- Email: [email protected]
Uterine defence mechanisms in the mare
AnimalReproduction Science 42 ( 19%) 197-204
Uterine defence mechanisms in the mare T. Katila Animal Reproduction, Faculty of Veterinary Medicine, University of Helsinki, 04840 Hautjiirvi, Finland
Abstract Breeding
or insemination
of mares is followed
by a transient
infection
and inflammation.
Normal mares eliminate bacteria and clear inflammatory by-products rapidly. Some mares are not capable of doing this. Studies on immunoglobulins, opsonins, chemotaxis and migration and phagocytosis of neutrophils have failed to show good grounds for susceptibility to uterine infections. The latest studies indicate that differences between these two kinds of mares are best explained by factors involved in uterine clearance: cervical dilation, myometrial activity and lymphatic drainage. Keywords: Mare; Endometritis; Uterine defence; Uterine drainage
1. Introduction infection and inflammation of the endometrium is an inevitable sequel to of mares. It has been believed that the uterus is grossly contaminated with bacteria during coitus (Bryans, 1962) and that bacteria elicit the acute inflammatory reaction in the uterus (Peterson et al., 1969). A recent study has shown that inflammation related to breeding is induced sooner by spermatozoa than by bacteria (Kotilainen et al., 1994). The quantity of bacteria after insemination (AI) or natural cover of normal mares was relatively low and quickly eliminated (Kotilainen et al., 1994; Katila, 1995). However, the intensity of the inflammation depended on the concentration of semen: frozen and centrifuged fresh semen caused the most intensive inflammation (Kotilainen et al., 1994). The defence mechanisms of the uterus against foreign invaders comprise complex interactions between different elements. The anatomical barriers have to be intact. Pneumovagina, urine pooling and cervical incompetency are severe predisposing factors to endometritis (Van Camp, 1986). The major barrier to ascending bacterial contamination may be the vulvovaginal fold (Himichs et al., 1988). Cellular components, immunoglobulins, bactericidal substances and mechanical factors all have their own important role to fulfil. Many of these factors are modified by steroid hormones. Transient
breeding
0378-4320/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved. PI1 SO378-4320(96)01507-2
198
2. Susceptibility
T. Karila/Animal Reproduction Science 4.2 (1996) 197-204
to uterine infections
Although the concept of a susceptible versus a resistant mare has been known for decades, it has not been clear what fails in the uterine defence of these mares. Numerous studies have approached this problem from various angles: immunoglobulins, chemotaxis, migration and phagocytosis of neutrophils, opsonins, components of uterine fluid, mechanical drainage, etc. In these studies, intrauterine inoculations of bacteria or some irritating agents, like oyster glycogen, have been commonly used. The vast majority of mares are fully capable of rapidly eliminating bacteria that have gained entrance to the uterus following breeding, parturition or intrauterine manipulations, but some other mares are not. Hughes and Loy (1969) and Peterson et al. (1969) showed that young mares were highly resistant to bacterial inoculations, whereas older or barren mares remained chronically infected. Mares prone to permanent infection are usually older, have a history of being barren or show clinical signs of endometritis and tend to belong to biopsy categories IIB or III (Kenney and Doig, 1986), but they cannot be classified as susceptible or resistant for these reasons only. The ultimate test is to infuse the mare’s uterus with bacteria. If she is able to clear the infection within 96 h on her own, she should be considered resistant to uterine infections (Troedsson, 1991). LeBlanc (1994) has suggested the infusion of charcoal into the uterus during oestrus. No charcoal is recovered in lavage done 48 h later in resistant mares, but those susceptible have retained charcoal in the uterus (LeBlanc, 1994).
