The curative effect of ozonated bidistilled water on Escherichia coli-induced endometritis in rats

ARA: designed the study, wrote the original draft, conducted the formal analysis, reviewed and edited the paper, and performed laparotomies, inoculations, and treatments. MK and GB: performed laparotomies, inoculations, and treatments and reviewed the original draft. ÖM: performed histopathological analyses. DÖ: prepared E. coli inoculum and performed bacteriological analyses.

All procedures involving the experimental animals were conducted in compliance with the guidelines of the Local Ethics Committee on Animal Experimentation of Burdur Mehmet Akif Ersoy University (approval no: 2015‐4).

No financial support was received for this study.

General anaesthesia or induced endometritis did not cause death in rats, while the inoculation process exhibited no observed complications. During the laparotomy performed 48 h after the intrauterine treatment similar changes were macroscopically observed in G1 and G2 inoculated uteruses as slight asymmetry between uterine horns and partial discoloration on serosal layers. In the samples taken for microbiological evaluation 48 h after intrauterine treatment, it was determined that the character of the fluids taken from the uterus was different from each other. In G3, all rats had an increase in volume and pus accumulation in the uterine horns, while in G2, all rats had more volume increase and fluid accumulation compared to G1. Additionally, in G1, mild asymmetry was observed in 5 rats, while in one rat, there was a general increase in volume and pus accumulation (Figure  2 ). Asymmetry in uterine horn after 48 h bacterial inoculation. Histopathological examinations. (a1) Marked inflammatory reaction (arrows) in uterus and necrotic debris at the lumen (arrow head), HE, Bar = 500 μm. (a2) Higher magnification of A1, inflammatory cell infiltrations (black arrows), completely ulcerative endometrial layer (red arrow), HE, Bar = 100 μm. (b1) Markedly decreased inflammatory reaction in uterus from G2 group, slight cellular debris in uterin lumen, (arrow head), HE, Bar = 500 μm. (b2) Higher magnification of B1, amelioration at the endometrial epithelial layer (blue arrow) and slight cellular debris in lumen of the uterus (blue arrow head), HE, Bar = 100 μm. (c1) Totally ameliorated uterin layers and completely empty uterin lumen (blue arrow head), HE, Bar = 500 μm. (c2) Higher magnification of c1, complete epithelisation at endometrial epithelial layer (blue arrow), HE, Bar = 100 μm. According to the microbiological cultures, E. coli was detected in the uterine tissues of all rats. An experienced pathologist who was uninformed about the groups evaluated the stained sections two times. The severity of metritis was documented based on hyperaemia, leukocyte infiltration, and necrosis. Histopathological examination revealed severe inflammation and necrosis were observed in G3. In this group, all uterine lumens were filled with pus, and a significant portion of the endometrial layer was lost. In G1 and G2, epithelial healing was observed to have started (Figure 3 ). In the semiquantitative analyses, significantly lower histopathological scores were observed for both the bidistilled water enriched with ozone and cephapirin treated endometritis groups compared to the control group ( p < 0.05) (Figure  4 ). After the intrauterine treatment, a laparotomy was performed 48 h later and, examined the asymmetry of the affected areas. The results showed that G3 had the most significant asymmetry, followed by G2 with mild asymmetry, and G1 had the least asymmetry when viewed macroscopically (Table  1 ). The histopathologic score of the groups. Different letters show a significant difference among groups ( p < 0.05). Condition of uterine horns after 48 h intrauterine treatment. ‐: no asymmetry; +: mild asymmetry; ++: moderate asymmetry; +++: severe asymmetry.

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.1223 .

