Animal Reproduction Science 159 (2015) 1–7

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Review article

Prophylaxis and therapeutic potential of ozone in buiatrics: Current knowledge Draˇzen Ðuriˇcic´ a , Hrvoje Valpotic´ b , Marko Samardˇzija b,∗ a b

– Veterinary practice Ðurdevac d.o.o., Croatia Faculty of Veterinary Medicine University of Zagreb, Croatia

a r t i c l e

i n f o

Article history: Received 5 March 2015 Received in revised form 6 May 2015 Accepted 29 May 2015 Available online 3 June 2015 Keywords: Ozone Prophylaxis Therapy Veterinary medicine Buiatrics

a b s t r a c t Ozone therapy has been in use since 1896 in the USA. As a highly reactive molecule, ozone may inactivate bacteria, viruses, fungi, yeasts and protozoans, stimulate the oxygen metabolism of tissue, treat diseases, activate the immune system, and exhibit strong analgesic activity. More recently, ozone has been used in veterinary medicine, particularly in buiatrics, but still insufficiently. Medical ozone therapy has shown effectiveness as an alternative to the use of antibiotics, which are restricted to clinical use and have been withdrawn from non-clinical use as in-feed growth promoters in animal production. This review is an overview of current knowledge regarding the preventive and therapeutic effects of ozone in ruminants for the treatment of puerperal diseases and improvement in their fertility. In particular, ozone preparations have been tested in the treatment of reproductive tract lesions, urovagina and pneumomovagina, metritis, endometritis, fetal membrane retention and mastitis, as well as in the functional restoration of endometrium in dairy cows and goats. In addition, the preventive use of the intrauterine application of ozone has been assessed in order to evaluate its effectiveness in improving reproductive efficiency in dairy cows. No adverse effects were observed in cows and goats treated with ozone preparations. Moreover, there is a lot of evidence indicating the advantages of ozone preparation therapy in comparison to the application of antibiotics. However, there are certain limitations on ozone use in veterinary medicine and buiatrics, such as inactivity against intracellular microbes and selective activity against the same bacterial species, as well as the induction of tissue inflammation through inappropriate application of the preparation. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Ozone use in veterinary medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 The use of ozone in buiatrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Treatment of reproductive tract lesions, urovagina and pneumovagina with ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Preventive intrauterine use of ozone for the improvement of reproductive efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Treatment of metritis and endometritis with ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Functional restoration of the endometrium through the intrauterine application of ozone pailletes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

∗ Corresponding author. Tel.: +385 1 2390 2321; Fax: +385 1 244 1390. E-mail address: [email protected] (M. Samardˇzija). http://dx.doi.org/10.1016/j.anireprosci.2015.05.017 0378-4320/© 2015 Elsevier B.V. All rights reserved.

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8. Treatment of fetal retained membrane with ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 9. Treatment of mastitis with ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 10. Perspectives and limitations of ozone use in veterinary medicine and buiatrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1. Introduction Ozone is a light blue gas of extraordinary odor which can be strongly sensed in the air during electrical discharges into the atmosphere (following thunder and lightning). Christian Friedrich Schönbein was the first to recognize ozone as a chemical substance in 1840, and named it after ˝ the Greek word o␨␧␫␯ (ozein), which means odor (Rubin, 2001). The chemical formula of ozone, O3 , was established by Jacques-Louis Soret (Soret, 1865). He proposed that this molecule, composed of 3 oxygen atoms, is an allotropic modification of oxygen. Ozone is a very strong oxidant, much stronger than O2 , and is unstable in higher concentrations, resulting in its decomposing into oxygen molecules. It is essential for life on Earth due to its ability to absorb wavelengths of harmful UV radiation coming from specters of sunlight (Di Paolo et al., 2004). Nikola Tesla, the brilliant inventor who originated from Croatia, patented the first ozone generator in 1896 in the USA (Tesla, 1896). Four years later, he founded the first firm, the “Tesla Ozone Company”, to use ozone for medical purposes (Mandhare et al., 2012). In 1898, Thauerkauf and Luth founded the Institute for Ozone Therapy in Berlin in Germany, where for the first time ozone injections were applied to an animal. In addition, for the first time ozone molecules were synthesized into a solid compound termed haemozon (today known as homozon). Afterwards, there followed a century of ozone use and improvements in its use for medical purposes. Ozone therapy is classified as an alternative medicine approach. Its therapeutic effect is recognized as a consequence of increased oxygen concentration on or in the body following the application of ozone in various preparation forms and by different routes. Today, ozone is used extensively in industry for sterilization and bleaching, the purification of water and swimming pools, and for the sterilization of operating rooms (Glaze, 1987; Rakness et al., 1993). In addition, its use in medical therapy has been found to be predominantly safe, with extremely rare side effects and negative effects on human health. Recently, the use of ozone has expanded, due to the fact that it is applied for medical purposes, i.e. for the oxygenation of tissue, as well as cosmetic purposes. There are also numerous diseases which can be successfully treated with bio-oxidative therapy with ozone, such as cardiovascular diseases and circulatory problems (Hernández et al., 1995; Martínez-Sánchez et al., 2012), arthritis (treated with intra-articular therapy, nucleolysis of intravertebral discs, paravertebral infiltration with ozone, periarticular infiltration with ozone) (Iliakis, 1995; Andreula, 2001; Ginanneschi et al., 2006), viral infections (such as hepatitis, Epstein-Barr virus, herpes, etc.), fungal infections, tissue regeneration (wounds and burns), dermatological diseases, and oral cavities and tooth diseases

