly detrimental effect on subsequent reproductive performance, causing endometrial damaging, delayed uterine involution, diminished conception rates and extended calving interval (Sheldon et al. 2009 ). The optimal management of uterine diseases following parturition aims to eradicate pathogens without suppressing the immune system, while also minimizing economic losses due to milk withdrawal. The current approach to treating reproductive diseases during the postpartum period predominantly relies on conventional antibiotic therapy (Mackeen et al. 2015 ). However, these allopathic medications have been linked to the presence of antibiotic residues and the escalating prevalence of multidrug-resistant pathogens (NASEM 2017). In light of these concerns, vegetal oils enriched with ozone (O 3 ) could be considered as an adjunctive or alternative approach for inflammation disorders and bacterial infections in cattle farming. O 3 therapy stimulates the synthesis of cellular growth factors (Bocci 1999 ) without inducing adverse effects (Travagli et al. 2007 ). In mares, the uterine insufflation with O 3 reduces the local contamination and inflammation (Ávila et al. 2022 ), in addition to trigging endometrial angiogenesis (Ferreira et al. 2021a ). Nevertheless, the widespread use of O 3 in large livestock herds is infeasible due to the instability of its gaseous form (Bocci 1999 ) and the need of O 3 -generator managing. Vegetable oils enriched with O 3 have emerged as potent oxidizers (Bocci 2012 , 1999 ) and efficient antimicrobial against bacteria (Sechi et al. 2001 ), fungi (Higa et al. 2022 ) and oomycetes (Ferreira 2021b). Moreover, ozonized oils exhibit anti-inflammatory action (Xiao et al. 2017 ), while their physicochemical properties remain unchanged for extended periods (Boland-Nazaret et al. 2016). Given the immunomodulatory and broad-spectrum antimicrobial properties of the ozonized sunflower oil, its infusion during the puerperium period may potentially support the uterine involution. However, there is a lack of knowledge regarding the local microscopic changes in response to ozonized oil therapy in postpartum cows. Therefore, the primary goal of present study was to characterize the endometrial response to the uterine infusion of ozonized sunflower oil during the puerperium. The specific objectives were: a) To describe the morphometric and morphologic changes of the endometrium exposed to ozonized oil during the firsts days after calving and b) to determine the antimicrobial effectiveness of the ozonized oil against uterine bacteria isolated from post-partum cows. Materials and Methods Animals Initially, 30 postpartum mixed breed cows aged between 30 and 36 months, weighing between 300 and 350 kg, were housed in an open shelter and outdoor paddock at the Research and Development Center located in Patrocínio Paulista, Sao Paulo, Brazil. Only first-calving heifers with eutocic births without retained placenta and without vaginal discharge were selected for the study. The management of the cows followed the guidelines provided in the Science Vet Guide for the Care and Use of Agricultural Animals in Research. All protocols were approved by the institutional Ethics Committee (protocol number #01/2024-CEUA). Treatments and experimental groups A total of 18 postpartum primiparous-cows were selected and divided in two groups according to the treatment (O 3 and non-O 3 groups; n= 9 cows/group). In the O 3 -oil group, a single uterine infusion of 50 mL of an ozonized sunflower oil at high peroxide index value (>600 mmol-meq kg -1 ) was performed with the aid of a sterile insemination pipette. In the non-O 3 group, a similar procedure was performed using 50 mL of non-ozonized sunflower oil (peroxide index value <20 mmol-meq kg -1 ). The treatments were done between days 10 and 12 (10.8±0.5) after calving by the same operator. Prior to treatment, the tail of the cow was wrapped and the perineal area was washed, rinsed with clean water and dried with disposable paper towels. The sterile insemination pipette was guided manually through the cervix and advanced into the uterine body. Endometrial sampling and processing Endometrial fragments were collected from the body–cornual junction using uterine biopsy forceps as previously described for cows (MADOZ et al. 2014). Biopsies were performed immediately before and 15 days after the uterine infusion (D0 and D15, respectively). Each endometrial fragment was then divided into two samples for analysis under light and scanning electron microscopy (SEM). The samples for light microscopy were fixed in 10% neutral buffered formalin and then processed for embedding in a plastic resin based on glycol methacrylate. Endometrial sections were cut and stained using the hematoxylin and eosin technique. The samples for SEM were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer. Subsequently, they underwent washes in 0.1M sodium cacodylate buffer and dehydration through a series of increasing ethanol concentrations until reaching absolute alcohol. Finally, the samples were metallized by depositing a thin layer of gold (20-30nm thick) onto them using an evaporation system (Sputter Coater SCD 050, BalTec). Light microscopy Light microscopy was employed to assess histomorphometry and histomorphology parameters of the endometrium. The microscopy analyzes were performed by two specialist pathologists without prior knowledge of the treatments. Histomorphometry procedures were conducted using software assistance following those previously described by Ferreira et al. (2021a). Five fields of each endometrial sample were photographed using a digital image analysis system (Exfocus – 0.5X) connected to an Opticam 0400S optical microscope. A total of six histomorphometry parameters were measured using the Java-based image processing program ImageJ (NIH): Height of the endometrial epithelium, height of the glandular epithelium, glandular diameter, glandular luminal area, number of endometrial glands and cells per endometrial gland (Figure 1). The tracing mode was utilized for glandular luminal area measurements. The height of the glandular epithelium was determined by considering the basal and apical membranes of the spherical spongy gland cells. The mean value of the five largest endometrial glands per field was used to calculate glandular parameters. Histomorphology was assessed subjectively using a scoring system adapted from Meira Jr et al. (2012). Scoring was conducted for the surface epithelium, lamina propria, endometrial glands, and vascular inflammatory status (Table 1). Six randomly chosen regions of interest (ROIs) were examined from each sample, with the score assigned to each parameter corresponding to the mean of the six examined ROIs. Scanning electron microscopy (SEM) SEM was conducted using a Quanta 200 model (FEI company), operated with a 30kV tungsten filament, SE detector, and EDS microanalysis capabilities. Four magnifications were employed for image capture (500x, 1500x, 1500x, and 3000x), and the morphological characteristics were documented in TIFF format. The lowest magnification was utilized to select areas for subsequent measurement at intermediate magnifications, while detailed images were captured at the highest magnification. SEM images were evaluated for the presence of cilia and microvilli on the apical surface of the epithelium. Uterine cytology Uterine cytology was performed immediately before each uterine biopsy procedure (D0 and D15) with the aid of a gynecological cytobrush attached to the sterile cytology forceps (Kasimanickam et al. 2004). All procedures were performed by the same operator. Once the cervix was passed, the cytobrush was exposed and rolled into the endometrium and then covered again with the protective forceps. After collecting, the cytobrush was removed from the pistol grip and rotated on a microscopic slide. In sequence, the slide was subjected to Panótico rapid staining (Laborclin, Brazil), following the manufacturer's instructions. The percentage of polymorphonuclear neutrophils (PMN) was established considering a total of 200 cells counted per microscopic immersion field (1000x magnification). The percentage of polymorphonuclear neutrophils (PMN) was determined by counting 200 cells per microscopic immersion field (1000x magnification). A cut-off value of ≥8% PMN was employed for diagnosing subclinical endometritis, as previously outlined for post-partum cows (Madoz et al. 2013). Antimicrobial analysis Antibiotic sensitivity testing (AST), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays were conducted to assess the antimicrobial efficacy of the ozonized sunflower oil against bacteria isolated from the uterine lumen of the cows previously on D0. A total of four bacteria were isolated: Streptococcus spp., Staphylococcus spp., Escherichia coli and Arcanobacterium pyogenes . AST involved measuring the diameter of bacterial inhibition zones around antibiotic disks and comparing them with interpretive criteria provided by the Clinical and Laboratory Standards Institute (CLSI 2012). MIC determination followed CLSI guidelines (2012), with adaptations utilizing resazurin as a microbial activity indicator (Sarker et al. 2007). MIC and MBC assays were conducted to confirm the bactericidal or bacteriostatic effects of the treatments against the respective pathogens, according to CLSI guidelines (2012). Similar MIC and MBC results indicated bactericidal action of the respective treatment against the bacteria tested, while disparate results suggested a bacteriostatic effect. Data analyses Data were initially tested for normality using the D’Agostino and Pearson normality test. Paired Student’s t-test was used to locate differences within and between the experimental groups for the histomorphometry parameters. Chi-square tests of independence were