Administration of ashwagandha and garlic extract in subclinical endometritis improves conception rate in cattle

Introduction

Infectious diseases of the female reproductive tract are among the major causes of impaired fertility in mammals, particularly in cattle, where they result in substantial reproductive and economic losses (Ross, 2002; Sheldon et al., 2006; Sheldon et al., 2020; Ribeiro et al., 2022) [32, 34, 36, 31] . The detrimental effects of these infections extend beyond local tissue damage, as they interfere with the normal physiological functions of both the uterus and ovaries, ultimately compromising reproductive performance. Over the past several years, considerable progress has been made in understanding the innate immune mechanisms that enable the reproductive tract to recognize and respond to invading microorganisms (O 'Neill, 2008; Akira et al., 2006; Beutler, 2004) [26,2,4] . Research has demonstrated that innate immune activity within the female reproductive tract is not confined to circulating immune cells. Endometrial and ovarian cells also possess the ability to detect pathogenic organisms through specific receptors and actively participate in local defence responses (Herath et al., 2006; Herath et al., 2007; Hernandez-Gonzalez et al., 2006) [13, 15, 16] . Although these responses are essential for controlling infection, microbial pathogens and their associated molecules may simultaneously disturb endocrine regulation by altering the synthesis of reproductive hormones and inflammatory mediators. Such alterations adversely influence ovarian function, uterine physiology and, consequently, fertility (Herath et al., 2006; Herath et al., 2007; Herath et al., 2009; Sheldon et al., 2002; Ribeiro et al., 2022; Sheldon and Owens, 2023) [13,15,14,37,31,35] . Among postpartum reproductive disorders, uterine infections remain one of the most frequently encountered conditions in dairy cattle. Metritis commonly develops during the first week after calving, although its reported prevalence varies depending on the diagnostic criteria adopted. Several epidemiological studies have indicated that approximately one-third to one-half of dairy cows may be affected, while a smaller proportion develops systemic manifestations such as fever and depression (Markusfeld, 1987; Zwald et al., 2004; Drillich et al., 2001; Benzaquen et al., 2007; LeBlanc, 2020; Sheldon and Owens, 2023) [24,44,7,3,21,35] . Failure of complete uterine recovery following calving predisposes animals to persistent uterine inflammation. Consequently, nearly 15-20% of cows develop clinical endometritis after the third postpartum week, whereas a considerable proportion experiences subclinical endometritis without obvious external signs (Sheldon et al., 2006; Gilbert et al., 2005; Ribeiro et al., 2022; LeBlanc, 2020) [34,12,31,21] . These postpartum uterine disorders delay uterine involution, impair ovarian cyclicity and significantly reduce subsequent reproductive efficiency, thereby increasing the economic burden on dairy production systems. Clinical endometritis is recognized by the presence of purulent uterine discharge after 21 days postpartum or mucopurulent discharge beyond 26 days postpartum, as described by Sheldon et al. (2006) [34] . In routine clinical practice, examination of vaginal mucus is widely used for diagnosis because its characteristics closely reflect the severity of uterine infection and inflammation (Williams et al., 2005; Sheldon and Owens, 2023) [43,35] . The nature of the discharge also provides valuable information regarding the likely bacterial involvement and the expected response to treatment. Unlike clinical endometritis, subclinical endometritis progresses without visible abnormalities in vaginal discharge and therefore requires cytological evaluation for diagnosis. The condition is generally identified by an increased proportion of polymorphonuclear neutrophils (PMNs) in endometrial cytology samples collected during the postpartum period (Kasimanickam et al., 2004; Santos et al., 2009; Ribeiro et al., 2022; LeBlanc, 2020) [17,33,31,21] . Despite the absence of apparent clinical signs, persistent inflammation adversely affects uterine function, lowers conception rates and prolongs the interval required to achieve successful pregnancy. Early diagnosis and appropriate management of both clinical and subclinical endometritis are therefore essential for improving reproductive performance in dairy cattle. The use of phytogenic feed additives has emerged as an important nutritional approach for improving animal health and reproductive performance in dairy cattle. Among the various medicinal plants, garlic (Allium sativum) has attracted considerable attention because of its antioxidant, antimicrobial, anti-inflammatory and immunomodulatory properties, which contribute to improved metabolic status, immune competence and reproductive efficiency in cattle (Puvača et al., 2020; El-Tarabany et al., 2021) [30,10] . Similarly, ashwagandha (Withania somnifera) is recognized for its adaptogenic and antioxidant activities and has been reported to alleviate stress, improve physiological functions and support reproductive performance in cattle (Kumar et al., 2022; Tewari et al., 2023) [19,42] . Although the individual biological effects of these phytogenic supplements have been documented, information regarding their use as nutritional interventions for improving reproductive performance under field conditions remains limited. Therefore, the present study was undertaken to evaluate the effect of dietary supplementation with garlic and ashwagandha extracts to treat the subclinical endometritis in cattle and to increase reproductive performance in cattle.