(Loncar et al., 2009; Bhateja, 2012). It is also used in complementary carcinoma therapy (Bocci, 2006; Elvis and Ekta, 2011). As a highly reactive molecule, ozone exhibits strong bactericidal, fungicidal, antivirus, anti-yeast and anti-protozoa activities. One of the major activities of medical ozone is the specific induction of the synthesis of cell membrane enzymes, such as superoxide dismutase, catalase and glutathione peroxidase, which protect the cell from the damaging effects of O2 free radicals (Hernández et al., 1995; Mandhare et al., 2012). Sensitivity to ozone is higher in Gram positive bacteria than in Gram negative bacteria. It has been recognized that oxidative processes induced by ozone may damage the capsule of bacteria through the activity of peroxidases and subsequently the cell membrane, and may also block replication of bacterial DNA. The oxidative mechanisms of ozone inhibition of fungal growth and its virucidal activity are performed through the inactivation of viruses following the destruction of lipid molecules in capsids and by blocking their replication. “Naked viruses” can be neutralized by the ability of ozone to produce protein hydroxides and hyperoxides. In addition, ozone may influence the immune system through the stimulation of lymphocytes, monocytes and neutrophils to release cytokines such as interferons ␣, ␤, ␥, tumor necrosis factor ␣, interleukins 1, 2, 4, 6, 8 and 10, granulopoietine and transforming growth factor ␤ (Ducusin et al., 2003; Larini and Bocci, 2005; Ohtsuka et al., 2006). These cytokines may re-establish cellular immunity during inflammatory processes (Travagli et al., 2009). Moreover, ozone may stimulate the proliferation of B and T lymphocytes (Korzun et al., 2008). 2. Ozone use in veterinary medicine There are some products with a special blend of vegetal ozoned oil with a lenitive, emollient, cicatrizing, antibacterial and hygienist action in the form of creams, gasses, syringes, pailletes, foam, boluses and obletes (Travagli et al., 2009). In addition, there are certain products (BIO OZOTECHTM ) patented according to the regulations of the European Conformity Medical Device (CEMD) and registered by the Italian Health Ministry in accordance with EU Directive 93/42/CEE. Although the use of ozone in veterinary medicine can be traced back more than 30 years (Altman, 2007), it is still insufficient and related only to certain specific areas, such as the treatment of mastitis, metritis, endometritis, fetal membrane retention, vaginitis, urovagina, enteritis and laminitis, as well as in the local treatment of various lesions and neuromuscular disorders, and in the intravenous application of ozone diluted in saline for flushing equine intestines (Marusi et al., 1999; Alves et al., 2004; Zobel et al., 2012; Ðuriˇcic´ et al., 2014; Zobel et al., 2014a;