used to examine the differences in frequency data for morphological parameters. A probability of P ≤0.05 indicated that a difference was significant, and probabilities between P >0.05 and ≤0.1 indicated that a difference approached significance. Data are presented as mean±S.E.M., unless otherwise indicated. Results Despite variations in treatment and time, all uterine samples exhibited a simple cylindrical epithelium supported by a network of collagen fibers, with moderate to mild epithelial detachment. The absence of active inflammatory cells as lymphocyte aggregates next to the detached epithelium suggests it was an artefact resulting from an extraneous factor. In both groups, severe and mild epithelial inflammatory infiltrate were visualized on D0 and D15, respectively. In D0, cows from both groups had similar intensity of granulomas and hemorrhagic spots in the lamina propria. However, only uterine samples from the O 3 -oil group showed a substantial decrease on visualization of the white blood cells clusters and blood extravasation 15 days after treatment. Hemorrhage and vascular dilatation were markedly found at D15 only in cows from non-O 3 group. In contrast, a greater accumulation of stroma between the endometrial glands was visualized in the endometrium 15 days after ozonized oil infusion. Histopathological changes associated with endometrial degeneration, such as periglandular fibrosis and endometrial atrophy, were not detected in the uteri from O 3 and non-O 3 groups. A minimal number of endometrial glands visualized before treatment in both groups (5.6±0.6 glands; P >0.1) was followed by a significant structural proliferation 15 days later ( P <0.1). However, this increase in number of endometrial glands at D15 was 3-fold greater ( P 0.1), the glandular epithelium height remained unchanged throughout the experiment ( P >0.1). However, the histomorphometry analysis revealed a significant impact of treatment ( P <0.01) on the dimensions of the endometrial glands (Table 2; Figure 2). While a minor increase ( P <0.1) in glandular luminal area was detected after treating the uterus with ozonized oil, the non-O 3 group exhibited an 8-fold expansion at D15 ( P <0.01). Moreover, an increase ( P <0.01) in the number of cells per endometrial gland was found only in the non-O 3 group (Table 2). SEM provided a detailed view of the apical surface of epithelial cells in the examined samples. SEM analysis revealed no morphological abnormalities induced by the uterine infusion of ozonized sunflower oil (Figures 3 and 4). Cells covered with microvilli and cilia were identified in all groups and evaluation times. Microvilli-covered cells were predominant, although within the same tissue section, some areas showed nearly all cells with microvilli, while others displayed a cratered apical surface with microvilli lining the junction between cells. About 30% of the observed fields showed a surface with less than 50% microvilli coverage. The presence of ciliated cells varied and was sporadic across different evaluation times. No apical protrusions corresponding to pinopodes were detected. Non-O 3 and O 3 groups had a similar ( P >0.1) and high PMN values at D0 (47.3±17.9% and 45.1±17.0%, respectively). A significant reduction ( P 0.1). On D15, only one cow from each group exceeded the cut-off value for diagnosing subclinical endometritis (17.5% and 45% for the non-O 3 and O 3 groups, respectively). AST revealed the antimicrobial effectiveness of ozonized sunflower oil against both Gram-positive and Gram-negative bacteria (Table 3). Compared to tetracycline, ozonized sunflower oil exhibited a larger zone of inhibition for bacterial growth. Furthermore, ozonized sunflower oil demonstrated bactericidal activity at concentrations below 1.50 µg mL -1 , while non-ozonized sunflower oil did not exhibit any antimicrobial properties. Details of the MIC and MBC assays for Streptococcus spp., Staphylococcus spp., E. coli and A. pyogenes are provided (Table 4). Nearly all females from the O 3 group (6 out of 7 cows; 85.7%) were pregnant by day 60 after calving, while 42.8% of the cows in the non-O 3 group remained non-pregnant. However, the small sampling per group did not allow statistical analysis of the pregnancy rate in the present study. Discussion This study marks the first documentation of the positive effects of ozonized sunflower oil on the uterine involution in postpartum primiparous cows. Additionally, our results confirm the germicidal action of the ozonized oil against both gram-positive and gram-negative bacteria isolated from the uterus of cows. Given the crucial role of a healthy uterus in reproductive success, our in vivo and in vitro findings suggest the benefits of puerperal treatment with ozonized oil in preventing uterine conditions that might lead to long-term infertility. In cattle, the presence of epithelial tissue debris and fluid during the two first weeks after calving makes the uterine environment favorable for microbial growth (Walker et al. 2015 ). Therefore, almost the totality of the cows is affected by uterine contamination at the early puerperium (Sheldon et al. 2009 ). In the present study, the ozonized oil showed greater inhibitory sensitivity than the Tetracycline against S. aureus , Streptococcus ssp. and E. coli . The antimicrobial action of the ozonized oil has been well documented against bacteria (Gentili et al. 2023 ; Ugazio et al. 2020 ), fungi (Celenza et al. 2020 ; Sehim et al. 2023 ), oomycetes (Carrijo et al. 2021 ; Ferreira et al. 2021b ) and multidrug resistant strains (Grandi et al. 2022 ; Oliveira et al. 2017 ). Similarly, dual bacteria/fungi and single multidrug resistant biofilms are also susceptible to ozonated oil (Gentili et al. 2023 ; Higa et al. 2021; Silva et al. 2020 ). Due to its potent antioxidant potential, ozonized oils are capable of degrading bacteria by interacting with the membrane, cytoplasmic and nuclear constituents (Lake et al. 2004 ; Khadre et al. 2001 ). The peroxidation compromises enzymatic functions and damages the genetic material, disrupting the metabolic and reproductive processes of the pathogen (reviewed by Liu et al. 2023 ). When in contact with fungi, the ozonides still disrupts critical cellular functions, inhibiting the production of fungal urease, amylase, alkaline phosphatase, lipase and keratin (Pages et al. 2020 ). Therefore, ozonated oils have been successfully used for the adjuvant treatment of superficial bacterial and fungal infections (Ouf et al. 2016 ; Song et al. 2018 ). The morphology of the apical surface may have a key role in governing pregnancy establishment. The surface of the luminal epithelium changes during the estrous cycle through a process that involves remodeling of the apical surface (Kumro et al. 2020 ). Acute postpartum endometritis is accompanied by ultrastructural changes in cattle. Dystrophic and necrobiotic processes in the parenchyma and endometrial stroma, exudative processes and disruption of microcirculatory bed integrity are common findings (Suleymanov et al. 2018 ). Destruction of microvilli, abundance of superficial coccal microflora, necrobiosis of epithelial cells and partial nuclear edema have also been reported in post-partum cows (Suleymanov et al. 2018 ). Even so, most of the current therapies for infectious endometritis did not consider their deleterious changes on the structural organization of the uterus. In contrast, the local treatment with ozonized sunflower (O 3 group) oil did not induce morphological abnormalities and prevented the occurrence of glandular dilations. O 3 stimulates complex regenerative mechanisms in addition to its direct action on microorganisms. Therefore, intrauterine treatment with O 3 and derivates been used successfully in ruminants for the complementary treatment of endometritis (Đuričić et al. 2014 ), uterine infections following dystocia, retained placenta (Đuričić et al. 2012 , 2016 ), and subfertility (Zobel et al. 2014 ) without causing local side effects. The antioxidant potential, immunomodulatory and angiogenic capacity of the O 3 may support the tissue healing process (Liu et al. 2023 ). It stimulates the fibroblast synthesis (Pchepiorka et al., 2020 ) and reduces the fibroblastic genotoxic damage (Akdeniz et al. 2018 ). In addition to a greater secretion of cytokines (Peden 2011 ) and the anti-inflammatory modulation via inhibition of NF-kB (Delgado-Roche et al. 2017 ), the O 3 intensify the local microcirculation (Ferreira et al. 2021a ; Pchepiorka et al. 2020 ). Finally, this adjuvant therapy also increases the number of leukocytes and the phagocytic capacity of granulocytes (Kucuksezer et al. 2014 ). Fifteen days after treatment (25–30 days post-partum), both groups showed percentage of PMN lower than the cut-off value for the diagnosis of subclinical endometritis in dairy cows (Madoz et al. 2013 ). In contrast, the histological changes associated with endometrial inflammation visualized at D15 in control cows (non-O 3 group) did not reflect on the cytological results. Our findings support the low agreement of endometrial biopsy with cytology for the diagnosis of subclinical endometriais (Madoz et al. 2014 ). The decrease in leukocyte count in O 3 group was expected since the endometrial PMN values ​​gradually decrease as the puerperium progresses in healthy cows (Santos et al. 2009 ). In opposite, reduced pregnancy rate is associated with endometrial PMN abundance in females suffering from uterine inflammatory diseases (Kasimanickam et al. 2004 ; Sicsic et al. 2018 ). The probability for getting normal uterine environment decreased 2% for every increasing percentage point in PMN, while the likelihood of isolating Trueperella pyogenes increases (Madoz et al. 2014 ). The antimicrobial and regenerative potential of the tested ozonized oil support its application as a