Materials and Methods

The present investigation was carried out in the Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science & Animal Husbandry, Mhow, in collaboration with the Central Semen Station, Bhopal.

Animals used for study

A total of 80 apparently healthy cows with no clinical signs of endometritis that calved between August 2017 and March 2018 were used for the study. Animals were maintained under an intensive housing system in cemented sheds with brick floor and stall-fed seasonally available green fodder, wheat straw, and concentrate. Supplements included mineral mixtures and ad libitum water. This management reflected standard private farming practices in the region. The cows were screened for subclinical endometritis using endometrial cytology and the Whiteside test for prevalence study. Blood was collected via jugular puncture during the last week pre-partum, the day of calving, and the first week postpartum for estimation of NEFA concentration using the copper soap solvent extraction method modified by Shipe et al. (1980) [38] , in healthy animals and animals affected with subclinical endometritis. From this population, 18 animals confirmed with subclinical endometritis (SCE) were selected and divided into three groups (n=6) to evaluate herbal therapeutic efficacy. Positive cows were randomly assigned to:

• Control group I (Group I, n=6): 20 ml intrauterine normal saline solution oid for 3 days.

•

Treatment II (Group II, n=6): 20 ml intrauterine ashwagandha extract oid for 3 days.

•

Treatment III (Group III, n=6): 20 ml intrauterine garlic extract oid for 3 days.

Treated animals were subjected to control breeding at 45 days using ovsynch protocol and conception rate were confirmed by per rectal examination at 60 days post artificial insemination.

Preparation of extract 1. Ashwagandha extract

Fresh Ashwagandha (Withania somnifera) leaves were dried in the dark, pulverized, and extracted with absolute alcohol using a Soxhlet apparatus until the solvent darkened. The extract was stored in sterile glass containers (Sunderam et al., 2011) [40] .

Garlic extract

Chopped Garlic (Allium sativum) bulbs were dried in the dark and subjected to successive Soxhlet extraction using different solvents such as Methanol. Extracts were concentrated and weighed according to Gangadhar et al. (2012) [11] .

Statistical analysis

Prevalence and conception rates were expressed as percentages. The data of PMN per cent were analysed statistically by two-way analysis of variance (ANOVA) and means were compared using Duncan's new multiple range test (DNMRT) with significance at p < 0.05.

Results a) Prevalence of sub-clinical endometritis in cattle

The prevalence of subclinical endometritis in cattle was determined by the formula mentioned below, when 41.13% animals were found positive for sub-clinical endometritis: 02. There was a highly significant difference (p ˂0.05) between the means of NEFA on all the three days, viz., a week prior to, on the day and a week after of calving in both healthy and diseased categories. However, no significant difference (p > 0.05) was found between healthy and SCE-positive animals.

