Prophylactic effect of intrauterine ozonated oils in augmenting the reproductive performances of postpartum crossbred dairy cows

Introduction

Post-partum uterine infections stand first among the reasons causing infertility in bovines (Lee and Kim, 2007) [16] . In addition to causing, decreased milk production (Dubuc et al., 2011) [8] and poor reproductive performances, postpartum uterine infections also demand high cost of diagnosis and treatment (Ganaie et al., 2018) [12] thereby affecting economic profitability of the farmers. Uterine infections occur more in postpartum period due to compromised uterine environment, high bacterial load and suppressed immune system (Gilbert et al., 2005; Sheldon et al., 2006) [14,20] , thereby causing poor reproductive performances. The most common way to treat postpartum uterine infections prophylactically is by using intrauterine or systemic antimicrobials (Zobel et al., 2014) [22] , intrauterine antiseptics, and hormonal therapy (Roberts, 1986) [19] . However, due to limitations of intrauterine antimicrobials such as, ineffectiveness in the presence of purulent lochia, antimicrobial resistance, residues in milk and meat and impairment in uterine defense mechanism (Dinsmore et al., 1996; Djuricic et al., 2012a; Zobel et al., 2014) [4,6,22] , alternative prophylactic therapies are gaining importance. Ozone molecule in various forms such as gas, spray and vegetable oils is gaining popularity as a prophylactic or therapeutic option for postpartum uterine infections as it does not produce antimicrobial resistance and residual effect in food, unlike antimicrobials. Ozone can be used as antibacterial (Duricic et al., 2015) [10] , fungicidal, viricidal (Korzun et al., 2008) [15] and immunomodulators (Ducusin et al., 2003; Ohtsuka et al., 2006) [9,18] using its oxidative properties. Studies on prophylactic effect of intrauterine ozone therapy on postpartum dairy cows showed that ozone has positive impact on improving reproductive performance in postpartum dairy cows by decreasing the days open (Djuricic et al., 2011; Djuricic et al., 2012; Zobel et al., 2014; Duricic et al., 2015; Escandón et al., 2020) [5,6,22,11] . Though the ozone in different forms is used for therapeutic management of various uterine disorders, limited information is available regarding the use of intrauterine ozonated oil as a prophylactic approach for postpartum uterine infections and

Materials and Methods

Thirty-six normally calved crossbred dairy cows with no history of dystocia, retained fetal membrane (RFM) and systemic illness were randomly divided into 3 groups with 12 each. Cows in Group I did not receive any treatment and served as control and cow in Group II and III received one time and two time intrauterine ozonated oil (100 mL) on day 5-7 and 14, respectively. All the study animals were inseminated following a voluntary waiting period of 45 days and their reproductive performances were assessed based on parameters such as, calving-first service interval (CFSI), number of inseminations per conception, calving-conception interval (CCI) and first service conception rate (FSCR). Mean values of reproductive parameters of all the study groups were analysed using one-way ANOVA. When F ratio was significant, Tukey's Multiple Comparison Test was used in order to find which pairs differed significantly.

Consent from the college level institutional Animal Ethics Committee (IAEC) was obtained to conduct the study (IAEC No.VCH/IAEC/2019/117; dated 03.12.2019.

Results and Discussion

The mean values of reproductive parameters of all the study group are presented (Table 1). Calving to first service interval (CFSI), Calving to conception interval (CCI) and number of inseminations per conception did not differ between control and preventive ozone infused groups and also between the preventive ozone infused groups.

Calving-first service interval (CFSI):

The present study revealed that calving to first service interval in Group I (control), Group II (one-time ozone infusion on day 7) and Group III (ozone infused twice on day 7 and 14 pp) are 60.58±0.49, 62.42±0.95 and 61.58±1.89 days, respectively. The CFSI between control and treated groups recorded in the present study are almost similar to the results recorded by Escandón et al. (2020) [11] (75.1 vs 66.1 days) but lesser than the values 81 recorded by Djuricic et al. (2011) [5] (131. 11 vs 118.20 days) and Djuricic et al. (2012) [6] (pearl vs spray vs control; 110.5 vs 105 vs 112 days).

Calving-conception interval (CCI):

Calving-conception interval (CCI) in the present study for Group 1 (control), Group II (one-time ozone infusion on day 7 pp) and Group III (ozone infused twice on day 7 and 14 pp) are 113.91±10.09, 110.75±11 and 109.91±10.91, respectively. Though the values are not statistically significant, lesser CCI have been observed in preventive ozone infused group compared to control group. The present study also revealed that CCI value recorded in control and preventive ozone infused group is almost similar to the results recorded by Duricic et al. (2015) [10] (112 vs110.5 days) but lesser than the values recorded by Zobel et al. (2014) [22] (98.03 vs 75.47). Higher values of CCI have been reported than the values recorded in the present study, by Djuricic et al. (2012) [6] (ozone pearl vs ozone spray vs control group; 145 vs 133 vs 150 days) and Escandón et al. (2020) [11] (control vs ozonized distilled water; 149.0 vs 126.2).