preventive treatment of puerperal uterine disturbances. The intrauterine infusion of ozonized solution in the postpartum period has been associated with increases in the conception rate and reproductive efficiency of dairy cows (Constantin and Bîrtoiu 2016; Đuričić et al. 2012 ; Zobel and Tkalcic 2013). In sheep, treatment with O 3 accelerated uterine regression in cases of retained placenta and dystocia (Đuričić et al. 2016 ). Ozonated vegetable oils are prepared by the direct reaction of O 3 with the carbon-carbon double bonds of their unsaturated fatty acids (Diaz Gomes et al. 2001). In contrast to the unstable O 3 gas (Kogelschatz et al. 1988 ), this ozonation process allows the storage of a O 3 derivate with high doses of ozonides products and prolonged biological action (Boland-Nazar et al. 2016 ). Therefore, the use of ozonized oil treatments is considered a cost-effective with proven efficiency and the absence of serious adverse effects (Serra et al. 2023). The main limitation of the present study was the experimental sampling for fertility analysis. Seven cows per group allowed a reliable microscopy analysis of the endometrial characteristics of the uterus subjected to ozonized oil. Still, the sampling does not have the statistical power required to confirm the benefits of the treatment for the fertility rates. However, it is important to highlight that the uterine infusion of ozonized oil did not interfere with the fertilization process in recently calved cows in the present study. Conclusion The findings of the present study suggest that infusion of ozonized sunflower oil during the puerperium improved the uterine involution in primiparous cows. Additionally, broad-spectrum germicidal efficacy of the ozonized oil against uterine bacteria supports the its use as an alternative therapeutic approach. Finally, the fertilization through the firsts weeks after calving of cows treated with ozonized oil implies a potential enhancement in fertility. However, further studies with bigger samples of animals are necessary to confirm the impact of uterine infusion of ozonized oil on the reproductive performance of cows. Declarations Orcid Jair C. Ferreira http://orcid.org/0000-0001-6908-3463 Conflicts of interest The authors declare that there are no conflicts of interest that would prejudice the impartiality of this scientific work. Author Contribution J.C.FERREIRA = Project administration.J.C.FERREIRA , Y.L.BOAKARI and M.A.RODRIGUES = Supervision, Visualization, Writing (original draft).J.C.FERREIRA and M.S.OLIVEIRA = Conceptualization, Methodology and Funding acquisition.M.S.OLIVEIRA, C.M.PP.FERREIRA, M.B.ALMEIDA and H.A.RODRIGUES = Data Curation, Analysis, Software analysis and Antimicrobial analysis.All authors reviewd the manuscritp Acknowledgment This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance code 001. References Akdeniz SS, Beyler E, Korkmaz Y, Yurtcu E, Ates U, Araz K, Sahin FI, Torun OY (2018). The effects of ozone application on genotoxic damage and wound healing in bisphosphonate-applied human gingival fibroblast cells. 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Silva V, Peirone C, Amaral JS, Capita R, Alonso-Calleja C, Marques-Magallanes JA, Martins A, Carvalho A, Maltez L, Pereira JE, Capelo JL, Igrejas G, Poeta P (2020) High efficacy of ozonated oils on the removal of biofilms produced by Methicillin-Resistant Staphylococcus aureus (MRSA) from infected diabetic foot ulcers. Molecules 25:3601. https://doi.org./10.3390/molecules25163601. Song M, Zeng Q, Xiang Y, Gao L, Huang J, Huang J, Wu K, Lu J (2018) The antibacterial effect of topical ozone on the treatment of MRSA skin infection. Molecular Medicine Reports 17:2449-2455. https://doi.org./10.3892/mmr.2017.8148. Suleymanov SM, Usha BV, Vatnikov YA, Sotnikova ED, Kulikov EV, Parshina VI, Bolshakova MV, Lyshko MU, Romanova EV (2018) Structural uterine changes in postpartum endometritis in cows. Veterinary World 11:1473-1478. https://doi.org./10.14202/vetworld.2018.1473-1478. Travagli V, Zanardi I, Silvietti A, Bocci V (2007) A physicochemical investigation on the effects of ozone on blood. International Journal of Biological Macromolecules 41:504-511. https://doi.org./10.1016/j.ijbiomac.2007.06.010. Ugazio E, Tullio V, Binello A, Tagliapietra S, Dosio F (2020) Ozonated oils as antimicrobial systems in topical applications. Their characterization, current applications, and advances in improved delivery techniques. Molecules 25:334. https://doi.org./10.3390/molecules25020334. Walker CG, Meier S, Hussein H, McDougall S, Burke CR, Roche JR, Mitchell MD (2015) Modulation of the immune system during postpartum uterine inflammation. Physiological Genomics 47:89-101. https://doi.org./10.1152/physiolgenomics.00098.2014. Xiao W, Tang H, Wu M, Liao Y, Li K, Li L, Xu X (2017) Ozone oil promotes wound healing by increasing the migration of fibroblasts via PI3K/Akt/mTOR signaling pathway. Bioscience Reports 37:BSR20170658. https://doi.org./10.1042/BSR20170658. Zobel R, Martinec R, Ivanović DID, Rošić NRN, Stančić ZSC, Žerjavić IZI, Flajsig BFB, Plavec HPH, Smolec O (2014) Intrauterine ozone administration for improving fertility rate intrauterine ozone administration for improving fertility rate in Simmental cattle. Veterinarski Arhiv 84:1–8. Tables Table 1. Histopathological criteria for analysis of endometrium from postpartum cows. Per high power field at the stated magnification (ppf; ×10 or ×40). Adapted from Meira Jr (2012). Variable Category Epithelium i. Height columnar cuboidal flattened ii. Epithelial damage absent mild moderate iii. Inflamatory cell type absent mononuclear polymorphonuclear iv. Infiltrate intensity absent mild (≤5 cells/hpf; x40) moderate (≥6-10cells/hpf; x40) severe (>10 cells/hpf; x40) Lamina própria i. Inflamatory cell type absent mononuclear polymorphonuclear ii. Infiltrate intensity normal (≤20 cells/hpf; x40) mild (≤21-40 cells/hpf; x40) moderate (≥41-70cells/hpf; x40) severe (>70 cells/hpf; x40) iii. Lynphocytic aggregates absent mild (≤3 aggregates/hpf; x40) moderate (≥4-5 aggregates/hpf; x40) severe (>6 aggregates/hpf; x40) Endometrial gland i. Atrophy or dilatation absent present ii. Fibrosis mild (1-3 layers/hpf; x40) moderate (4-5 layers/hpf; x40) severe (>6 layers/hpf; x40) Vascular i. Vessel degeneration absent present ii. Hemorrhage absent present iii. Hemosiderin macrophages absent present Table 2. Endometrial histomorphometry exam from puerperal primiparous cows receiving uterine infusion of ozonized or non-ozonized sunflower oil (O 3 and non-O 3 groups; n=9 cows/group). Moment immediately before and 15 days after treatment were considered D0 and D15, respectively. a, b, c Different letters within a parameter are different ( P < 0.01). O 3 group non-O 3 group Parameter D0 D15 D0 D15 Number of glands 5.1±0.5 a 32.6±2.5 c 6.1±2.2 a 11.0±1.0 b Number of cells per gland 33.2±1.3 a 34.1±2.2 a 26.7±1.2 a 57.7±2.9 b Epithelial height (µm) 67.3±6.5 57.9±5.0 44.1±4.1 54.3±3.7 Gland height (µm) 33.2±1.3 34.1±2.2 33.2±2.0 30.9±2.7 Gland diameter (µm) 111.0±10.9 a 111.9±10.8 a 108.3±5.5 a 2013.5±20.9 b Gland luminal area (µm 2 ) 1,750±303 a 2,376±830 b 1,713±300 a 15,008±1,757 c Table 3. Antibiotic sensitivity testing (AST) based on the diameter of inhibition zone of ozonized and non-ozonized sunflower oils (O 3 and non-O 3 oils, respectively) against bovine uterine bacteria. Tetracyclin and untreated were considered controls groups. Sensibility and resistance of the bacteria to the treatment are indicated, respectively, by the letter “S” and “R”. Diameter of zone inhibition (mm) Bacteria O 3 oil non-O 3 oil Untreated Tetracycline Escherichia coli 33.5±3.5 a (S) 0.0±0.0 c (R) 0.0±0.0 c (R) 16.7±1.8 b (S) Staphylococcus ssp. 41.3±4.9 a (S) 0.0±0.0 c (R) 0.0±0.0 c (R) 24.3±2.3 b (S) Streptococcus ssp. 38.0±1.2 a (S) 0.0±0.0 c (R) 0.0±0.0 c (R) 14.3±2.6 b (S) Arcanobacterium pyogenes 33.2±1.3 a (S) 0.0±0.0 c (R) 0.0±0.0 c (R) 23.7±2.2 b (S) a, b, c different letters on the same line are different ( P <0.05) Table 4. Minimum inhibitory concentration and minimum bactericidal concentration (MIC and MBC; µg mL -1 ) of ozonized and non-ozonized and sunflower oils (O 3 and non-O 3 oils) against bovine uterine bacteria ( Escherichia coli , Staphylococcus ssp., Streptococcus ssp . and Arcanobacterium pyogenes ). O 3 oil non-O 3 oil untreated Tetracycline Bacteria MIC MBC MIC MBC MIC MBC MIC MBC E. coli 0.05 0.09 >5.9 >5.9 >5.9 >5.9 0.05 0.09 Staphylococcus ssp. 0.09 0.18 >5.9 >5.9 >5.9 >5.9 0.18 0.36 Streptococcus ssp. 0.09 0.18 >5.9 >5.9 >5.9 >5.9 0.33 0.97 A. pyogenes 0.18 0.18 >5.9 >5.9 >5.9 >5.9 0.18 0.36 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7520703","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511100629,"identity":"b1643c2f-904d-4d94-b9f7-8894d02da949","order_by":0,"name":"Jair Camargo Ferreira","email":"data:image/png;base64,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","orcid":"","institution":"Universidade de Franca","correspondingAuthor":true,"prefix":"","firstName":"Jair","middleName":"Camargo","lastName":"Ferreira","suffix":""},{"id":511100631,"identity":"80645fc8-8cb4-4f48-9741-e0aa2dec39c7","order_by":1,"name":"Matheus dos Santos Oliveira","email":"","orcid":"","institution":"Universidade de Franca","correspondingAuthor":false,"prefix":"","firstName":"Matheus","middleName":"dos Santos","lastName":"Oliveira","suffix":""},{"id":511100633,"identity":"407c1568-a871-4502-9103-0d536a912337","order_by":2,"name":"Cristiane Marisa Piacitelli Prado Ferreira","email":"","orcid":"","institution":"Universidade de Franca","correspondingAuthor":false,"prefix":"","firstName":"Cristiane","middleName":"Marisa Piacitelli Prado","lastName":"Ferreira","suffix":""},{"id":511100634,"identity":"ec45bea2-1b73-4235-b9c4-662d9b98f959","order_by":3,"name":"Maysa Barbosa Almeida","email":"","orcid":"","institution":"Universidade