Discussion

In the present study, the relatively high prevalence observed indicates that subclinical uterine inflammation remains a common reproductive disorder during the postpartum period. Since affected animals did not exhibit obvious clinical signs, diagnosis through Whiteside test and endometrial cytology proved useful for identifying infected animals at an early stage. Similar observations were reported by Kasimanickam et al. (2004) [17] , who demonstrated that a considerable number of postpartum cows without visible vaginal discharge were cytologically positive for endometritis, indicating that clinical examination alone is insufficient for diagnosis. Likewise, Gilbert et al. (2005) [12] reported an overall prevalence of approximately 53% in postpartum Holstein cows and observed that affected animals required a significantly longer period to conceive than healthy cows. Dubuc et al. (2010) [8] also reported that nearly one-fifth of postpartum dairy cows suffered from cytological endometritis, emphasizing that uterine inflammation frequently persists even in apparently healthy animals. Recent reviews by Parikh et al. (2022) [28] and Pascottini et al.

(2023) [29] have further emphasized that postpartum uterine diseases remain one of the major causes of reduced fertility and economic losses in dairy cattle, particularly when diagnosis and treatment are delayed. Therefore, early diagnosis followed by timely therapeutic intervention is essential to restore uterine health and improve reproductive performance. [27] and Chapinal et al. (2011) [5] , who demonstrated that elevated NEFA concentrations during the transition period are associated with increased fat mobilization and a greater risk of postpartum metabolic and reproductive disorders. LeBlanc et al. (2011) [22] further suggested that excessive lipid mobilization may impair immune function, thereby reduce uterine defence mechanisms and increase susceptibility to postpartum uterine infections. Although serum NEFA concentration was numerically higher in the subclinical endometritis group in the present study, the absence of a significant difference between healthy and diseased animals indicates that NEFA alone cannot be considered a reliable diagnostic marker for subclinical endometritis. Development of uterine inflammation is influenced not only by metabolic status but also by bacterial contamination, immune competence and uterine defence mechanisms. Similar conclusions have been drawn by Pascottini et al. (2023) [29] , who reported that postpartum uterine diseases result from the combined effects of metabolic stress, microbial infection and host immune response rather than any single biochemical parameter. Ashwagandha (Withania somnifera) contains withanolides, sitoindosides and alkaloids that possess potent antioxidant, anti-inflammatory and immunomodulatory properties. These compounds reduce oxidative stress, regulate inflammatory cytokines and improve cellular immune responses, thereby promoting repair of the damaged endometrium and facilitating elimination of pathogenic microorganisms (Mirjalili et al., 2009; Abd El-Hack et al., 2022) [25,1] . Improved immune competence also enhances uterine defence mechanisms during the postpartum period, leading to faster resolution of subclinical infection. Similarly, Garlic (Allium sativum) contains allicin and other organosulphur compounds that exhibit broad-spectrum antibacterial, antifungal and antioxidant activities. Allicin inhibits bacterial multiplication, suppresses inflammatory mediators and improves phagocytic activity of neutrophils and macrophages, thereby accelerating uterine involution and recovery (El-Saber Batiha et al., 2020; Chen et al., 2021) [9,6] . The reduction in Whiteside test positivity observed in the Garlic-treated group in the present study supports these pharmacological properties. The present findings are in close agreement with Lawange et al. (2019) [20] , who evaluated Garlic extract with and without Ashwagandha in repeat breeding cattle associated with uterine infection. They reported a significantly higher recovery rate in animals receiving the combined herbal treatment, accompanied by conversion of Whiteside test-positive animals to negative status and improvement in conception rate. Likewise, Swelum et al. (2021) [41] suggested that phytogenic feed additives improve reproductive efficiency primarily through their antioxidant, antimicrobial and immunomodulatory effects. The superior response observed in the Ashwagandha-treated group may be attributed to its immunomodulatory activity, which improves both innate and adaptive immune responses while simultaneously reducing oxidative stress within uterine tissues. Reduced production of reactive oxygen species and inflammatory mediators promotes regeneration of the endometrial epithelium and restores normal uterine function (Abd El-Hack et al., 2022) [1] . Garlic supplementation also contributed to reduction in PMN percentage through its antimicrobial activity, which limits bacterial growth and minimizes tissue damage caused by persistent infection (El-Saber Batiha et al., 2020) [9] . The findings of the present investigation are consistent with those of Kasimanickam et al. (2004) [17] , who reported that reduction in PMN percentage following treatment was associated with improved uterine health and higher fertility. Similarly, Gilbert et al. (2005) [12] demonstrated that persistence of elevated PMN levels was associated with reduced conception and prolonged days open. Pascottini et al.