No. of inseminations per conception:

Average no. of insemination per conception recorded in present study for Group I (control), Group II (one-time ozone infusion on day 7 pp) and Group III (ozone infused twice on day 7 and 14 pp) are 3.66±0.49, 3.66±0.49 and 3.41±0.54, respectively. The recorded values in the control group are almost similar to the values recorded by Escandón et al. (2020) [11] (3.1 straws) but higher than the values recorded by Zobel et al. (2014) [22] (2.42 straws). However, in the same study, the average number of inseminations per conception for preventive intrauterine ozone infused group recorded by Zobel et al. (2014) [22] (1.68) and Escandón et al. (2020) [11] (2.1) are much lower than present study observation.ys).

First service conception rate (FSCR):

The present study revealed that first service conception rate for Group 1(control), Group II (one-time ozone infused on day 7 pp) and Group III (ozone infused twice on day 7 and 14 pp) are 25.00, 33.33 and 33.33 percent, respectively (Table2). Escandón et al. (2020) [11] (ozone vs control; 16.2 vs 50%) recorded highest conception rate for preventive ozone infused group compared to control group. Whereas, the conception rate between preventive ozone infused group and control groups reported by Djuricic et al. (2012) [6] and Duricic et al. (2015) [10] are 27.80 vs 20.40 percent and 14.60 vs 14 percent respectively. The values recorded in the present study lies in between the values reported by above authors (25 vs 33.33%). Observations recorded with respect to the above-mentioned reproductive parameters between preventive ozone infused and control group in the present study, did not show any significant difference. However, many authors have reported significant differences in the above reproductive parameters between preventive ozone infused and control group (Djuricic et al., 2011; Zobel et al., 2014; Constantin and Birtoiu, 2016 and Escandón et al., 2020) [5,22,3,11] . Different authors have reported that reproductive performances of the postpartum cows are influenced by various factors such as season, uterine infections, nutrition, management, lactation, bacterial load and endotoxins by interfering with endocrine status of the ovary (Suzuki et al., 2001; Maizon et al., 2004; Gilbert et al., 2005 and Gautam et al., 2009) [21,17,14,13] . Similarly, the voluntary waiting period, followed by different authors in their respective study were also varied.

These two facts may be the probable reason for wide variations in the values of reproductive parameters recorded in the present study compared to values reported by other authors. Djuricic et al. (2012) [6] reported that the forms of ozone preparations and the amount of active substance in it are decisive factor to determine its efficacy. The author also reported that ozone can be used in different formulations like pearl, spray, paillets, gas and foam among which gas and spray are having more expanding capacity and less residual effect in uterus. However, ozonated oil used in the present study were having less expanding capacity due to its high viscosity (16.2 cSt) and more residual effect in the uterus which could be the probable reason for the nonsignificant difference of the reproductive parameters recorded in the present study. The non-significant results in the present study can be attributed to multiple factors influencing the same. Usually, the peroxide value of the ozonated oil determined its acidic value and viscosity (Carata, 2018) [2] . The ozonated oil used in this study was highly viscous (16.2 cSt) having high peroxide index (1280 mEq/kg) and acidic index (0.561 mgKOH/g) when compared to the formulation of other authors. The peroxide value of ozonated sunflower oil formulated in the study, conducted by Carata (2018) [2] and Campanati et al. (2013) [1] were 200 mEq/kg and 75-100 mEq/kg, respectively which is lower than the present study formulation. So, the higher peroxide content of the present study formulation could be the reason for non-significant results. Similarly, volume of ozonated oil infused intrauterine and form of the ozone (ozonised oil having more viscosity) used might have failed to spread the ozone evenly throughout the postpartum uterine lumen thereby failed to exert its effect efficiently. Many authors have reported beneficial effects of ozone in the form of sprays, gases, pearls and pailletes on reproductive performances in dairy cows (Djuricic et al., 2011 and Zobel et al., 2014) [5,22] . However, very less work on ozonated oil have been reported and hence, more work needs to be done to standardize the ozonated oil in terms of peroxide values and dosage to be infused so as to use it effectively in bovine reproduction. Conclusion: The effect of ozonated oil in cows with normal puerperium under study on reproductive performances is non-significant. So, studies using ozonated oil in abnormal puerperium may throw light on its efficacy in improving reproductive performances by combating uterine infections. Further, standardization of ozonated oil in terms of volume, formulation and characterization of oil is needed for better utilisation of ozonated oils in bovine reproduction and also evaluation of the ozonated oil on a larger number of cows is required before adopting it for field use.