de Franca","correspondingAuthor":false,"prefix":"","firstName":"Maysa","middleName":"Barbosa","lastName":"Almeida","suffix":""},{"id":511100635,"identity":"f92978f7-8d0e-47c8-aaf0-d7a1b09ba553","order_by":4,"name":"Heloisa Andrade Rodrigues","email":"","orcid":"","institution":"Universidade de Franca","correspondingAuthor":false,"prefix":"","firstName":"Heloisa","middleName":"Andrade","lastName":"Rodrigues","suffix":""},{"id":511100636,"identity":"c8ddfa40-4359-4dae-9543-911a688d765e","order_by":5,"name":"Yatta Linhares Boakari","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Yatta","middleName":"Linhares","lastName":"Boakari","suffix":""},{"id":511100637,"identity":"bad306a5-4c82-4d6f-97c9-c18e8c7726f1","order_by":6,"name":"Marcela Aldrovani Rodrigues","email":"","orcid":"","institution":"Universidade de Franca","correspondingAuthor":false,"prefix":"","firstName":"Marcela","middleName":"Aldrovani","lastName":"Rodrigues","suffix":""}],"badges":[],"createdAt":"2025-09-02 19:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7520703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7520703/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91868265,"identity":"090ec34f-5575-41bb-abe4-1906bb223a98","added_by":"auto","created_at":"2025-09-22 13:36:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":875870,"visible":true,"origin":"","legend":"\u003cp\u003eHistomorphometric analysis of the bovine endometrial stroma before and 15 days afeter the uterine infusion of ozonized sunflower oil. Original and color-marked of microscopic light images (A and B, respectively) are shown. Measurements of glandular luminal area (yellow), height of the glandular epithelium (black line) and diameter of the gland (black dotted line) were measured immediately before and 15 days after treatment. (H-E 100×).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7520703/v1/e2d801317382187fe7126f57.png"},{"id":91868269,"identity":"6bc7d94d-17e0-4a64-8ceb-42b54522dbf3","added_by":"auto","created_at":"2025-09-22 13:36:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1230905,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections of endometrial stroma from primiparous post-partum cows immediately before (D0) and 15 days (D15) after uterine infusion of non-ozonized and ozonized oil (non-O\u003csub\u003e3\u003c/sub\u003e and O\u003csub\u003e3\u003c/sub\u003e group, respectively). The local ozone therapy prevents (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) the dilatation of the endometrial gland dilatations (H-E 100×).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7520703/v1/b3d06965d049444f6cb4cb31.png"},{"id":91869865,"identity":"31e690bd-6dc1-4dda-a7d2-9c5a0c3b3817","added_by":"auto","created_at":"2025-09-22 13:44:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":872640,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy showing the apical surface of the luminal uterine epithelium of the puerperal uterus immediately before (A and C) and 15 days after (B and D) a single infusion of ozonized oil in post-partum primiparous cows.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7520703/v1/002361b0cb0e3c31c35e127b.png"},{"id":91868266,"identity":"2c965200-8403-48af-b6df-64914d4e45ee","added_by":"auto","created_at":"2025-09-22 13:36:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":938861,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy showing the apical surface of the luminal uterine epithelium of the puerperal uterus immediately before (A and C) and 15 days after (B and D) a single infusion of non-ozonized oil in post-partum primiparous cows.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7520703/v1/188e15aa07af5dfaec17db62.png"},{"id":92624722,"identity":"19e9f22a-86cb-4602-b275-d00b0a081fd2","added_by":"auto","created_at":"2025-10-01 22:01:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4887225,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7520703/v1/7745e0ab-b216-4b3b-b77a-59d0e4948e18.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Scanning Electron and Light Microscopic Characterization of the Endometrium Exposed to Ozonated Sunflower Oil at High Peroxide Index Value in Postpartum Primiparous Cows.","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eOzonated sunflower oil infusion enhanced postpartum uterine involution in cows.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eOzone therapy intensified the endometrial gland proliferation after calving.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003eThe surface microstructure of the bovine uterus is not compromised by the infusion of ozonized oil.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"4\"\u003e\n \u003cli\u003eOzonized oil has germicidal action against uterine bacteria isolated from post-partum cows.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eEndometritis ranks among the most prevalent disorders observed during the postpartum period in cattle (Foldi et al. 2006). In addition to localized inflammation, approximately 90% of cows experience uterine infection within the initial days following calving (Sheldon et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Clinical and subclinical endometritis are also detected in 20 and 30% of the cows, respectively, beyond three weeks postpartum (Cheong et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Gilbert et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Chronic endometritis exerts a profoundly detrimental effect on subsequent reproductive performance, causing endometrial damaging, delayed uterine involution, diminished conception rates and extended calving interval (Sheldon et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe optimal management of uterine diseases following parturition aims to eradicate pathogens without suppressing the immune system, while also minimizing economic losses due to milk withdrawal. The current approach to treating reproductive diseases during the postpartum period predominantly relies on conventional antibiotic therapy (Mackeen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, these allopathic medications have been linked to the presence of antibiotic residues and the escalating prevalence of multidrug-resistant pathogens (NASEM 2017). In light of these concerns, vegetal oils enriched with ozone (O\u003csub\u003e3\u003c/sub\u003e) could be considered as an adjunctive or alternative approach for inflammation disorders and bacterial infections in cattle farming.\u003c/p\u003e\u003cp\u003eO\u003csub\u003e3\u003c/sub\u003e therapy stimulates the synthesis of cellular growth factors (Bocci \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) without inducing adverse effects (Travagli et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In mares, the uterine insufflation with O\u003csub\u003e3\u003c/sub\u003e reduces the local contamination and inflammation (\u0026Aacute;vila et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in addition to trigging endometrial angiogenesis (Ferreira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Nevertheless, the widespread use of O\u003csub\u003e3\u003c/sub\u003e in large livestock herds is infeasible due to the instability of its gaseous form (Bocci \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and the need of O\u003csub\u003e3\u003c/sub\u003e-generator managing.\u003c/p\u003e\u003cp\u003eVegetable oils enriched with O\u003csub\u003e3\u003c/sub\u003e have emerged as potent oxidizers (Bocci \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and efficient antimicrobial against bacteria (Sechi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), fungi (Higa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and oomycetes (Ferreira 2021b). Moreover, ozonized oils exhibit anti-inflammatory action (Xiao et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), while their physicochemical properties remain unchanged for extended periods (Boland-Nazaret et al. 2016).\u003c/p\u003e\u003cp\u003eGiven the immunomodulatory and broad-spectrum antimicrobial properties of the ozonized sunflower oil, its infusion during the puerperium period may potentially support the uterine involution. However, there is a lack of knowledge regarding the local microscopic changes in response to ozonized oil therapy in postpartum cows. Therefore, the primary goal of present study was to characterize the endometrial response to the uterine infusion of ozonized sunflower oil during the puerperium. The specific objectives were: a) To describe the morphometric and morphologic changes of the endometrium exposed to ozonized oil during the firsts days after calving and b) to determine the antimicrobial effectiveness of the ozonized oil against uterine bacteria isolated from post-partum cows.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, 30 postpartum mixed breed cows aged between 30 and 36 months, weighing between 300 and 350 kg, were housed in an open shelter and outdoor paddock at the Research and Development Center located in Patroc\u0026iacute;nio Paulista, Sao Paulo, Brazil. Only first-calving heifers with eutocic births without retained placenta and without vaginal discharge were selected for the study. The management of the cows followed the guidelines provided in the Science Vet Guide for the Care and Use of Agricultural Animals in Research. All protocols were approved by the institutional Ethics Committee (protocol number #01/2024-CEUA).