(2023) [29] emphasized that successful treatment of subclinical endometritis should primarily be reflected by reduction in cytological inflammation rather than only disappearance of clinical signs. Following herbal treatment, the reduction in uterine inflammation, as confirmed by the Whiteside test and PMN cytology, probably restored normal endometrial function, thereby increasing the probability of successful conception. Ashwagandha has been reported to improve antioxidant defence mechanisms and modulate immune responses, while Garlic enhances antimicrobial activity and reduces oxidative stress through sulphur-containing bioactive compounds such as allicin (El-Saber Batiha et al., 2020; Abd El-Hack et al., 2022) [9,1] . The present findings are comparable with those reported by Lawange et al. (2019) [20] , who evaluated Garlic extract alone and in combination with Ashwagandha in repeat breeding cattle suffering from uterine infection. They observed a significantly higher conception rate in animals receiving herbal therapy than in untreated animals and concluded that improvement in uterine health following herbal treatment contributed directly to enhanced fertility. Similarly, LeBlanc et al. (2011) [22] reported that successful resolution of postpartum uterine inflammation substantially improves reproductive efficiency in dairy cattle. More recently, Swelum et al. (2021) [41] suggested that phytogenic feed additives improve reproductive performance by reducing oxidative stress, enhancing immune competence and maintaining reproductive tract health. [9,1] . The present findings are consistent with previous reports indicating that phytogenic feed additives improve metabolic health and immune response in livestock. Chen et al. (2021) [6] reported that Garlic supplementation enhances antioxidant status and improves nutrient utilization in farm animals. Likewise, Kholif et al.

(2021) [18] emphasized that herbal feed additives improve metabolic efficiency and overall health by reducing oxidative stress and enhancing rumen and immune function.

Conclusion

The present study demonstrated that subclinical endometritis is an important postpartum reproductive disorder in cattle that adversely affects uterine health and fertility. The findings revealed that supplementation with Garlic (Allium sativum) and Ashwagandha (Withania somnifera) extracts effectively reduced uterine inflammation, improved endometrial health and enhanced reproductive performance. Both herbal treatments decreased uterine inflammatory changes, improved cytological findings and increased conception rate compared to the untreated control animals.

The beneficial effects of these herbal extracts may be attributed to their antimicrobial, antioxidant, antiinflammatory and immunomodulatory properties, which promote uterine recovery and improve the uterine environment for successful conception. Overall, the study suggests that Garlic and Ashwagandha can be considered safe and promising herbal alternatives or supportive therapies for the management of subclinical endometritis and improvement of reproductive efficiency in cattle.

Acknowledgement

AcknowledgementThe financial support received from RKVY and UPCAR, Lucknow for carrying out the research is thankfully acknowledged.