3. Effect of ovarian steroids It is commonly agreed that mares are more susceptible to infectious endometritis during progesterone dominance than when under the influence of oestrogen or with no hormonal influence at all. The modifying effects of steroids on various uterine functions have been studied in intact cycling mares or in hormone-treated ovariectomised mares. In many instances, the results are conflicting. One explanation is the different techniques used in the collection of uterine fluid. The most common method has been the lavage technique, which does not take into account how much fluid there is in the uterine lumen before saline injection. Concentrations of various components of uterine fluid can be accurately measured by absorbing uterine fluid into tampons (Katila et al., 19901, but with this method the total uterine content of a substance remains obscure, because the volume of uterine fluid is not measured. Troedsson (1991) combined these two techniques to allow a more accurate estimation of the total amount of a certain substance in the uterine fluid. Sampling techniques may also be a good explanation for discrepancies between the studies that have compared normal mares and mares with chronic endometritis. Susceptible mares accumulated six times more fluid in the uterus after bacterial challenge than did normal mares (Troedsson and Liu, 1992). Administmtion of hormones did not affect the phagocytic capacity of circulating neutrophils (Washburn et al., 1982). After bacterial challenge, the situation changed. At 2 days after experimental infection, circulating polymorphonuclear leukocytes (PMN) from oestrogen-treated mares showed better phagocytic response and streptocidal activ-
T. Katila / Animal
Reproduction
Science 42 (1996)
197-204
199
ity than progesterone-treated or control mares (Washburn et al., 1982). Random migration of blood PMNs and phagocytosis by uterine neutrophils were reduced in progesterone-treated mares 18 h after intrauterine infusion of Streptococcus zooepidemicus (Watson et al., 1987~). It is not known whether progesterone is a primary cause of this apparent neutrophil deficiency. Neutrophils may also be adversely affected by the persistence of bacteria in the progesterone-dominated uterus (Watson et al., 1987~). When looking at physical drainage of the uterus, the effects of hormones become obvious. Under the influence of progesterone the cervix is closed, whereas under oestrogen dominance it stays open. Myometrial activity of the uterus changes with the stage of oestrous cycle (Troedsson, 1991). Uterine epithelial cells from mares treated with oestradiol demonstrate significantly lower bacterial adherence than cells collected from mares treated with progesterone (Watson et al., 1988). It is probable that all these factors contribute to the marked difference between mares treated with progesterone and those treated with oestrogen in the clearance of bacteria and non-antigenic markers after intra-uterine inoculation (Evans et al., 1986).
4. Immunoglobulins Immunoglobulins were detected in equine uterine flushings for the first time by Kemrey and Khaleel (1975), who isolated six classes of immunoglobulins: IgG,, IgG,, IgG,, IgT, IgA, and IgM. The equine endometrium is considered part of the mucosal immune system, which has the potential for local immunoglobulin synthesis (Widders et al., 1985). The early assumption that problem mares did not have enough IgA, led to the use of colostrum as a source of immunoglobulins in the treatment of endometritis. However, Asbury et al. (1980) had previously reported that mares, who were not able to clear bacteria after intrauterine inoculation, had, in fact, higher concentrations of antibodies in uterine secretions than resistant mares. Although immunoglobulins are an essential component of defence, the susceptibility of some mares to uterine infections does not result from immunoglobulin deficiency.
5. Migration of neutrophils into the uterus The influx of PMNs into tissues is the hallmark of the acute inflammatory response. In normal mares, the first neutrophils enter the uterus within 1 h after AI (Katila, 1995) or experimental infection (Watson et al., 1987d). After AI (Katila, 1995) or bacterial inoculation (Williamson et al., 1987; Katila et al., 1990), the highest numbers of neutrophils are found around 6-12 h. Peak neutrophil numbers do not differ between susceptible and resistant mares (Liu et al., 1986; Williamson et al., 1987), but in persistently infected mares, PMNs stay at a higher level for a longer period of time. In healthy mares, neutrophils gradually diminish in numbers, and 48 h after AI they have disappeared (Katila, 1995).
200
T. Katila/Animal Reproduction Science 42 (19%) 197-204
6. Chemotaxis and neutrophil migration in vitro Chemotactic agents, which enhance the migration of peripheral neutrophils, include serum (Blue et al., 1984; Strzemienski et al., 19841, uterine fluid (Strzemienski et al., 1984) and especially serum (Watson et al., 1987b) and uterine fluid (Blue et al., 1984) from infected mares. This is obvious, since uterine fluid after bacterial infusion contains many chemoattractive components, e.g. certain metabolites of arachidonic acid (Watson et al., 1987a). The first studies using uterine neutrophils and chemotactic chambers indicated that PMNs collected 12 h after bacterial infusion from susceptible mares did not respond to chemotactic agents and were nondeformable (Liu et al., 1985). A later study suggested that a premature migration dysfunction occurred 12 h after infection in mares belonging to biopsy category III (Liu et al., 1986). The same study also showed that in susceptible mares, there is a continued recruitment of PMNs from the peripheral circulation in response to the presence of bacteria. When these mares were sampled later than 12 h post inoculation, different and new populations of neutrophils were obtained (Liu et al., 1986). Watson et al. (1987c) discovered that uterine neutrophils did not migrate under agarose. Neutrophils that have already phagocytosed may run out of plasma membrane for locomotion (Watson et al., 1987c.j. It can be concluded from the above studies that there are no real differences between susceptible and resistant mares in the migration of PMNs.