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTreatments and experimental groups\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 18 postpartum primiparous-cows were selected and divided in two groups according to the treatment (O\u003csub\u003e3\u003c/sub\u003e and non-O\u003csub\u003e3\u003c/sub\u003e groups; n= 9 cows/group). In the O\u003csub\u003e3\u003c/sub\u003e-oil group, a single uterine infusion of 50 mL of an ozonized sunflower oil at high peroxide index value (\u0026gt;600 mmol-meq kg\u003csup\u003e-1\u003c/sup\u003e) was performed with the aid of a sterile insemination pipette. In the non-O\u003csub\u003e3\u003c/sub\u003e group, a similar procedure was performed using 50 mL of non-ozonized sunflower oil (peroxide index value \u0026lt;20 mmol-meq kg\u003csup\u003e-1\u003c/sup\u003e). The treatments were done between days 10 and 12 (10.8\u0026plusmn;0.5) after calving by the same operator.\u003c/p\u003e\n\u003cp\u003ePrior to treatment, the tail of the cow was wrapped and the perineal area was washed, rinsed with clean water and dried with disposable paper towels. The sterile insemination pipette was guided manually through the cervix and advanced into the uterine body.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEndometrial sampling and processing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndometrial fragments were collected from the body\u0026ndash;cornual junction using uterine biopsy forceps as previously described for cows (MADOZ et al. 2014). Biopsies were performed immediately before and 15 days after the uterine infusion (D0 and D15, respectively). Each endometrial fragment was then divided into two samples for analysis under light and scanning electron microscopy (SEM). \u003c/p\u003e\n\u003cp\u003eThe samples for light microscopy were fixed in 10% neutral buffered formalin and then processed for embedding in a plastic resin based on glycol methacrylate. Endometrial sections were cut and stained using the hematoxylin and eosin technique.\u003c/p\u003e\n\u003cp\u003eThe samples for SEM were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer. Subsequently, they underwent washes in 0.1M sodium cacodylate buffer and dehydration through a series of increasing ethanol concentrations until reaching absolute alcohol. Finally, the samples were metallized by depositing a thin layer of gold (20-30nm thick) onto them using an evaporation system (Sputter Coater SCD 050, BalTec).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLight microscopy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLight microscopy was employed to assess histomorphometry and histomorphology parameters of the endometrium. The microscopy analyzes were performed by two specialist pathologists without prior knowledge of the treatments.\u003c/p\u003e\n\u003cp\u003eHistomorphometry procedures were conducted using software assistance following those previously described by Ferreira et al. (2021a). Five fields of each endometrial sample were photographed using a digital image analysis system (Exfocus \u0026ndash; 0.5X) connected to an Opticam 0400S optical microscope. A total of six histomorphometry parameters were measured using the Java-based image processing program ImageJ (NIH): Height of the endometrial epithelium, height of the glandular epithelium, glandular diameter, glandular luminal area, number of endometrial glands and cells per endometrial gland (Figure 1). The tracing mode was utilized for glandular luminal area measurements. The height of the glandular epithelium was determined by considering the basal and apical membranes of the spherical spongy gland cells. The mean value of the five largest endometrial glands per field was used to calculate glandular parameters.\u003c/p\u003e\n\u003cp\u003eHistomorphology was assessed subjectively using a scoring system adapted from Meira Jr et al. (2012). Scoring was conducted for the surface epithelium, lamina propria, endometrial glands, and vascular inflammatory status (Table 1). Six randomly chosen regions of interest (ROIs) were examined from each sample, with the score assigned to each parameter corresponding to the mean of the six examined ROIs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eScanning electron microscopy (SEM)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSEM was conducted using a Quanta 200 model (FEI company), operated with a 30kV tungsten filament, SE detector, and EDS microanalysis capabilities. Four magnifications were employed for image capture (500x, 1500x, 1500x, and 3000x), and the morphological characteristics were documented in TIFF format. The lowest magnification was utilized to select areas for subsequent measurement at intermediate magnifications, while detailed images were captured at the highest magnification. SEM images were evaluated for the presence of cilia and microvilli on the apical surface of the epithelium.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eUterine cytology \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUterine cytology was performed immediately before each uterine biopsy procedure (D0 and D15) with the aid of a gynecological cytobrush attached to the sterile cytology forceps (Kasimanickam et al. 2004). All procedures were performed by the same operator.\u003c/p\u003e\n\u003cp\u003eOnce the cervix was passed, the cytobrush was exposed and rolled into the endometrium and then covered again with the protective forceps. After collecting, the cytobrush was removed from the pistol grip and rotated on a microscopic slide. In sequence, the slide was subjected to Pan\u0026oacute;tico rapid staining (Laborclin, Brazil), following the manufacturer\u0026apos;s instructions. \u003c/p\u003e\n\u003cp\u003eThe percentage of polymorphonuclear neutrophils (PMN) was established considering a total of 200 cells counted per microscopic immersion field (1000x magnification). The percentage of polymorphonuclear neutrophils (PMN) was determined by counting 200 cells per microscopic immersion field (1000x magnification). A cut-off value of \u0026ge;8% PMN was employed for diagnosing subclinical endometritis, as previously outlined for post-partum cows (Madoz et al. 2013).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAntimicrobial analysis \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibiotic sensitivity testing (AST), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays were conducted to assess the antimicrobial efficacy of the ozonized sunflower oil against bacteria isolated from the uterine lumen of the cows previously on D0. A total of four bacteria were isolated: \u003cem\u003eStreptococcus \u003c/em\u003espp., \u003cem\u003eStaphylococcus \u003c/em\u003espp., \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eArcanobacterium pyogenes\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAST involved measuring the diameter of bacterial inhibition zones around antibiotic disks and comparing them with interpretive criteria provided by the Clinical and Laboratory Standards Institute (CLSI 2012). MIC determination followed CLSI guidelines (2012), with adaptations utilizing resazurin as a microbial activity indicator (Sarker et al. 2007).\u003c/p\u003e\n\u003cp\u003eMIC and MBC assays were conducted to confirm the bactericidal or bacteriostatic effects of the treatments against the respective pathogens, according to CLSI guidelines (2012). Similar MIC and MBC results indicated bactericidal action of the respective treatment against the bacteria tested, while disparate results suggested a bacteriostatic effect.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were initially tested for normality using the D\u0026rsquo;Agostino and Pearson normality test. Paired Student\u0026rsquo;s t-test was used to locate differences within and between the experimental groups for the histomorphometry parameters. Chi-square tests of independence were used to examine the differences in frequency data for morphological parameters. A probability of \u003cem\u003eP\u003c/em\u003e\u0026le;0.05 indicated that a difference was significant, and probabilities between \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05 and \u0026le;0.1 indicated that a difference approached significance. Data are presented as mean\u0026plusmn;S.E.M., unless otherwise indicated.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDespite variations in treatment and time, all uterine samples exhibited a simple cylindrical epithelium supported by a network of collagen fibers, with moderate to mild epithelial detachment. The absence of active inflammatory cells as lymphocyte aggregates next to the detached epithelium suggests it was an artefact resulting from an extraneous factor.\u003c/p\u003e\n\u003cp\u003eIn both groups, severe and mild epithelial inflammatory infiltrate were visualized on D0 and D15, respectively. In D0, cows from both groups had similar intensity of granulomas and hemorrhagic spots in the lamina propria. However, only uterine samples from the O\u003csub\u003e3\u003c/sub\u003e-oil group showed a substantial decrease on visualization of the white blood cells clusters and blood extravasation 15 days after treatment. \u003c/p\u003e\n\u003cp\u003eHemorrhage and vascular dilatation were markedly found at D15 only in cows from non-O\u003csub\u003e3\u003c/sub\u003e group. In contrast, a greater accumulation of stroma between the endometrial glands was visualized in the endometrium 15 days after ozonized oil infusion. Histopathological changes associated with endometrial degeneration, such as periglandular fibrosis and endometrial atrophy, were not detected in the uteri from O\u003csub\u003e3\u003c/sub\u003e and non-O\u003csub\u003e3\u003c/sub\u003e groups.\u003c/p\u003e\n\u003cp\u003eA minimal number of endometrial glands visualized before treatment in both groups (5.6\u0026plusmn;0.6 glands; \u003cem\u003eP\u003c/em\u003e\u0026gt;0.1) was followed by a significant structural proliferation 15 days later (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.1). However, this increase in number of endometrial glands at D15 was 3-fold greater (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) in the uterus exposed to ozonized oil when compared to non-O\u003csub\u003e3\u003c/sub\u003e group (32.6\u0026plusmn;2.5 and 11.0\u0026plusmn;1.0 glands, respectively; Table 2).