Total of 21 female Wistar Albino rats with 12 months (Burdur Animal Production and Research Laboratory) were used for this study. The animals were housed in a room with controlled environmental conditions of 25 ± 2°C and a 12‐h light/12‐h dark cycle. They had access to standard nonmedicated laboratory rodent chow and water ad libitum. All procedures involving the experimental animals were conducted in compliance with the guidelines of the Local Ethics Committee on Animal Experimentation of Burdur Mehmet Akif Ersoy University (approval no: 2015‐4). For creating an experimental endometritis model, Escherichia coli ( E. coli ) was used. The E. coli strain inoculated into the uterus was obtained from samples collected from the cervix of a cow diagnosed with endometritis. The samples were collected using a sterile swab and E. coli was isolated from the swab sample at the Department of Microbiology, Burd University. Following a previous study that had created an experimental endometritis model in rats, the E. coli strain was diluted to a concentration of 1.0 × 10 9 cfu/mL to prepare the inoculum (Xu et al., 2015 ). The endometritis model was applied to all groups. General anaesthesia was induced using isoflurane for all rats. A median incision was selected as the laparotomy site. Cutaneous, subcutaneous and muscle layers were incised via a 5 mm midline vertical incision by the scalpel blade and, abdominal cavity opened then the uterus has been found. Using a laparotomic approach, 0.3 mL of inoculum containing E. coli at a concentration of 1.0 × 10 9 cfu/mL was injected into the right horn of the uterus using a syringe (Xu et al., 2015 ) (Figure  1a ). The horn of the uterus, where the inoculation was performed, was ligated using a 4/0 absorbable suture near the bifurcation uteri to prevent the leakage of the inoculum (Figure  1b ). The incision site was closed with simple separate sutures. Inoculation process of E. coli into uterus. ( a ) Inoculation of E. coli into uterine lumen and ( b ) ligation of uterine horn. Forty‐eight hours after inoculation, the second laparotomy operation was performed under general anaesthesia to evaluate the asymmetry and volume increase of the uterine horns. It was confirmed macroscopically that experimental endometritis was induced in all individuals. The rats were randomly divided into three groups. In the uterine horns where inoculation was performed; in group 1 (G1, n = 7), 0.3 mL bidistilled water enriched with ozone at a concentration of 5–80 μg/mL using the Vetozon COMPACT device (Herrmann Apparatebau GmbH), 0.3 mL antibiotic solution (11.2 mg cephapirin, Metricure®) was applied in group 2 (G2, n = 7), and 0.3 mL 0.9% NaCl was applied in group 3 (G3, n = 7). All rats in the groups were sacrificed in deep anaesthesia 48 h after intrauterine treatment for macroscopic, microbiological and histopathological examinations The two horns of the uterus with and without inoculation were examined for symmetry. If there was no asymmetry between the two horns, it was considered as no asymmetry (‐), if the inoculated horn had a growth of less than half the volume of the healthy horn, it was considered as mild asymmetry (+), if the inoculated horn had a growth of half or more of the volume of the healthy horn, it was considered as moderate asymmetry (++), if the inoculated horn had a growth of at least the volume of the healthy horn, it was considered as severe asymmetry (+++). For this purpose, sterile swabs were taken from the uterine lumen of each individual. The samples were evaluated for colony formation on MacConkey agar under aerobic conditions at 37°C. The uterine tissues were fixed in 10% formalin solution for 2 days and then embedded in paraffin wax to prepare paraffin blocks. Sections (5 μm) were cut from the blocks and stained with haematoxylin‐eosin (H&E) solutions. The sections were then examined under a microscope to observe any histopathological changes. Endometrial degeneration, necrotic debris in uterine lumen and inflammatory cell infiltration were assessed for scoring criteria. Each criterion was scored semi‐quantitatively as follows: 0 = none, 1 = mild and 2 = severe, with the maximum total score being 6. The scoring system was designed according to the recommendations of Schafer et al. ( 2018 ). Statistical analysis was performed using the MINITAB 16.0 (MINITAB 16.0 for windows; Minitab Ltd). After confirming the normal distribution of the data using the Kolmogorov–Smirnov test, the one‐way analysis of variance (ANOVA) test was applied, with the data being presented as mean ± standard deviation (SD) and a p < 0.05 indicating a significant difference.