7. Opsonisation and phagocytosis The first papers on opsonisation in the mare’s uterus demonstrated that the addition of serum to uterine flushings significantly enhanced opsonisation of bacteria (Asbury et al., 1982) and that this was a complement-dependent event (Asbury et al., 1984). These results, and the initial finding that susceptible mares had a deficiency in the release of opsonins, initiated the use of intra-uterine plasma therapy (Asbury, 1984). Later studies did not support the early hypothesis that susceptible mares are not effective in opsonisation (Brown et al., 1985). In fact, haemolytic complement activity was significantly greater in uterine washings from susceptible than resistant mares (Watson et al., 1987b). This is understandable because of the persistent nature of inflammation in these mares. No opsonins were detected prior to uterine infection, but after bacterial inoculation they accumulated in the uterine lumen (Brown et al., 1985). Opsonic activity in the uterus was approximately of the same magnitude as that in the circulation (Magnusson and Jonsson, 19911, and it involved both complement and antibody (Brown et al., 1985; Watson, 1988). Release of opsonins is an important component of uterine defence, but susceptibility to uterine infections does not depend on defective opsonisation. Neutrophils derived from the uterus are more effective in phagocytosis than those derived from blood, perhaps because they have already been activated in the inflamed uterus (Watson et al., 1987c). Studies on the phagocytosing ability of uterine PMNs comparing resistant and susceptible mares have produced conflicting results. One has to bear in mind that uterine neutrophils are not a homogeneous population of cells, and
T. Katila/Animal Reproduction Science 42 (19%) 197-204
201
thus, not as easy to study as blood PMNs. They have already responded to chemotactic signals by the time they have migrated into the uterus. They come from an environment which is full of bacteria and various inflammatory by-products. Some of them have already shown phagocytic activity, which has changed the cell. The ability of PMNs to phagocytose and kill yeast blastospores-a test used to measure phagocytosis-decreases after bactericidal activity (Watson et al., 1987b). The ability to migrate may have been lost because of plasma membrane changes. The lifetime of a neutrophil is only 12-15 h. In the continued presence of bacteria, new PMNs are recruited from the circulation (Liu et al., 1986). When sampling mares with endometritis, old and dying PMNs and new ones are obtained in the same collection.
8. Mechanical clearance During recent years, increasing attention has focused on mechanical drainage through the cervix. The most common pathogen in equine endometritis, Streptococcus zooepidemicus has the ability to adhere to endometrial epithelial cells, which may help the bacteria to combat physical clearance mechanisms (Watson et al., 1988). Evans et al. (1986) showed that physical clearance mechanisms are an important factor in the persistence of infections. Young mares cleared inoculated bacteria, charcoal, and microspheres more rapidly than older mares (Evans et al., 1987). Progesterone treatment made previously resistant mares susceptible to bacterial challenge (Evans et al., 1987). Using scintigraphy, LeBlanc et al. (1994) showed that susceptible mares were more likely to retain radiocolloid in the uterus than resistant mares during oestrus or 48 h after ovulation. However, some nulliparous mares did not clear radiocolloid, nor did normal mares during dioestrus. These workers concluded that poor cervical dilation may delay uterine clearance. Pycock (cited in Allen, 1993) also considered the functioning of the cervix to be critical in the drainage of the uterus: mares with intrauterine fluid collections often had an abnormal cervix. Manual dilation of the cervix together with oxytocin and intrauterine treatments started early in oestrus helped these mares to conceive (Allen, 1993). Troedsson and his group studied the role of myometrial activity in physical clearance. Troedsson et al. (1993) showed that in all mares there was a visible increase in myometrial activity following bacterial infusion, but resistant mares demonstrated a greater myometrial activity in frequency, duration and intensity than susceptible mares. The researchers concluded that susceptible mares had impaired electrical myometrial activity. Troedsson et al. (1993) also assumed that prostaglandins, released from activated PMNs, may be responsible for increased myometrial activity during infection. In a subsequent study, the same group showed that PGF,,, PGE, and oxytocin induced myometrial activity (Troedsson et al., 1995). When uterine clearance was measured in mares with scintigraphy, both oxytocin and PGF*, stimulated clearance of intrauterine radiocolloid (Cadario et al., 1995). The postbreeding treatment of problem mares with oxytocin has become a commonly used therapeutic regimen in the stud farm practice (LeBlanc, 1994).