\u003c/p\u003e\n\u003cp\u003eRegardless of the group (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.1), the glandular epithelium height remained unchanged throughout the experiment (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.1). However, the histomorphometry analysis revealed a significant impact of treatment (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) on the dimensions of the endometrial glands (Table 2; Figure 2). While a minor increase (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.1) in glandular luminal area was detected after treating the uterus with ozonized oil, the non-O\u003csub\u003e3\u003c/sub\u003e group exhibited an 8-fold expansion at D15 (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Moreover, an increase (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) in the number of cells per endometrial gland was found only in the non-O\u003csub\u003e3\u003c/sub\u003e group (Table 2).\u003c/p\u003e\n\u003cp\u003eSEM provided a detailed view of the apical surface of epithelial cells in the examined samples. SEM analysis revealed no morphological abnormalities induced by the uterine infusion of ozonized sunflower oil (Figures 3 and 4). Cells covered with microvilli and cilia were identified in all groups and evaluation times. Microvilli-covered cells were predominant, although within the same tissue section, some areas showed nearly all cells with microvilli, while others displayed a cratered apical surface with microvilli lining the junction between cells. About 30% of the observed fields showed a surface with less than 50% microvilli coverage. The presence of ciliated cells varied and was sporadic across different evaluation times. No apical protrusions corresponding to pinopodes were detected. \u003c/p\u003e\n\u003cp\u003eNon-O\u003csub\u003e3\u003c/sub\u003e and O\u003csub\u003e3\u003c/sub\u003e groups had a similar (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.1) and high PMN values at D0 (47.3\u0026plusmn;17.9% and 45.1\u0026plusmn;17.0%, respectively). A significant reduction (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) in PMN percentage was observed 15 days later in both groups (5.9\u0026plusmn;3.3%; \u003cem\u003eP\u003c/em\u003e\u0026gt;0.1). On D15, only one cow from each group exceeded the cut-off value for diagnosing subclinical endometritis (17.5% and 45% for the non-O\u003csub\u003e3\u003c/sub\u003e and O\u003csub\u003e3\u003c/sub\u003e groups, respectively).\u003c/p\u003e\n\u003cp\u003eAST revealed the antimicrobial effectiveness of ozonized sunflower oil against both Gram-positive and Gram-negative bacteria (Table 3). Compared to tetracycline, ozonized sunflower oil exhibited a larger zone of inhibition for bacterial growth. Furthermore, ozonized sunflower oil demonstrated bactericidal activity at concentrations below 1.50 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e, while non-ozonized sunflower oil did not exhibit any antimicrobial properties. Details of the MIC and MBC assays for \u003cem\u003eStreptococcus\u003c/em\u003e spp., \u003cem\u003eStaphylococcus\u003c/em\u003e spp., \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA.\u003c/em\u003e \u003cem\u003epyogenes\u003c/em\u003e are provided (Table 4).\u003c/p\u003e\n\u003cp\u003eNearly all females from the O\u003csub\u003e3\u003c/sub\u003e group (6 out of 7 cows; 85.7%) were pregnant by day 60 after calving, while 42.8% of the cows in the non-O\u003csub\u003e3\u003c/sub\u003e group remained non-pregnant. However, the small sampling per group did not allow statistical analysis of the pregnancy rate in the present study.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study marks the first documentation of the positive effects of ozonized sunflower oil on the uterine involution in postpartum primiparous cows. Additionally, our results confirm the germicidal action of the ozonized oil against both gram-positive and gram-negative bacteria isolated from the uterus of cows. Given the crucial role of a healthy uterus in reproductive success, our \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e findings suggest the benefits of puerperal treatment with ozonized oil in preventing uterine conditions that might lead to long-term infertility.\u003c/p\u003e\u003cp\u003eIn cattle, the presence of epithelial tissue debris and fluid during the two first weeks after calving makes the uterine environment favorable for microbial growth (Walker et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, almost the totality of the cows is affected by uterine contamination at the early puerperium (Sheldon et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In the present study, the ozonized oil showed greater inhibitory sensitivity than the Tetracycline against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e ssp. and \u003cem\u003eE. coli\u003c/em\u003e. The antimicrobial action of the ozonized oil has been well documented against bacteria (Gentili et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ugazio et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), fungi (Celenza et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sehim et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), oomycetes (Carrijo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ferreira et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) and multidrug resistant strains (Grandi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similarly, dual bacteria/fungi and single multidrug resistant biofilms are also susceptible to ozonated oil (Gentili et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Higa et al. 2021; Silva et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDue to its potent antioxidant potential, ozonized oils are capable of degrading bacteria by interacting with the membrane, cytoplasmic and nuclear constituents (Lake et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Khadre et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The peroxidation compromises enzymatic functions and damages the genetic material, disrupting the metabolic and reproductive processes of the pathogen (reviewed by Liu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). When in contact with fungi, the ozonides still disrupts critical cellular functions, inhibiting the production of fungal urease, amylase, alkaline phosphatase, lipase and keratin (Pages et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, ozonated oils have been successfully used for the adjuvant treatment of superficial bacterial and fungal infections (Ouf et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe morphology of the apical surface may have a key role in governing pregnancy establishment. The surface of the luminal epithelium changes during the estrous cycle through a process that involves remodeling of the apical surface (Kumro et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Acute postpartum endometritis is accompanied by ultrastructural changes in cattle. Dystrophic and necrobiotic processes in the parenchyma and endometrial stroma, exudative processes and disruption of microcirculatory bed integrity are common findings (Suleymanov et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Destruction of microvilli, abundance of superficial coccal microflora, necrobiosis of epithelial cells and partial nuclear edema have also been reported in post-partum cows (Suleymanov et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Even so, most of the current therapies for infectious endometritis did not consider their deleterious changes on the structural organization of the uterus.\u003c/p\u003e\u003cp\u003eIn contrast, the local treatment with ozonized sunflower (O\u003csub\u003e3\u003c/sub\u003e group) oil did not induce morphological abnormalities and prevented the occurrence of glandular dilations. O\u003csub\u003e3\u003c/sub\u003e stimulates complex regenerative mechanisms in addition to its direct action on microorganisms. Therefore, intrauterine treatment with O\u003csub\u003e3\u003c/sub\u003e and derivates been used successfully in ruminants for the complementary treatment of endometritis (Đuričić et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), uterine infections following dystocia, retained placenta (Đuričić et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and subfertility (Zobel et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) without causing local side effects.