Studies conducted with experimentally induced inflammation are crucial in the prevention of some diseases or disorders and, the development of new treatments. One of the most commonly used animal models for this purpose is rats. In this study, the application of routine antibiotic therapy and ozone‐enriched bidistilled water in the treatment of endometritis was compared. The usability of ozone therapy has been determined in many other diseases such as proctitis (Gültekin et al., 2013 ), peritonitis (de Souza et al., 2010 ) and pyelonephritis (Caliskan et al., 2011 ) using animal models. In this study, there was no difference microbiologically between the results of the application of ozone‐enriched bidistilled water and antibiotics in the treatment of experimental endometritis, it was found that the histological healing was at a better level in G1 and G2 compared to G3. The histopathological examination of the uterus in cows with acute endometritis reveals degenerative areas in the lamina epithelialis, neutrophil leukocyte infiltration, oedema, and hyperaemia in the lamina propria (Doğruer & Güler, 2010 ). Suleymanov et al. ( 2018 ) have identified oedema and exudative inflammatory sites in the endometrial glands. In some endometrial sites, they noted that the epithelial layer of the uterine glands cannot maintain its integrity, microvilli are destroyed, and necrosis occurs in the epithelial layer. Similarly, in studies conducted on pigs and rats, after intrauterine infusion of E. coli , the uterine horn was significantly enlarged with grey‐white mucosal exudate accumulation containing dense pus and thickening of the uterus wall and blood vessels, as well as redness in the endometrium (Demirel et al., 2019 ; Jana et al., 2004 ). In the presented study, similar to the endometritis seen in cows and pigs, after inoculation of E. coli in rats, serious pathological findings such as pus or serous fluid accumulation and asymmetry in the uterine horns were observed at 96 h in G3. Histopathological examination revealed that all uterine lumens were filled with pus, a significant portion of the endometrial layer was lost, and severe inflammation and necrosis were present in the group without intrauterine treatment (G3). Thus, the successful induction of endometritis was supported both macroscopically and microscopically. There is currently no ‘gold standard’ method for diagnosing uterine diseases. This makes it challenging to evaluate clinical findings and histological observations (Sheldon et al., 2006 ). However, evaluating the character and odour of vaginal mucus reflects the bacterial count and acute‐phase protein response in the uterus (Williams et al., 2005 ). Therefore, there is a relationship between vaginal discharge characteristics, bacterial count and inflammation. In the presented study, the presence of pus accumulation and the isolation of E. coli in all exudates from the uterine horns in the control group (G3) support this view. Dar et al. ( 2016 ) evaluated the pathogenic potential of E. coli isolated from postpartum buffaloes to induce endometritis in 30 Swiss Albino mice in the diestrus phase of their oestrous cycle. They inoculated the mice intravaginally with 1.5 × 10 4 , 10 5 and 10 6 cfu/mL E. coli to induce acute endometritis, which was observed in 0%, 50% and 66.7% of the mice, respectively. In another study conducted in rats, it was revealed that E. coli inoculation into the uterine lumen at 10 5 cfu/rat caused endometritis (Demirel et al., 2019 ). In the present study, after intrauterine inoculation of 1 × 10 9 cfu/mL E. coli , asymmetry and increased volume were observed in all groups after 48 h. After intrauterine treatment (96 h after inoculation), endometritis occurred in the G3. Additionally, the present study showed that ligating the uterine horn at a location close to the bifurcation with an absorbable suture prevented leakage and dosing errors during inoculation. Antimicrobial resistance has become a growing global health problem in recent years. Resistant bacteria found in dairy cows make it more difficult to treat them, and as a result, the risk of bacterial contamination increases, leading to serious problems. Failures in antibiotic treatment can lead to more complications, higher veterinary and drug expenses, prolonged illnesses, or deaths. Therefore, studies investigating the effectiveness of alternative treatment methods in animal models are rapidly increasing. The presented study concluded that while both antibiotic treatment (G2) and ozone (G1) produced similar microbiological and histopathological results. Another advantage of using ozone is that it does not create resistance mechanisms in animal‐derived food products, and there is no residue period for milk. In a study conducted by Kasimanickam et al. (2005), the efficacy of cloprostenol and