202
T. Katila /Animal
Reproduction
Science 42 (1996) 197-204
Besides cervical dilation and myometrial contractions, lymphatic drainage may be important in uterine clearance. Lymphatic vessels and lymph nodes drain the equine uterine submucosa and uterine lumen of excess fluid. The efficacy and rate of lymphatic drainage were decreased in susceptible mares compared with resistant mares (LeBlanc et al., 1995). Many susceptible mares have lymphatic lacunae in endometrial biopsies (LeBlanc, 1994). LeBlanc (1994) proposed the following hypothesis for the pathogenesis of endometritis in mares. If the uterus is not cleared before the cervix is closed, lymphatics drain the uterine contents in normal mares. In susceptible mares, inflammatory by-products remain in the uterine lumen, producing more inflammation and irritation of the endometrium. Removing intrauterine fluid containing inflammatory by-products by saline lavage and inducing uterine contractions with oxytocin improved conception rates considerably in a treatment trial, which included mares with a history or evidence of endometritis (LeBlanc, 1994). In conclusion, all aspects of neutrophil function are vital in the defence against bacterial invaders: migration in sufficient numbers into the desired site in response to chemotaxis, coating of bacteria by opsonins (complement, IgG and others), phagocytosis and intracellular killing. There is no conclusive evidence, however, that any of these are deficient in mares susceptible to endometritis. Increasing evidence shows that differences in the mechanical drainage of the uterus may provide the best explanation for susceptibility to uterine infections. Adequate cervical dilation, myometrial contractions and intact lymphatic drainage are required for the maintenance of uterine health.
References Allen, W.R., 1993. Proceedings of the John P. Hughes International Workshop on Equine Endometritis. Equine Vet. J.. 25: 184-193. Asbury, A.C., 1984. Uterine defense mechanisms in the mare: The use of intrauterine plasma in the management of endometritis. Theriogenology, 21: 387-393. Asbury, A.C., Halliwell, R.E.W., Foster, G.W. and Longino, S.J., 1980. Immunoglobulins in uterine secretions of mares with differing resistance to endometritis. Theriogenology, 4: 299-308. Asbury, AC., Schultz, K.T., Klesius, P.H., Foster, G.W. and Washburn, SM., 1982. Factors affecting phagocytosis of bacteria by neutrophils in the mare’s uterus. J. Reprcd. Fertil., Suppl., 32: 151-159. Asbury, A.C., Gorman, N.T. and Foster, G.W., 1984. Uterine defense mechanisms in the mare: Serum opsonins affecting phagccytosis of Streptococcus zooepidemicus by equine neutrophils. Theriogenology, 21: 375-385. Blue, H.B., Blue, M.G., Kemrey, R.M. and Merritt, T.L., 1984. Chemotactic properties and protein of equine uterine fluid. Am. J. Vet. Res., 45: 1205-1208. Brown, A.E., Hansen, P.J. and Asbury, A.C., 1985. Opsonization of bacteria by uterine secretions of cyclic mares. Am. J. Reprod. Immunol., 9: 119-123. Bryans, J.T., 1962. Research on bacterial diseases of horses. Lectures for Stud Managers’ Course, Lexington, Kentucky, pp. 155- 156. Cadario, M.E., Thatcher, M.J.D. and LeBlanc, M.M., 1995. Relationship between prostaglandin and uterine clearance of radiocolloid in the mare. Biol. Reprod. Mono, 1: 495-500. Evans, M.J., Hamer, J.M., Gason, L.M., Graham, C.S., Asbury, A.C. and Irvine, C.H.G., 1986. Clearance of bacteria and non-antigenic markers following intra-uterine inoculation into maiden mares: effect of steroid hormone environment. ‘lheriogenology, 26: 37-50. Evans, M.J., Hamer, J.M., Gason, L.M. and Irvine, C.H.G., 1987. Factors affecting uterine clearance of inoculated materials in mares. J. Reprod. Fertil., Suppl., 35: 327-334.