\u003c/p\u003e\u003cp\u003eThe antioxidant potential, immunomodulatory and angiogenic capacity of the O\u003csub\u003e3\u003c/sub\u003e may support the tissue healing process (Liu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It stimulates the fibroblast synthesis (Pchepiorka et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and reduces the fibroblastic genotoxic damage (Akdeniz et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition to a greater secretion of cytokines (Peden \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and the anti-inflammatory modulation via inhibition of NF-kB (Delgado-Roche et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the O\u003csub\u003e3\u003c/sub\u003e intensify the local microcirculation (Ferreira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; Pchepiorka et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Finally, this adjuvant therapy also increases the number of leukocytes and the phagocytic capacity of granulocytes (Kucuksezer et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFifteen days after treatment (25\u0026ndash;30 days post-partum), both groups showed percentage of PMN lower than the cut-off value for the diagnosis of subclinical endometritis in dairy cows (Madoz et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In contrast, the histological changes associated with endometrial inflammation visualized at D15 in control cows (non-O\u003csub\u003e3\u003c/sub\u003e group) did not reflect on the cytological results. Our findings support the low agreement of endometrial biopsy with cytology for the diagnosis of subclinical endometriais (Madoz et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe decrease in leukocyte count in O\u003csub\u003e3\u003c/sub\u003e group was expected since the endometrial PMN values ​​gradually decrease as the puerperium progresses in healthy cows (Santos et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In opposite, reduced pregnancy rate is associated with endometrial PMN abundance in females suffering from uterine inflammatory diseases (Kasimanickam et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sicsic et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The probability for getting normal uterine environment decreased 2% for every increasing percentage point in PMN, while the likelihood of isolating \u003cem\u003eTrueperella pyogenes\u003c/em\u003e increases (Madoz et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe antimicrobial and regenerative potential of the tested ozonized oil support its application as a preventive treatment of puerperal uterine disturbances. The intrauterine infusion of ozonized solution in the postpartum period has been associated with increases in the conception rate and reproductive efficiency of dairy cows (Constantin and B\u0026icirc;rtoiu 2016; Đuričić et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zobel and Tkalcic 2013). In sheep, treatment with O\u003csub\u003e3\u003c/sub\u003e accelerated uterine regression in cases of retained placenta and dystocia (Đuričić et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOzonated vegetable oils are prepared by the direct reaction of O\u003csub\u003e3\u003c/sub\u003e with the carbon-carbon double bonds of their unsaturated fatty acids (Diaz Gomes et al. 2001). In contrast to the unstable O\u003csub\u003e3\u003c/sub\u003e gas (Kogelschatz et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), this ozonation process allows the storage of a O\u003csub\u003e3\u003c/sub\u003e derivate with high doses of ozonides products and prolonged biological action (Boland-Nazar et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the use of ozonized oil treatments is considered a cost-effective with proven efficiency and the absence of serious adverse effects (Serra et al. 2023).\u003c/p\u003e\u003cp\u003eThe main limitation of the present study was the experimental sampling for fertility analysis. Seven cows per group allowed a reliable microscopy analysis of the endometrial characteristics of the uterus subjected to ozonized oil. Still, the sampling does not have the statistical power required to confirm the benefits of the treatment for the fertility rates. However, it is important to highlight that the uterine infusion of ozonized oil did not interfere with the fertilization process in recently calved cows in the present study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings of the present study suggest that infusion of ozonized sunflower oil during the puerperium improved the uterine involution in primiparous cows. Additionally, broad-spectrum germicidal efficacy of the ozonized oil against uterine bacteria supports the its use as an alternative therapeutic approach. Finally, the fertilization through the firsts weeks after calving of cows treated with ozonized oil implies a potential enhancement in fertility. However, further studies with bigger samples of animals are necessary to confirm the impact of uterine infusion of ozonized oil on the reproductive performance of cows.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eOrcid\u003c/h2\u003e\u003cp\u003eJair C. Ferreira \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://orcid.org/0000-0001-6908-3463\u003c/span\u003e\u003cspan address=\"http://orcid.org/0000-0001-6908-3463\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that there are no conflicts of interest that would prejudice the impartiality of this scientific work.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.C.FERREIRA = Project administration.J.C.FERREIRA , Y.L.BOAKARI and M.A.RODRIGUES = Supervision, Visualization, Writing (original draft).J.C.FERREIRA and M.S.OLIVEIRA = Conceptualization, Methodology and Funding acquisition.M.S.OLIVEIRA, C.M.PP.FERREIRA, M.B.ALMEIDA and H.A.RODRIGUES = Data Curation, Analysis, Software analysis and Antimicrobial analysis.All authors reviewd the manuscritp\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e\u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES) \u0026ndash; Finance code 001.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkdeniz SS, Beyler E, Korkmaz Y, Yurtcu E, Ates U, Araz K, Sahin FI, Torun OY (2018). 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Veterinary World 11:1473-1478. https://doi.org./10.14202/vetworld.2018.1473-1478. \u003c/li\u003e\n\u003cli\u003eTravagli V, Zanardi I, Silvietti A, Bocci V (2007) A physicochemical investigation on the effects of ozone on blood. International Journal of Biological Macromolecules 41:504-511. https://doi.org./10.1016/j.ijbiomac.2007.06.010. \u003c/li\u003e\n\u003cli\u003eUgazio E, Tullio V, Binello A, Tagliapietra S, Dosio F (2020) Ozonated oils as antimicrobial systems in topical applications. Their characterization, current applications, and advances in improved delivery techniques. Molecules 25:334. https://doi.org./10.3390/molecules25020334. \u003c/li\u003e\n\u003cli\u003eWalker CG, Meier S, Hussein H, McDougall S, Burke CR, Roche JR, Mitchell MD (2015) Modulation of the immune system during postpartum uterine inflammation. Physiological Genomics 47:89-101. https://doi.org./10.1152/physiolgenomics.00098.2014. \u003c/li\u003e\n\u003cli\u003eXiao W, Tang H, Wu M, Liao Y, Li K, Li L, Xu X (2017) Ozone oil promotes wound healing by increasing the migration of fibroblasts via PI3K/Akt/mTOR signaling pathway. Bioscience Reports 37:BSR20170658. https://doi.org./10.1042/BSR20170658. \u003c/li\u003e\n\u003cli\u003eZobel R, Martinec R, Ivanović DID, Ro\u0026scaron;ić NRN, Stančić ZSC, Žerjavić IZI, Flajsig BFB, Plavec HPH, Smolec O (2014) Intrauterine ozone administration for improving fertility rate intrauterine ozone administration for improving fertility rate in Simmental cattle. Veterinarski Arhiv 84:1\u0026ndash;8. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eHistopathological criteria for analysis of endometrium from postpartum cows. Per high power field at the stated magnification (ppf; \u0026times;10 or \u0026times;40). Adapted from Meira Jr (2012).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"473\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCategory\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eEpithelium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; i. Height\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003ecolumnar\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003ecuboidal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eflattened\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; ii. Epithelial damage\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emild\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; iii. Inflamatory cell type\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emononuclear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003epolymorphonuclear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; iv. Infiltrate intensity\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emild (\u0026le;5 cells/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emoderate (\u0026ge;6-10cells/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003esevere (\u0026gt;10 cells/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eLamina pr\u0026oacute;pria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; i. Inflamatory cell type\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emononuclear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003epolymorphonuclear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; ii. Infiltrate intensity\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003enormal (\u0026le;20 cells/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emild (\u0026le;21-40 cells/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emoderate (\u0026ge;41-70cells/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003esevere (\u0026gt;70 cells/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; iii. Lynphocytic aggregates\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emild (\u0026le;3 aggregates/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emoderate (\u0026ge;4-5 aggregates/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003esevere (\u0026gt;6 aggregates/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eEndometrial gland\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; i. Atrophy or dilatation\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003epresent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; ii. Fibrosis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emild (1-3 layers/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003emoderate (4-5 layers/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003esevere (\u0026gt;6 layers/hpf; x40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eVascular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; i. Vessel degeneration\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003epresent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; ii. Hemorrhage\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003epresent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; iii. Hemosiderin macrophages\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eabsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003epresent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eEndometrial histomorphometry exam from puerperal primiparous cows receiving uterine infusion of ozonized or non-ozonized sunflower oil (O\u003csub\u003e3\u003c/sub\u003e and non-O\u003csub\u003e3\u003c/sub\u003e groups; n=9 cows/group). Moment immediately before and 15 days after treatment were considered D0 and D15, respectively. \u003csup\u003ea, b, c\u0026nbsp;\u003c/sup\u003eDifferent letters within a parameter are different (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"643\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 220px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eO\u003csub\u003e3\u003c/sub\u003e group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cstrong\u003enon-O\u003csub\u003e3\u003c/sub\u003e group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003eNumber of glands\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;5.1\u0026plusmn;0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e32.6\u0026plusmn;2.