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Djuricic et al., 2015). Ozone also acts as a strong analgesic when applied post-operatively following ovariohysterectomy in bitches (Teixeira et al., 2013). 3. The use of ozone in buiatrics The reproductive efficiency of cows is related to genetic/paragenetic factors, environmental factors, management and herd health status (Gröhn and Rajala-Schultz, 2000). This efficiency is correlated to the monitoring of the following parameters: days open until first service, days open until pregnancy, the intercalving period, the conception rate and the non-return rate (Prandi et al., 1999; Koˇcila et al., 2009). However, among these parameters the most important for successful cattle production are shortening of the days open until pregnancy and the intercalving period. The conception rate is a reliable parameter of fertility, and represents the ratio of the number of all artificial inseminations applied and the number of all pregnant cows (Samardˇzija et al., 2008). Economic losses in cattle production are related to diseases which significantly decrease reproductive efficiency during the transition and late puerperium periods. These diseases include hypocalcemia, fetal membrane retention, metritis, endometritis, metabolic disorders and hoof diseases (Maizon et al., 2004; Gautam et al., 2009). More recently, the use of ozone as a preventive and therapeutic agent in the treatment of ruminant puerperal diseases and for the improvement of reproductive efficiency has been considered (Djuricic et al., 2011a, 2012a,b; Ðuriˇcic´ et al., 2014; Zobel et al., 2014b; Djuricic et al., 2015). 4. Treatment of reproductive tract lesions, urovagina and pneumovagina with ozone Reproductive tract lesions, such as injuries of the vulva, vagina or urogenital tract may be observed following dystocia. These lesions can be treated with ozone spray supplied with a catheter, which has to be pushed into the vagina in order to apply the preparation for 3–5 s. Such treatment produces a foam which fills the vagina and vestibule of the vagina and stimulates cicatrization and the regeneration of injuries through the proliferation of granular tissue. Intravaginal treatment with ozone spray may also shorten the days open until pregnancy and improve reproductive efficiency in cows due to the fact that urovagina and pneumovagina can be a reason for prolonged days open until pregnancy, repeat breed syndrome and infertility (Zobel et al., 2012). 5. Preventive intrauterine use of ozone for the improvement of reproductive efficiency Preventive intrauterine ozone application had different effects on reproductive performance in dairy cows. The cows treated with either ozone foaming spray or pearls 24–48 h after calving in comparison to the non-treated control cows: became pregnant earlier (110.5 days or 105 days vs. 112 days, respectively), had better first service conception rate (FSCR; 20.4% or 27.8% vs. 14.6%, respectively) and all service conception rate (ASCR; 39.2% or 44.6% vs. 36.2%, respectively).

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It is assumed that this improvement can be ascribed to the ozone preparation in foaming spray and the amount of active substance in it. In addition, ozone pearls (a prescribed single dose of 6 pearls) are very small paraffin obletes, moist and crude, and thus cannot expand throughout the whole uterus, while ozone foaming spray fills the entire uterus (Djuricic et al., 2011a, 2012a). Nevertheless, the preventive intrauterine use of ozone in either foaming spray or in paraffin pearls improves the reproductive efficiency of dairy cows of the Holstein (n = 404) and Simmental breeds (n = 120) (Djuricic et al., 2011a, 2012a). 6. Treatment of metritis and endometritis with ozone It is well known that the uterus following calving is particularly exposed to bacterial colonization resulting in a rapid proliferation of pathogenic species after delivery. The immune system is then usually compromised (Sheldon and Dobson, 2004; Gilbert et al., 2005). Sheldon and Dobson (2004) assume that systemic immunosuppression prior to calving probably leads to decreased defense mechanism operation in the endometrium, such as the decreased functional ability of neutrophils, and consequently may increase the risk of uterus inflammation, particularly in cows with fetal membrane retention. It has been established that the number of uteruses colonized with pathogenic bacteria subsequently decreases during puerperium. About 78% of uteruses are colonized by pathogenic bacteria 16–30 days post-partum, 50% of uteruses are colonized by pathogenic bacteria 31–45 days post-partum and only 9% of uteruses are colonized by pathogenic bacteria 46–60 days post-partum. Acute metritis and endometritis are commonly caused by coliform and Gram negative anaerobic bacteria, Arcanobacterium pyogenes and other bacteria (Bekana et al., 1994; Dohmen et al., 2000; Azawi, 2008). Coliform bacteria producing endotoxins are almost always found in lochia in cows following dystocia and fetal membrane retention. These endotoxins may directly affect bovine neutrophils with their cytotoxic effect and thus facilitate infection with bacterial species such as A. pyogenes, Escherichia coli, streptococci and staphylococci, which are very frequent during early puerperium. These infections are also observed in about 65% of cows with endometritis (Djuricic et al., 2011b). The transition period is critical for the incidence of puerperal disorders (Prandi et al., 1999). These disorders have been diagnosed in 10–50% of cows with uterus infections (Gilbert et al., 2005). On average, 32.6% of cows have various forms of cervicovaginal discharge (from muco-purulent to purulent) during the first two months post-partum, but only about 25.9% of these cows have visible clinical signs. More than 61% of these cows exhibit a lower conception rate and prolonged days open until pregnancy of up to 205 days (Gautam et al., 2009). Endometritis may prolong the days open until first service, days open until pregnancy, the intercalving period, the conception rate and the risk of cows being culled due to infertility (LeBlanc et al., 2002; Maizon et al., 2004; Gilbert et al., 2005). The incidence of clinical or subclinical endometritis in North America and Ireland has

