Post partum fertility has a profound im-pact on the economic viability of dairy industry. Post-partum period is the most crucial transitory phase in bovine life when various physiological, gynaecologi-cal, biochemical and immunological changes are occurring. During this phase, cattle are exposed to increased risk of infection of uterus, as during parturition the anatomical barriers are broached and remain open for several days (Goff & Horst, 1997).
Infection of the uterus is largely influenced by the balance between bacterial contamination, the local and systemic immune status during pregnancy and parturition. Infectious diseases are more prevalent during this period because of an impaired immune status before and immediately after parturition (LeBlanc et al., 2002). The major classes of immunoglobulins (IgM, IgA and IgG), either by passive diffusion or local production, play an important protective role in the uterus by acting as opsonins to enhance phagocytosis, stimulating the complement pathways or blocking pathogens from adhering to mucous surfaces. Other cellular components activated in the uterus during this period include neutrophils, lymphocytes, eosinophils, mast cells and macrophages. A scientific approach towards reproductive health management of animals during this crucial phase had always been emphasized by the scientific community and a lot of research has been done to potentiate overall immune response of cows during postpartum phase (Wheeler & Wait, 1993). Immune response at local sites with the help of cellular invasion such as B- and T-lymphocytes, neutrophils, macrophages and other polymorhonuclear cells is an important defense mechanism for cows against various post partum pathogenic infections.
Indiscriminate usage of antibiotics for treatment of uterine infections has led to emergence of resistant strains. As a result of overuse and misuse, attention is now focused towards alternative treatments viz. herbal medicine (Hemiaiswarya et al., 2008). Many herbs are known for their strong immunopotentiating properties. Hu et al. (1992) have studied the influence of medicinal herbs on phagocytosis by bovine neutrophils. Herbal preparations as Uterotone, Exapar, Replanta, Involon etc. are claimed to have therapeu-tic efficacy (Nehra, 1987; Srinivas et al., 1998). The drugs capable of inducing myometrial contractions, potentiating immune response, may be useful for hastening the expulsion or to prevent the retention of foetal membranes (Singal, 1996; Tandle et al., 2002).
Exapar Liquid is a unique combina-tion of potent medicinal herbal extracts that tones up uterus for better post partum reproductive efficiency. Cleansing and restorative effects therefore, appears to be a safe and effective option both therapeu-tically and prophylactically. It is a combi-nation of herbs with documented action profile e.g. Plumbago zeylanica (Oyed-apo, 1996), Aloe barbadensis (Hutter et al, 1996), Aristolochia indica (Chopra et. al., 1982), Gloriosa superba (Tiwari et. al., 1967), Lepidium sativum (Nadkarni, 1954), Leptadenia reticulata (Satyavati et. al., 1976), Rubia cordifolia (Nadkarni, 1954), and others.
The current study was undertaken to explore the efficacy of the polyherbal formulation Exapar Liquid in improving the local immune response in cattle. Some earlier reports have also validated efficacy of Exapar in expulsion of membranes, regulation of lochial discharge, treatment of various uterine and involution of uterus in bovines (Singal, 1996) and in improvement of reproductive efficiency in buffaloes (Khanna et al., 1997).
MATERIALS AND METHODS
The research programme was conducted in the department of Gynaecology & Ob-stetrics, College of Veterinary and Animal Sciences, Bidar, Karnataka, India. The methodology of research trial and protocol was legally approved by the Committee for the purpose of control and supervision on experiments in animals (CPCSEA) No. 164/2005 (India) and Institutional Ethics Committee, C.V.S, Bi-dar, Karnataka, India. Eighteen crossbred cows in late gestation of second to fourth lactation were selected from a university dairy farm, for the experiment and were randomly allocated to two groups, control (n=6) and treatment (n=12). The cows from the treatment group were administered Exapar Liquid herbal uterine tonic and cleanser (supplied by M/S Ayurvet Ltd, Baddi, India) at a dose of 100 mL/cow twice daily orally on the day of calving followed by 50 mL Exapar Liquid daily from 2nd to 5th post partum days.