References

1. Abd El-Hack ME, Alagawany M, Abdel-Moneim AME, Mohammed NG, Khafaga AF, Bin-Jumah M, et al. Impacts of phytogenic feed additives on animal health and production: A review. Animals. 2022;12(5):587.
2. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124(4):783-801.
3. Benzaquen ME, Risco CA, Archbald LF, Melendez P, Thatcher MJ, Thatcher WW. Rectal temperature, calvingrelated factors, and the incidence of puerperal metritis in postpartum dairy cows. J Dairy Sci. 2007;90(6):28042814.
4. Beutler B. Inferences, questions and possibilities in Tolllike receptor signalling. Nature. 2004;430(6996):257263.
5. Chapinal N, Carson M, Duffield TF, Capel M, Godden S, Overton M, et al. The association of serum metabolites with clinical disease during the transition period. J Dairy Sci. 2011;94(10):4897-4903.
6. Chen J, Wang F, Yin Y, Ma X. The nutritional applications of garlic (Allium sativum) as natural feed additives in animals. PeerJ. 2021;9:e11934.
7. Drillich M, Beetz O, Pfützner A, Sabin M, Sabin HJ, Kutzer P, et al. Evaluation of a systemic antibiotic treatment of toxic puerperal metritis in dairy cows. J Dairy Sci. 2001;84(9):2010-2017.
8. Dubuc J, Duffield TF, Leslie KE, Walton JS, LeBlanc SJ. Definitions and diagnosis of postpartum endometritis in dairy cows. J Dairy Sci. 2010;93(11):5225-5233.
9. El-Saber Batiha G, Magdy Beshbishy A, Wasef LG, Elewa YHA, Al-Sagan AA, Abd El-Hack ME, et al. Chemical constituents and pharmacological activities of garlic (Allium sativum L.): A review. Nutrients. 2020;12(3):872.
10. El-Tarabany MS, El-Tarabany AA, Atta MA. Dietary phytogenic feed additives and productive and reproductive performance in dairy cattle: A review. Animals. 2021;11(11):3165.
11. Gangadhar KN, Pai KSR, Sreenivasa GM. Phytochemical evaluation and antimicrobial activity of Allium sativum extracts. Int J Pharm Pharm Sci. 2012;4(3):650-654.
12. 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.
13. Herath S, Fischer DP, Werling D, Williams EJ, Lilly ST, Dobson H, et al. Expression and function of Toll-like receptor 4 in the endometrial cells of the uterus. Endocrinology. 2006;147(1):562-570.
14. Herath S, Lilly ST, Fischer DP, Williams EJ, Dobson H, Bryant CE, et al. Bacterial lipopolysaccharide induces an endocrine switch from prostaglandin F2α to prostaglandin E2 in bovine endometrium. Endocrinology. 2009;150(4):1912-1920.
15. Herath S, Williams EJ, Lilly ST, Gilbert RO, Dobson H, Bryant CE, et al. Ovarian follicular cells have innate immune capabilities that modulate their endocrine function. Reproduction. 2007;134(5):683-693.
16. Hernandez-Gonzalez I, Gonzalez-Robayna I, Shimada M, Wayne CM, Ochsner SA, White L, et al. Gene expression profiles of cumulus cell-oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: Does this expand their role in the ovulation process? Mol Endocrinol. 2006;20(6):13001321.
17. Kasimanickam R, Duffield TF, Foster RA, Gartley CJ, Leslie KE, Walton JS, et al. Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows. Theriogenology. 2004;62(1-2):9-23.
18. Kholif AE, Olafadehan OA, Matloup OH, Anele UY. Essential oils and phytogenic feed additives in animal nutrition: Effects on performance, health and metabolism. Anim Feed Sci Technol. 2021;276:114914.
19. Kumar S, Singh AK, Sharma RK, Yadav S. Role of herbal feed additives in improving health and reproduction of dairy cattle: A review. Indian J Anim Sci. 2022;92(10):1181-1188.
20. Lawange SR, Markandeya NM, Kumawat BL, Sawale AG, Anbhule RS, Kadam PD. Efficacy of garlic extract with and without ashwagandha for treatment of repeat breeding associated with uterine infection in cattle. Indian J Anim Reprod. 2019;40(1):31-34.
21. LeBlanc SJ. Monitoring uterine health in dairy cattle. Anim Reprod. 2020;17(3):e20200095.
22. LeBlanc SJ, Osawa T, Dubuc J. Reproductive tract defense and disease in postpartum dairy cows. Theriogenology. 2011;76(3):409-423.