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;6.1\u0026plusmn;2.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e11.0\u0026plusmn;1.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003eNumber of cells per gland\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e33.2\u0026plusmn;1.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e34.1\u0026plusmn;2.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e26.7\u0026plusmn;1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e57.7\u0026plusmn;2.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003eEpithelial height (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e67.3\u0026plusmn;6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e57.9\u0026plusmn;5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e44.1\u0026plusmn;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e54.3\u0026plusmn;3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003eGland height (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e33.2\u0026plusmn;1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e34.1\u0026plusmn;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e33.2\u0026plusmn;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e30.9\u0026plusmn;2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003eGland diameter (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e111.0\u0026plusmn;10.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e111.9\u0026plusmn;10.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e108.3\u0026plusmn;5.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e2013.5\u0026plusmn;20.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003eGland luminal area (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e1,750\u0026plusmn;303\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e2,376\u0026plusmn;830\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e1,713\u0026plusmn;300\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u0026nbsp; 15,008\u0026plusmn;1,757\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eAntibiotic sensitivity testing (AST) based on the diameter of inhibition zone of ozonized and non-ozonized sunflower oils (O\u003csub\u003e3\u003c/sub\u003e and non-O\u003csub\u003e3\u003c/sub\u003e oils, respectively) against bovine uterine bacteria. Tetracyclin and untreated were considered controls groups. Sensibility and resistance of the bacteria to the treatment are indicated, respectively, by the letter \u0026ldquo;S\u0026rdquo; and \u0026ldquo;R\u0026rdquo;.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"636\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 454px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiameter of zone inhibition (mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eO\u003csub\u003e3\u003c/sub\u003e oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003enon-O\u003csub\u003e3\u003c/sub\u003e oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUntreated\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTetracycline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp;33.5\u0026plusmn;3.5\u003csup\u003ea\u003c/sup\u003e (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e16.7\u0026plusmn;1.8\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus\u003c/em\u003e ssp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e41.3\u0026plusmn;4.9\u003csup\u003ea\u003c/sup\u003e (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e24.3\u0026plusmn;2.3\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus\u003c/em\u003e ssp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e38.0\u0026plusmn;1.2\u003csup\u003ea\u003c/sup\u003e (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e14.3\u0026plusmn;2.6\u003csup\u003eb\u003c/sup\u003e (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cem\u003eArcanobacterium pyogenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e33.2\u0026plusmn;1.3\u003csup\u003ea\u003c/sup\u003e (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u0026plusmn;0.0\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e23.7\u0026plusmn;2.2\u003csup\u003eb\u003c/sup\u003e (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea, b, c\u003c/sup\u003e different letters on the same line are different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003eMinimum inhibitory concentration and minimum bactericidal concentration (MIC and MBC; \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e) of ozonized and non-ozonized and sunflower oils (O\u003csub\u003e3\u003c/sub\u003e and non-O\u003csub\u003e3\u003c/sub\u003e oils) against bovine uterine bacteria (\u003cem\u003eEscherichia coli\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eStaphylococcus\u0026nbsp;\u003c/em\u003essp.,\u003cem\u003e\u0026nbsp;Streptococcus\u003c/em\u003e ssp\u003cem\u003e.\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eArcanobacterium pyogenes\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"594\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eO\u003csub\u003e3\u003c/sub\u003e oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003enon-O\u003csub\u003e3\u003c/sub\u003e oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003euntreated\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTetracycline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus\u003c/em\u003e ssp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus\u003c/em\u003e ssp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cem\u003eA. pyogenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026gt;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cattle, endometrial gland, integrative medicine, ozone, puerperium, uterus","lastPublishedDoi":"10.21203/rs.3.rs-7520703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7520703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe goals of the present study were to characterize the uterine response to the infusion of ozonized sunflower oil in first-calf heifers and to determine the \u003cem\u003ein vitro\u003c/em\u003e antimicrobial action of the therapy. A total of 18 cows were submitted to a single treatment with ozonized or non-ozonized oil (O\u003csub\u003e3\u003c/sub\u003e and non-O\u003csub\u003e3\u003c/sub\u003e groups, respectively; n=9 cows/group) between days 10 and 12 after calving.\u0026nbsp; Scanning electron and light microscopy were performed immediately before and 15 days after treatment (D0 and D15, respectively) to assess morphometric and morphological parameters of the endometrium. Additionally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the treatments were done against uterine bacteria (\u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e ssp., \u003cem\u003eStreptococcus\u003c/em\u003e ssp. and \u003cem\u003eArcanobacterium pyogenes\u003c/em\u003e) isolated at D10. On D15, the number of endometrial glands was greater (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) in the O\u003csub\u003e3\u003c/sub\u003e group (32.6±2.5) than in non-O\u003csub\u003e3\u003c/sub\u003e group (11.0±1.0). Endometrial glands from O\u003csub\u003e3\u003c/sub\u003e group had reduced (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) number of cells (34.1±2.2), diameter (111.9±10.8 µm) and luminal area (2376±830 µm\u003csup\u003e2\u003c/sup\u003e) after treatment, when compared to the non-O\u003csub\u003e3\u003c/sub\u003e group (57.7±2.9, 2013.5±20.9 µm and 15008±1757 µm\u003csup\u003e2\u003c/sup\u003e, respectively). Scanning electron microscopy indicated the absence of surface structure abnormalities in the endometrium of the O\u003csub\u003e3\u003c/sub\u003e group. Ozonized sunflower oil inactivated (MIC and MBC ≤0.18 µg mL\u003csup\u003e-1\u003c/sup\u003e) all isolated uterine bacteria. In conclusion, the infusion of ozonized oil at high peroxide index value enhanced the endometrial gland proliferation and the uterine involution through the first weeks after calving in cows. In addition, the ozonized oil had a broad-spectrum germicidal action against uterine bacteria.\u003c/p\u003e","manuscriptTitle":"Scanning Electron and Light Microscopic Characterization of the Endometrium Exposed to Ozonated Sunflower Oil at High Peroxide Index Value in Postpartum Primiparous Cows.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 13:36:28","doi":"10.21203/rs.3.rs-7520703/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"402abb09-8112-452c-a972-925b55c90f3a","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-01T21:53:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-22 13:36:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7520703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7520703","identity":"rs-7520703","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}