References

1. Campanati A, De Blasio S, Giuliano A, Ganzetti G, Giuliodori K, Pecora T, et al. Topical ozonated oil versus hyaluronic gel for the treatment of partial-to fullthickness second-degree burns: a prospective, comparative, single-blind, non-randomized, controlled clinical trial. Burns. 2013;39(6):1178-1183.
2. Carata E, Tenuzzo BA, Dini L. Powerful properties of ozonated extra virgin olive oil. In: Msomi ZN, Simelane BCM, editors. Herbal Medicine. IntechOpen; 2018. p. 230-245.
3. Constantin TB, Prelliminary IA. Preliminary study on ozone therapy in postpartum endometritis of dairy cows. Agric Agric Sci Procedia. 2016;10:384-389.
4. Dinsmore RP, Stevens RD, Cattell MB, Salman MD, Sundlof SF. Oxytetracycline residues in milk after intrauterine treatment of cows with retained fetal membranes. J Am Vet Med Assoc. 1996;209(10):17531755.
5. Djuricic D, Vince S, Grizelj J, Dobranic T, Lipar M, Samardzija M. Influence of different therapy approaches with or without manual extraction of retained placenta on reproductive performance in Simmental cows. Acta Vet. 2011;61:599-608.
6. Djuricic D, Vince S, Grizelj J, Dobranic T, Lipar M, Samardzija M. Intrauterine ozone treatment of retained fetal membrane in Simmental cows. Anim Reprod Sci. 2012;134(3-4):119-124.
7. Djuricic D, Valpotic H, Samardzija M. The intrauterine treatment of retained foetal membrane in dairy goats by ozone: novel alternative to antibiotic therapy. Reprod Domest Anim. 2015;50:236-239.
8. Dubuc J, Duffield TF, Leslie KE, Walton JS, Le Blanc SJ. Impact of postpartum uterine diseases on milk production and culling in dairy cows. J Dairy Sci. 2011;94(3):1339-1346.
9. Ducusin RJT, Nishimura M, Sarashina T, Uzuka Y, Tanab S, Otani M. Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro. J Vet Med Sci. 2003;65(4):535539.
10. Duricic D, Valpotic H, Samardzija M. Prophylaxis and therapeutic potential of ozone in buiatrics: current knowledge. Anim Reprod Sci. 2015;159:1-7.
11. Escandón BM, Espinoza JS, Perea FP, Quito F, Ochoa R, López GE, et al. Intrauterine therapy with ozone reduces subclinical endometritis and improves reproductive performance in postpartum dairy cows managed in pasture-based systems. Trop Anim Health Prod. 2020;52:2523-2528.
12. Ganaie BA, Sultan F, Dar RR, Ahmad F. Uterine infection in dairy animals and its ameliorative measures: a review. J Pharmacogn Phytochem. 2018;7:194-199.
13. Gautam G, Nakao T, Yusuf M, Koike K. Prevalence of endometritis during the postpartum period and its impact on subsequent reproductive performance in two Japanese dairy herds. Anim Reprod Sci. 2009;116:175187.
14. Gilbert RO, Shin ST, Guard CL, Erb HN, Frajblat M. Prevalence of endometritis and its effects on reproductive performance of dairy cows. Theriogenology. 2005;64(9):1879-1888.
15. Korzun W, Hall J, Sauer R. The effect of ozone on common environmental fungi. Clin Lab Sci. 2008;21:107-111.
16. Lee SC, Jeong JK, Choi IS, Kang HG, Jung YH, Park SB, et al. Cytological endometritis in dairy cows: diagnostic threshold, risk factors, and impact on reproductive performance. J Vet Sci. 2018;19(2):301308.
17. Maizon DO, Oltenacu PA, Gröhn YT, Strawderman RL, Emanuelson U. Effects of diseases on reproductive International Journal of Advanced Biochemistry Research https://www.biochemjournal.com performance in Swedish Red and White dairy cattle. Prev Vet Med. 2004;66:113-126.
18. Ohtsuka A, Ogata N, Terasaki M, Koiwa S. Changes in leukocyte population after ozonated autohemoadministration in cows with inflammatory diseases. J Vet Med Sci. 2006;68:175-178.
19. Roberts SJ. Involution or regression of the uterus. In: Veterinary Obstetrics and Genital Diseases. 3rd ed. Woodstock (VT): Published by Woodstock; 1986. p. 256-259.
20. Sheldon IM, Lewis GS, Le Blanc S, Gilbert RO. Defining postpartum uterine disease in cattle. Theriogenology. 2006;65(8):1516-1530.
21. Suzuki C, Yoshioka K, Iwamura S, Hirose H. Endotoxin induces delayed ovulation following endocrine aberration during the proestrus phase in Holstein heifers. Domest Anim Endocrinol. 2001;20:267-278.
22. Zobel R, Martinec R, Ivanovic D, Rosic N, Stancic Z, Zerjavic I, et al. Intrauterine ozone administration for improving fertility rate in Simmental cattle. Vet Arh. 2014;84(1):1-8.