cephapirin (intrauterine) in subclinical endometritis in cows was investigated, and both treatment methods reduced open days compared to the control group and increased conception rates. However, this diagnosis was not supported microbiologically. In the presented study, cephapirin (Metricure®) and ozonated bidistilled water were found to provide similar improvement in rats. On the other hand, Ahmadi et al. ( 2005 ) observed in their study that the effectiveness of the drug containing the active ingredient cephapirin (Metri‐Care, Afarin Daroo) was lower than that of other antibiotics used. This was predicted to be due to the inadequacy of the 10 mL syringes containing the drug in covering the entire volume of the uterus. In the presented study, it is thought that intrauterine cephapirin and ozonated distilled water can be administered in sufficient amounts even in smaller uterus volumes. Commercially, ozone is available in different forms. Djuricic et al. ( 2012 ) applied different ozone preparations in intrauterine form as a protective measure in dairy cows during the physiological puerperium period, such as ovules (Ripromed ovuli O3, Novagen) and foam (Riger spray, Novagen), and investigated their reproductive efficiency. It is believed that the form and the amount of active substance of the ozone preparation are decisive. They concluded that the intrauterine application of ozone for protective purposes during the puerperal period, especially in the form of foam spray, increases reproductive efficiency in dairy cows. Bocci ( 2007 ) suggested that ozone doses within a defined range (10–80 μg/mL) can stimulate the organism's antioxidant capacity. In the presented study, bidistilled water enriched with ozone at a density of 5–80 μg/mL was applied in a volume that covered the entire uterus. Additionally, contact of the ozone‐enriched water with infected tissue was provided by gentle palpation. Bialoszewski et al. ( 2011 ) investigated the bactericidal activity of ozonated water and ozone‐oxygen mixture against biofilms. They indicated that ozonated water was an effective bactericidal agent against biofilms after 30 s of exposure, but the exposure time to the gaseous disinfectant had to be extended up to 40 min. In the presented study, ozone‐enriched bidistilled water was used for histological regeneration and effective immunomodulatory effect, taking into consideration the effect time mentioned by Bialoszewski et al. ( 2011 ). The effect of commercial ozone preparations or enriched ozone obtained from ozone generators on different animal species and different reproductive disorders has been evaluated. The effectiveness of the treatment is generally based on clinical and reproductive parameters, as demonstrated by Drucic et al. (2014), Durrani et al. (2017) and Zobel et al. ( 2012 , 2013 , 2014 ). Durrani et al. (2017) compared the efficacy of gentamicin sulphate and a commercial ozone (Sanafoam®) preparation against different bacterial agents in repeat breeder cows with abnormal uterine discharge. They found that ozone therapy was more effective than antibiotic therapy against E. coli isolated from cows. Similarly, Đuričić et al. ( 2014 ) investigated the effectiveness of intrauterine ozone therapy (Riger spray G; Novagen®) and foam tablets containing oxytetracycline (Geomisin F®) on sheep with retention of placenta. They found that ozone therapy was more effective than the control group in both normal calving and difficulty. In addition, ozone therapy reduced the open days, number of inseminations and culling rate in cows with retention or urovagina problems during the postpartum period (Zobel et al., 2012 , 2013 , 2014 ). However, none of these studies included data on uterus histopathology. Chethan et al. ( 2015 ) evaluated the histopathology of uterus pathologies such as cytological and purulent endometritis, hydrometra and mucometra in buffaloes using slaughterhouse materials. Although some researchers have used live animal biopsy materials, there is no histopathological study of ozone therapy in any animal model. Therefore, the presented study is the first to demonstrate histopathological improvement after ozone therapy in a rat model of E. coli ‐induced endometritis. The presented study utilised both antibiotic and ozone‐enriched bidistilled water as treatment for the experimentally induced endometritis model. Results showed that both treatments had similar effects on a histopathological and microbiologically. As a result, it has been observed that ozone‐enriched bidistilled water can be used in the treatment of experimentally induced endometritis in rats, and can provide effective microbiological and histopathological improvement. Further studies involving larger populations of different species are needed to determine the medical suitability of this new treatment.