To study the efficacy of the poly-herbal formulation Exapar in improving local immune response, samples of lochial discharge and whole blood were collected before administration of test drug (day 0) and on 6th, 15th and 21st day post calving. Various non-specific immunity parameters such as phagocytic index, total and differential leukocyte counts (TLC, DLC) were assessed in whole blood, while more specific parameters for cell mediated immune response, IgA and IgG were assessed in blood serum and lochial discharge. Phagocytic index was assessed by the nitroblue tetrazolium as-say (NBT) as per Park et al. (1968). TLC and DLC was estimated by method of Nambiar (1960). IgA and IgG were estimated by Ouchterlony technique of agar gel immunodiffusion using anti-bovine immunoglobulins raised as per the procedure described by Hari Babu & Panda (1993).
The data was statistically analyzed using one-way ANOVA as described by Snedecor & Cochran (1980).
Fig. 1. Average phagocytic index (NBT-test) in post parturient cows, treated with Exapar (n=12) and untreated controls (n=6).
The results revealed a remarkable increase in phagocytic index from day 0 (22.25± 1.36) to day 21 (58.42±2.92) intreated cows (P≤0.05) as compared to controls, in which a non-significant varia-tion in phagocytic index value was ob-served from day 0 (26.33±2.59) to day 21st (31.67±2.17) (Fig. 1).
Total and differential leukocyte counts in the treated group exhibited a significant increase from day 0 to 21, vs. the insig-nificant increase in untreated controls (Table 1).
Lochial discharge, collected on 21st day after calving, showed 91.67% of positive reactors for IgA content vs 16.67% in control cows (Table 2). There was not however much significant differ-ence in serum IgA content in both treated and control groups. In the lochial dis-charges of the treated group, the IgA positive reactors increased from day 0 (66.67%) to day 21st (91.67%) while con-trols showed 66.67% positive reactors on day 0 followed by a decrease up to 16.67% on day 21. The serum samples showed 83.33% positive reactors in treated group and 66.67% in controls.
Table 1. Total and differential leukocyte counts in post parturient cows, treated with Exapar (n=12) and untreated controls (n=6). Data are presented as mean ± SEM
Table 2. Number (percentage) of immunoglobulin A positive reactors in lochial and serum samples from- post parturient cows, treated with Exapar (n=12) and untreated controls (n=6) in the agar gel immunodiffusion test
IgG levels in both lochial discharge and serum samples exhibited a trend, similar to that of IgA (Table 3).
The salient findings of the current ex-perimental study are relevant and have established a unique property of poly-herbal formulation Exapar Liquid in im-proving local immune response in dairy cattle, in addition to its uterine cleansing activity.
Significantly increased phagocytic index is desirable for the sequestration of pathogenic organisms and is an important parameter of local immunity (Lee et al., 1992). The observed increase in the phagocytic index in treated cows is suggestive of the probable role of Exapar Liquid in immunoprotection. The results are quite encouraging and there is no literature available to compare present find-ings on the PI values after administration of Exapar. The constituent herbs of Exapar Liquid: Plumbago zeylanica, Citrullus colocynthis, Aloe barbadensis, Tribulus terristris etc. are well known for their antibacterial and immunostimulant activ-ity (Satyawati et al., 1987; Hutter et al., 1996). The total and differential leukocyte counts were determined to study the effect of Exapar Liquid on non-specific immunity.
Table 3. Number (percentage) of Immunoglobulin G positive reactors in lochial and serum samples from- post parturient cows, treated with Exapar (n=12) and untreated controls (n=6) in the agar gel immunodiffusion test
It can be suggested that administration of Exapar Liquid increases polymor-phonuclear cells, particularly leukocytes and neutrophils, thus potentiating non-specific immune response. Neutrophils are known to play a primary role in the defense of the uterus against infection. Influx of neutrophils into the uterus is thought to be mediated by chemoattrac-tants, chemokines and adhesion molecules, such as β2-integrin and l-selectin (Tizard, 2000). The results are in accordance with that of Gilbert et al. (1993) revealing prophylactic use of herbal preparations in increasing the polymor-honuclear counts. Hu et al. (1992) also recorded accelerated phagocytosis by an increase of blood neutrophils after administration of a herbal drug in bovines. The herbal ingredients of Exapar liquid namely Piper longum, Tribulus terristris, Cyperus rotundus and many more are well known for their potent anti-inflammatory activity (Hussain et al., 1992).