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been reported in 17%, 24% and 54% of cows (LeBlanc et al., 2002; Rajala-Schultz and Frazer, 2003; Gilbert et al., 2005; McDougall, 2006). In the case of metritis, ozone is applied into the uterus as foaming spray by catheter for 8–10 s. The same procedure is used for the treatment of endometritis, but the uterus of cows has to be in the follicular (proliferative) phase. Following the induction of oestrus by prostaglandins, the cervix of the uterus opens and the application of ozone spray by catheter is feasible. Based on vaginal findings after three weeks, during the next oestrus it would be easier to decide whether to repeat the application of the preparation alone or in combination with the antibiotic ceftiofur. After the first and second intrauterine applications of ozone in cows with endometritis caused by Arcanobacterium pyogenes, 40% and more than 80% cured animals have been recorded, respectively (Scrollavezza et al., 1997). Since the puerperal period considerably affects the fertility of cows, intrauterine ozone treatment in cases of metritis and endometritis act beneficially on overall fertility by shortening the days open until first service and the days open until pregnancy in Holstein cows (Ðuriˇcic´ et al., 2014). Antibacterial broad spectrum agents targeted for intrauterine application, in comparison to ozone preparations, lose activity in lochia in the presence of pus and in anaerobic conditions, and residua in milk and meat and increased bacteria resistance may appear (Djuricic et al., 2012b). On the other hand, intrauterine therapy with ozone may have beneficial effects on the incidence of metritis and endometritis, and consequently on the improvement of reproductive efficiency, as well as lowering the costs of treatment (Scrollavezza et al., 1997; Marusi et al., 1999; Marusi et al., 2000; Djuricic et al., 2012a). 7. Functional restoration of the endometrium through the intrauterine application of ozone pailletes The best results of therapy through the intrauterine application of ozone pailletes can be attained 20–40 days post-partum. It has been observed through ultrasound examination that the uterus is not sufficiently reduced in size 12 h before breeding or 24 h after breeding, or in oestrus synchronization protocols (Ovsync) at the moment of the second GnRH dose application (Scrollavezza et al., 1997). 8. Treatment of fetal retained membrane with ozone In the majority of cases (77.3%), the placenta of cows is expelled 6–8 h after calving (Van Werven et al., 1992). The occurrence of retention of the fetal membrane is considered when the placenta is not expelled within 12 h of delivery (Tomaˇskovic´ et al., 2007; Noakes et al., 2009). The incidence of fetal membrane retention in cows is 5–10%, depending on the herd (LeBlanc, 2008), or on average 8.6% (ranging from 1.3 to 39.2%) (Kelton et al., 1998). There are numerous predisposing factors influencing the separation of the placenta, such as premature birth, genetic, nutritional, immunological and pathological processes (such as