Endometritis is a condition that occurs in cows when there is inflammation in the lining of the uterus. It is a common cause of infertility in cows (Sheldon et al., 2008 ). Endometritis in cows can be caused by a variety of factors, including bacterial (Sheldon et al., 2006 ) and viral infections (Donofrio et al., 2007 ). The inflammatory response can interfere with the implantation of the embryo (Semambo et al., 1991 ). And also, cows with endometritis experience several reproductive issues, including slower follicle growth, impaired ovulation and delayed and irregular ovarian cyclicity (Sheldon et al., 2002 ). Consequently, endometritis can have a significant impact on the reproductive performance of cows negatively and can lead to infertility (Sheldon et al., 2006 ). It is essential for farmers and veterinarians to be aware of the condition and to take appropriate measures to prevent and treat it in order to maximise fertility and reproductive success in their herds (Tzelos et al., 2020 ). Antibiotics are commonly used in the treatment of endometritis in cows. And, penicillin, third‐generation cephalosporins, oxytetracycline and gentamicin as the most commonly used antibiotics in the treatment of endometritis (Nak et al., 2011 ). These antibiotics are administered via intrauterine infusion. However, use of antibiotics in veterinary medicine may result in the future development of antibiotic resistance, which could make the treatment of bacterial infections more challenging (Haimerl & Heuwieser, 2014 ). Antibiotic resistance occurs when bacteria evolve to become resistant to the drugs used to treat them, usually as a result of overuse or misuse of antibiotics (Tenover, 2006 ). In addition to the problem of antibiotic resistance, the use of antibiotics in food‐producing animals can also lead to the presence of antibiotic residues in milk. These residues can pose a risk to human health if consumed in high enough quantities and can also lead to trade barriers and other economic consequences (Virto et al., 2022 ). Therefore, alternatives to antibiotics, such as probiotics, immunomodulators or other substances are also being investigated for their potential to treat endometritis in cows while minimising the use of antibiotics (Rosales & Ametaj, 2021 ). Ozone therapy has been considered as an alternative to antibiotic treatment due to its potential to avoid the issues of antibiotic residue and antibiotic resistance. Ozone is an allotrope of oxygen. It is known to possess antimicrobial and anti‐inflammatory properties, making it a potential therapeutic agent for treating endometritis (Polat et al., 2015 ). In recent years, several studies have investigated the use of ozone in the treatment of endometritis in cows (Escandón et al., 2020 ; Polat et al., 2015 ; Zobel et al., 2014 ). Ozone therapy has been shown to possess antimicrobial properties against a wide range of bacteria, viruses, and fungi (Varga & Szigeti 2016 ), making it a potential alternative to antibiotics in the treatment of endometritis. Additionally, ozone therapy has been shown to be effective against antibiotic‐resistant bacteria, which is a significant advantage over conventional antibiotic treatment (Rowen, 2019 ). Therefore, ozone therapy does not pose a risk of antibiotic residue in food products or contribute to the development of antibiotic resistance. The aim of this study was to examine the efficacy of ozone‐enriched distilled water, which has not been used previously for therapeutic purposes, in the treatment of experimentally induced endometritis in rats. The study will also explore how ozone therapy can reduce inflammation and promote healing in the endometrium. Additionally, the study's findings suggest that ozone therapy may play a role in histological recovery in cows, making this study a preliminary investigation into this hypothesis.

The authors have no conflict of interest to declare.