The ingredients of Exapar Liquid viz. Uraria picta, Tribulus terristris, Plum-bago zeylanica, Leptidium sativum etc. are scientifically proven to have adapto-genic and immunomodulatory properties (Satyavati et al., 1987). The results in present study are concomitant with those of Kumar et al., (1999) who studied the immunomodulatory effect of Rasayanas – a herbal drug. Administration of Rasa-yanas significantly enhanced the prolif-eration of spleen cells and increased the number of antibody forming cells thereby improving humoral immune response through high titre of circulating antibo-dies.
The increase in immunoglobulin lev-els are compared to the normal levels of IgG and IgA in cattle as reported by Ogunrinde et al. (1984). Results revealed that treated post-partum cows exhibited significantly higher IgG levels in serum than in lochial discharge indicating an improvement in specific immune response by opsonization.
Evaluation of specific immune parameters in lochial discharge and blood serum has shown augmentation of both local and systemic immune response in post partum cow uterus after treatment with Exapar Liquid. There was a remar-kable increase in polymorphonuclear cells, phagocytic index and total serum immunoglobulins in the group treated with Exapar Liquid. The immunoprotec-tive and immunomodulatory activity of Exapar Liquid can be attributed to the various herbal constituents present in the formulation which make it a unique choice for veterinarians and farmers for enhancing local immunity and prevention of various reproductive disorders in post-partum dairy cattle.
The authors are thankful to KVAFSU and Dean, Veterinary College, Bidar for infrastruc-ture facilities & Ayurvet Limited, for provi-ding samples to conduct the research.
Chopra, R. N., S. L. Naye, & I. C. Chopra, 1956. Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Re-search, New Delhi.
Gilbert, R. O., Y. T. Grohn, C. L. Guard, V. Surman, N. Neilsen & D. O. Slauson, 1993. Impaired post partum neutrophil function in cows which retain foetal mem-branes. Research in Veterinary Science, 55, No 1, 15−19.
Goff, J. P. & R. L. Horst, 1997. Physiological changes at parturition and their relation-ship to metabolic disorders. Journal of Dairy Science, 80, 1260–1268.
Hari Babu, Y. & B. K. Panda, 1993. Immu-nostimulating effect of Livol against New-castle Disease virus in chicken. Indian Journal of Indigenous Medicine, 10, No 2, 9−21.
Hussain, A.., O. P.Virmain, S. P. Popli, L. N. Mishra, M. M. Gupta, G. N. Srivastava, Z. Abraham & A. K. Singh, 1992. Dictionary of Indian Medicinal Plants, CIMAP, Lucknow, India.
Hemiaiswarya, S., S. K. Kruthiventi & M. Doble, 2008. Synergism between natural products and antibiotics against infectious diseases. Phytomedicine, 15, 639−652.
Hu, S. H., W. M. Cai, Z. J. Qian & Z. M. Sun, 1992. Influence of medicinal herbs on phagocytosis by bovine neutrophils. Jour-nal of Veterinary Medicine, 39, No 8, 593−599.
Hutter, J. A., M. Salman, W. B. Stavinoha, N. Satsangi, R. F. Williams, R. T. Streeper & S. T. Weintraub, 1996. Anti-inflammatory C-glucosyl hormone from Aloe barbaden-sis. Journal of Natural Products, 59, No 5, 541−543.
Khanna, S., K. L. Khurana, V. N. Tripathi & A. Manuja, 1997. Effect of Exapar on some parameters of reproductive effi-ciency in buffaloes. Indian Journal of Animal Reproduction 18, 41−43.