inflammation of the placenta), and thus the real cause of retention of the fetal membrane is still not clear. However, the main role is attributed to structural changes in fetoplacental connections and hormonal disturbances. Cows with retained fetal membrane belong to a high risk group prone to the appearance of metritis (Djuricic et al., 2011b). The appearance of endometritis or metritis following delivery is a consequence of retained fetal membrane in about 25% of cases. It has been reported that cows with retained fetal membrane have numerous bacteria in utero more frequently, such as E. coli, Clostridium spp. and Gram-negative anaerobes, than cows with normal, physiological puerperium (Noakes et al., 2009). In cases of retained fetal membrane in cows, the use of ozone in either spray form or as paraffin obletes has shown very positive therapeutic effects. Recent findings suggest that the manual extraction of the retained fetal membrane is not recommended, and thus therapy with foaming antibiotic preparations is used more often. Due to the well-recognized detrimental side effects of antibiotic use, it is urgent to find a safe and effective alternative to antibiotic therapy for the treatment of retained fetal membrane in ruminants. With the use of ozone 12–24 h (or even 36 h) after birth, the duration of the days open until first service and the days open until pregnancy are comparable with those recorded in cows that have physiological puerperium (Djuricic et al., 2012b). In addition, following therapy with ozone preparations for the treatment of the uterus in cows, shortening has been recorded of the days open until first service, days open until pregnancy and the intercalving period, improvement of the conception rate, a decreased number of doses of deep frozen bull semen applied, and also a decrease in long-term negative economic impact due to the risk of cows being culled because of infertility (Djuricic et al., 2012b). Apart from cows, ozone has been used for the first time in dairy goats with retained fetal membrane, and has had similar positive effects (Djuricic et al., 2015). Major reproductive disorders in the periparturient period of goats are abortion and retention of the fetal membrane(s) (Ahmad et al., 2007). The fetal membranes in does are expelled 1–4 h after kidding. Retained fetal membranes in does are defined as the failure of their expulsion within 6–8 h after kidding (Noakes et al., 2009; Samardzija et al., 2010). In this regard, veterinary attention should be sought if retained fetal membranes are not expelled within 12 h of delivery. After this time, immediate veterinary intervention is necessary (Matthews, 2009). The incidence of retained fetal membranes in does varies from 2 to 10%, depending on the herd (Durrani and Kamal, 2009; Mude et al., 2010; Ameen and Ajayi, 2013). Dairy goats from 7 selected farms in Croatia were observed during early puerperium and were divided into animals without retained fetal membranes (n = 522) and those with retained fetal membranes (n = 41), which were treated with either ozone foam spray (n = 21) or foaming oxytetracycline tablets (n = 20). The does with retained fetal membranes were mated successfully and became pregnant next kidding season, regardless of the treatment applied. Treatment with ozone attained similar results to the standard antibiotic therapy, indicating that it could be a novel potential alternative therapy for

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retained fetal membranes in dairy goats (Djuricic et al., 2015). 9. Treatment of mastitis with ozone To control the incidence of mastitis, it is necessary to be aware of the causative agents of inflammation of the udder, and particularly to follow up on new knowledge regarding the etiology, prevalence, and clinical course of the disease, the economic aspects of its prevention and control, and new approaches to the prevention and treatment of udder inflammation (Oliver et al., 2004; Roberson, 2012). There are several reports regarding the very beneficial effects of ozone preparations in the treatment of udder inflammation in dairy cows and goats through the syringe application of the preparation into the affected udder or quarter (Ogata and Nagahata, 2000; Ioffe and Chernova, 2013). It appears that the effect of pure ozone without a carrier attains better therapeutic results in the treatment of mastitis within 3–6 h of application than registered preparations with carriers, due to the assumption that pure ozone may more easily spread within the milk cistern and alveoli of the udder (Ogata and Nagahata, 2000; Ohtsuka et al., 2006). The treatment of acute mastitis during lactation represents a rather big economic loss in cattle production. This is related to the costs of medication (veterinary services and drugs), decreased production and milk price, as well as the increased number of somatic cell counts in bulk milk samples, an increased number of culled cows, and increased losses due to the findings of residual antibiotics in milk, meat, etc. (Zadoks et al., 2001; Pitkälä et al., 2004; Swinkels et al., 2005). There are no products approved by the US Food and Drug Administration that can be used for the treatment of mastitis on organic dairy farms, since the use of unapproved products is not in accordance with Food and Drug Administration guidelines. Farmers that have adopted organic management have consistently reported fewer cases of clinical mastitis, but do not use the same criteria for its detection. European dairy farmers that have adopted organic management report the use of different conventional and alternative therapies for the treatment and control of mastitis. Organic farmers have treated clinical mastitis using a variety of alternative therapies, including whey-based products, botanicals, vitamin supplements, and homeopathy. Some associations between organic management and the antimicrobial susceptibility of Gram positive mastitis pathogens have been noted, but overall few mastitis pathogens from both conventional and organic dairy herds of cows demonstrate resistance to the antibiotics commonly used for mastitis control (Ruegg, 2009). The infusion of ozone into the inflamed quarter of cows with clinical mastitis via a teat canal using ozone gas generating equipment has been performed and the efficacy of the ozone therapy evaluated. Sixty percent of cows with acute clinical mastitis treated with ozone therapy did not require any antibiotics for recovery. This newly developed ozone therapy method has proven to be effective, safe, and cost effective, posing no risk of drug residues in milk (Ogata and