23. Madoz LV, Giuliodori MJ, Migliorisi AL, Jaureguiberry M, de la Sota RL. Endometrial cytology for the diagnosis of subclinical endometritis in dairy cows. J Dairy Sci. 2014;97(7):4123-4131.
24. Markusfeld O. Periparturient traits in seven high dairy herds. Incidence rates, association with parity, and interrelationships among traits. J Dairy Sci. 1987;70(1):158-166.
25. Mirjalili MH, Moyano E, Bonfill M, Cusidó RM, Palazón J. Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules. 2009;14(7):23732393.
26. O'Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: 10 years of progress. Immunol Rev. 2008;226(1):10-18.
27. Ospina PA, Nydam DV, Stokol T, Overton TR. Associations of elevated nonesterified fatty acids and βhydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle. J Dairy Sci. 2010;93(4):15961603.
28. Parikh SC, Garcia-Guerra A, Santos JEP. Uterine disease and reproductive performance in dairy cattle: Current perspectives. J Dairy Sci. 2022;105:1-15.
29. Pascottini OB, LeBlanc SJ, Gnemi G, Leroy JLMR, Opsomer G. Genesis of clinical and subclinical endometritis in dairy cows. Reproduction. 2023;166:R15R30.
30. Puvača N, Pelić DL, Kostadinović LJ, Lević J, Đuragić O, Bursić V. Garlic (Allium sativum) as a natural feed additive: A review. Worlds Poult Sci J. 2020;76(2):343358.
31. Ribeiro ES, Gomes GC, Greco LF, Cerri RLA, VieiraNeto A, Monteiro PLJ Jr, et al. Uterine disease, immunity and fertility in dairy cows: Current concepts and future perspectives. J Dairy Sci. 2022;105(5):3935-3956.
32. Ross JDC. An update on pelvic inflammatory disease. Sex Transm Infect. 2002;78(1):18-19.
33. Santos NR, Lamb GC, Brown DR, Gilbert RO. Postpartum endometrial cytology in beef cows. Theriogenology. 2009;71(5):739-745.
34. Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO. Defining postpartum uterine disease in cattle. Theriogenology. 2006;65(8):1516-1530.
35. Sheldon IM, Owens SE. Postpartum uterine disease and infertility in dairy cattle: Advances in pathogenesis, immunity and management. Theriogenology. 2023;206:1-12.
36. Sheldon IM, Cronin JG, Bromfield JJ. Tolerance and innate immunity shape the postpartum uterus and fertility in dairy cattle. Reprod Fertil Dev. 2020;32(1):1-13.
37. Sheldon IM, Noakes DE, Rycroft AN, Pfeiffer DU, Dobson H. Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle. Reproduction. 2002;123(6):837-845.
38. Shipe WF, Senyk GF, Fountain KB. Modified copper soap solvent extraction method for measuring free fatty acids in milk. J Dairy Sci. 1980;63(2):193-198.
39. Singh J, Honparkhe M, Ghuman SPS, Dhindsa SS, Kumar A, Chandra M, et al. Diagnosing sub-clinical endometritis through uterine cytobrush cytology and its treatment with proteolytic enzymes in buffaloes. In: Proceedings of the 29 th Annual Convention and National Symposium on Frontier Reproductive Biotechnologies for Enhancing Animal Fertility and Fecundity: Global Perspective; 2014. p. 147.
40. Sunderam S, Muthukumar S, Ramesh S. Phytochemical screening and antimicrobial activity of Withania somnifera leaf extracts. Int J Pharm Sci Res. 2011;2(10):2569-2573.
41. Swelum AA, Hashem NM, Abdelnour SA, Taha AE, Ohran H, Khafaga AF, et al. Effects of phytogenic feed additives on the reproductive performance of animals. Saudi J Biol Sci. 2021;28(10):5816-5822.
42. Tewari D, Stankiewicz AM, Mocan A. Withania somnifera (Ashwagandha): Biological activities and therapeutic potential. Phytother Res. 2023;37:1050-1072.
43. Williams EJ, Fischer DP, England GCW, Dobson H, Pfeiffer DU, Sheldon IM. Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the inflammatory response to endometritis in cattle. Theriogenology. 2005;63(1):102-117.
44. Zwald NR, Weigel KA, Chang YM, Welper RD, Clay JS. Genetic selection for health traits using producerrecorded data. I. Incidence rates, heritability estimates, and sire breeding values. J Dairy Sci. 2004;87(12):42874294.