Kumar, V. P., R. Kuttan & G. Kuttan, 1999. Effect of 'Rasayanas', a herbal drug prepa-ration on immune responses and its sig-nificance in cancer treatment. Indian Jour-nal of Experimental Biology, 37, No 1, 27−31.
LeBlanc, S. J., T. F. Duffield, K. E. Leslie, K. G. Bateman, G. P. Keefe, J. S. Walton & W. H. Johnson, 2002. Defining and diag-nosing postpartum clinical endometritis and its impact on reproductive perform-ance in dairy cows. Journal of Dairy Sci-ence, 85, 2223–2236.
Lee, C. S., E. Meeusen & M. R. Brandon, 1992. Local immunity in the mammary gland. Veterinary Immunology and Im-munopathology, 32, No 1−2, 1−11.
Nadkarni, A. K., 1954. Indian Materia Medica. Bombay: Popular Prakashan Pvt. Ltd, pp. 736−737.
Nehra, R. P. S., 1987. Studies on retention of foetal membranes in crossbred cattle (Sa-hiwal × Holstein). Part II. Prophylaxis by indigenous drugs and therapeutic ap-proach. The Journal of Remount and Vete-rinary Crops, 25, No 2, 81−87.
Oyedapo, O. O., 1996. Studies on the bioactiv-ity of the root extract of Plumbago zeylanica. International Journal of Pharma-cognosy, 34, No 5, 365− 369.
Ogunrinde, E. B., A. Otosile & M. F. Obsaju, 1984. Normal serum and colostrum levels of IgG1, IgM and IgA in indeginous exotic and crossbred cattle in Nigeria. British Veterinary Journal, 140, 374− 379.
Park, B. H., S. M. Fikrig & E. M. WickSmith, 1968. Infection and nitroblue tetrazolium reduction by neutrophils. Lancet, 7, 532.
Satyavati, G. V., A. K. Gupta & N. Tandon, 1976. Medicinal Plants of India, Vol. 1, ICMR Publications, New Delhi, India.
Satyavati, G. V., A. K. Gupta & N. Tandon, 1987. Medicinal Plants of India, Vol. 2, ICMR Publications, New Delhi, India.
Singal, S. P., 1996. Efficacy of Exapar in post-parturient disorders with retained placenta in bovines. Indian Journal of Animal Re-production, 17, No 2, 109−110.
Snedecor, R. W. & W. G. Cochran, 1980. Statistical Methods. 7th edn., Oxford and IBH publishing Co., Calcutta, pp. 32− 62.
Srinivas, T., K. S. Naidu, K. V.Brahmaiah, T. S. C. S. Rao & P. R. Kumar, 1998. Re-tained foetal membranes in crossbred cows − herbal treatment and uterine involution. Indian Journal of Animal Reproduction, 19, 26−28.
Tandle, M. K., S. Honnogopal, V. K. Sunda-ravadanan, S. D. Sonwane, A. Murugeppa, Mohd., Amanullah & V. B. Shettar, 2002. Efficacy of herbal preparations in improv-ing reproductive performance in post-partum cows. In: Proceedings of the 9th In-ternational Congress on Biotechnology in Animal Reproduction, December 24, 2002, Chennai, p. 267.
Tiwari, P., D. N. Prasad, C. Chaturvedi & P. K. Das, 1967. Preliminary studies on uteri-ne activity of Gloriosa superba Linn., and its adulterant Costus speciosus Sm. Jour-nal of Research in Indian Medicine, 1, 196−202.
Tizard, I. R., 2000. Innate immunity: Inflam-mation. In: Veterinary Immunology, 6th edn, ed. I. R. Tizard, W. B. Saunders Company, Philadelphia, PA, pp. 36–46.
Wheeler G. E. & C. Wait, 1993. Use of herbal medicines in modern dairy farming − a breeding efficiency programme. Acta Hor-ticulturae, 333, 299−308.