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Nagahata, 2000). There are also formulated preparations based on ozone in the treatment of udder inflammation, but they are only applied in the treatment of acute mastitis. The application of ozone by syringe should be performed every 12 h (using a maximum of 3 syringes), similar to the application of certain antibiotics in the treatment of mastitis (Scrollavezza et al., 1997). 10. Perspectives and limitations of ozone use in veterinary medicine and buiatrics There have been no observed negative effects of intrauterine, intravaginal and intramammary applications of ozone preparations in dairy cows and goats according to all the related cited reports in the current review. Moreover, there are a lot of facts in these reports which underline the advantages of ozone preparations over antibiotics applied for the same preventive and therapeutic purposes (Ogata and Nagahata, 2000; Ohtsuka et al., 2006; Djuricic et al., 2011a, 2012a,b; Zobel et al., 2012; Ioffe and Chernova, 2013; Ðuriˇcic´ et al., 2014; Zobel et al., 2014a,b; Djuricic et al., 2015). The advantages of ozone use in relation to the use of antibiotics in the treatment of different pathological conditions are: usage without prescription, absence of adverse effects, absence of residues in milk, meat and other tissues, and consequently there are no currencies or microbe resistance (Djuricic et al., 2012a; Zobel et al., 2014a,b). Furthermore, the effects of ozone following application to a particular site in the organism are multiple and result in achieving an almost sterile microenvironment (due to its bactericidal, antivirus and fungicidal effects), whereas certain antibiotics may or may not be effective, and have been found to be effective only regarding bacterial pathogens. The effectiveness of ozone does not decrease in milk, intestinal contents or in the uterus, while effectiveness decreases or is totally absent in the case of certain antibiotics. Ozone does not exhibit any adverse interactions with other drugs or with gastrointestinal microbiota, while most antibiotics unpredictably do. There are no allergic reactions observed following the use of therapeutic concentrations of ozone preparations, while conversely all antibiotics may induce adverse reactions to a certain degree. For instance, penicillin may often cause specific or general allergic reactions. The milk of diseased animals treated with antibiotics should not be consumed for at least 3 to 5 days following treatment, whereas in the case of affected animals treated with ozone, milk can be utilized immediately for industrial or nutritional purposes. In addition, following antibiotic therapy there are residua retained in meat (for 3 to 30 days or more), while for ozone therapy there is no withdrawal time (Scrollavezza et al., 1997). However, there are certain limitations on ozone use in veterinary medicine and/or buiatrics. In spite of extremely strong disinfection activity, ozone is not able to inactivate intracellular bacteria and viruses, but may stimulate the immune system by activating neutrophils and enhancing the release of cytokines (Bocci and Di Paolo, 2004). In addition, ozone may induce tissue inflammation, as demonstrated in experiments in animals and humans (Kerhl et al., 1987; Zobel et al., 2014a). However, these

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findings were disproved after the proper application of ozone by the other author (Bocci, 2005; Bocci, 2006). Other disadvantages of ozone therapy have been observed in the treatment of metritis and endometritis in cows with certain commercial preparations showing insufficient effectiveness. Thus, treatment had to be repeated 1 to 2 times in 60% of cows, and sometimes combined with ceftiofur (Scrollavezza et al., 1997). In addition, in the treatment of mastitis, ozone has shown selective inactivating effects, The intramammary application of ozone preparations has been effective against certain bacteria (for example, Streptococcus dysgalactiae, Streptococcus uberis) but only partially effective or ineffective against other bacteria (for example, Staphylococcus aureus, E. coli, Arcanobacterium pyogenes) (Scrollavezza et al., 1997). Furthermore, we have concerns regarding the treatment of urovagina and pneumovagina with ozone due to the fact that these disorders are mainly caused by anatomical changes (upward tipping of the pelvis in old cows). Thus, we assume that ozone efficiency can be of more relevance in the treatment of accompanying vaginitis. Recently, ozone has frequently been used in veterinary medicine and buiatrics, but still insufficiently. It has exhibited very promising results as an alternative to the use of antibiotics, which are restricted or withdrawn in the production of food animals. Concerns about potential risks to human health due to the use/misuse of antibiotics in the production of food animals (Dewey et al., 1997) have led to their ban throughout the EU (Regulation EC no. 1831/2003). To accommodate the withdrawal of antibiotics (since 2006 in the EU), it is now urgent for Croatia as a Member State and for the Croatian scientific community to follow up forthcoming EU regulations and keep up with scientific trends in veterinary medicine in general (Alves et al., 2004; Teixeira et al., 2013) and in buiatrics (Djuricic et al., 2015), in order to provide relevant health criteria and scientifically-based recommendations for alternatives to antibiotics.

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