T. Katila /Animal
Reproduction Science 42 (1996) 197-204
203
Hinrichs, K., Cummings, M.R., Sertich, P.L. and Kenney, R.M., 1988. Clinical significance of aerobic bacterial flora of the uterus, vagina, vestibule, and clitoral fossa of clinically normal mares. J. Am. Vet. Med. Assoc., 193: 72-75. Hughes, J.P. and Loy, R.G., 1969. Investigations on the effect of intrauterine inoculations of Streptococcus zooepidemicus in the mare. Proc. 15th Annual Convention of the American Association of Equine Practitioners, pp. 289-292. Katila, T., 1995. Onset and duration of uterine inflammatory response of mares after insemination with fresh semen. Biol. Reprod. Mono, 1: 515-5 17. Katila, T., Lock, T.F., Hoffman, W.E. and Smith, A.R., 1990. Lysozyme, alkaline phosphatase, and neutrophils in uterine secretions of mares with differing resistance to endometritis. Tberiogenology, 33: 723-732. Kenney, R.M. and Doig, P.A., 1986. Equine endometrial biopsy. In: D.A. Morrow (Editor), Current Therapy in Theriogenology. W.B. Saunders, Philadelphia, PA, pp. 723-729. Kenney, R.M. and Khaleel, S.A., 1975. Bacteriostatic activity of the mare uterus: A progress report on immuno-globulins. J. Reprod. Fertil., Suppl., 23: 357-358. Kotilainen, T., Huhtinen, M. and Katila, T., 1994. Sperm-induced leukocytosis in the equine uterus. Theriogenology, 41: 629-636. LeBlanc, M., 1994. Breakdown in uterine defense mechanisms in the mare: Is a delay in physical clearance the culprit? Proc. of the Annual Meeting of the Society of Theriogenology, Kansas City, MO, pp. 121-129. LeBlanc, M.M., Neuwirth, L., Asbury, A.C., Tran, T., Mauragis, D. and Klapstein, E., 1994. Scintigraphic measurement of uterine clearance in normal mares and mares with recurrent endometritis. Equine Vet. J., 26: 109-l 13. LeBlanc, M.M., Johnson, R.D., Caldenvood Mays, M.B. and Valderrama, C., 1995. Lymphatic clearance of India ink in reproductively normal mares and mares susceptible to endometritis. Biol. Reprod. Mono, I: 501-506. Liu, I.K.M., Cheung, A.T.W., Walsh, E.M., Miller, M.E. and Lindenberg, P.M., 1985. Comparison of peripheral blood and uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis: Chemotactic and cell elastimetry analysis. Am. J. Vet. Res., 46: 917-920. Liu, I.K.M., Cheung, A.T.W., Walsh, E.M. and Ayin, S., 1986. The functional competence of uterine-derived polymorpho-nuclear neutrophils (PMN) from mares resistant and susceptible to chronic uterine infection: A sequential migration analysis. Biol. Reprod., 35: 1168-l 174. Magnusson, U. and Jonsson, K., 1991. A method for the accurate measurement of opsonic activity in uterine secretions of the mate. Theriogenology, 36: 737-747. Peterson, F.B., McFeely, R.A. and David, J.S.E., 1969. Studies on the pathogenesis of endometritis in the mare. hoc. 15th Annual Convention of the American Association of Equine Practitioners, pp. 279-287. Strzemienski, P.J., Do, D. and Kenney, R.M., 1984. Anti-bacterial activity of mare uterine fluid. Biol. Reprod., 31: 303-311. Troedsson, M.H.T., 1991. Uterine defense mechanisms in the mare. Ph.D. Thesis, University of California, Davis, USA. Troedsson, M.H.T. and Liu, I.K.M., 1992. Measurement of total volume and protein concentration of intrauterine secretion after intrauterine inoculation of bacteria in mares that were either resistant or susceptible to chronic infection. Am. J. Vet. Res., 53: 1641- 1644. Troedsson, M.H.T., Liu, I.K.M., Ing, M., Pascoe, J. and Thurmond, M., 1993. Multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. J. Reprod. Fertil., 99: 307-313. Troedsson, M.H.T., Liu, I.K.M., Ing, M. and Pascoe, J., 1995. Smooth muscle electrical activity in the oviduct and the effect of oxytocin, prostaglandin F,, and Prostaglandin E, on the myometrium and the oviduct of the cycling mare. Biol. Reprod. Mono, 1: 475-488. Van Camp, S., 1986. Breeding soundness examination of the mare and common genital abnormalities encountered. In: D.A. Morrow (Editor), Current Therapy in Theriogenology. W.B. Saunders, Philadelphia, PA, pp. 654-661. Washburn, S.M., Klesius, P.H., Ganjam, V.K. and Brown, B.G., 1982. Effect of estrogen and progesterone on the phagocytic response of ovariectomized mares infected in utero with P-hemolytic streptococci. Am. J. Vet. Res., 43: 1367-1370.
204
T. Katila/Animal Reproduction Science 42 (1996) 197-204
Watson, E.D.,‘1988. Opsonins in uterine washings influencing in vitro activity of equine neutrophils. Equine Vet. J., 20: 435-437. Watson, E.D., Stokes, C.R. and Boume, F.J., 1987a. Influence of arachidonic acid metabolites in vitro and in uterine washings on migration of equine neutrophils under agarose. Res. Vet. Sci., 43: 203-207. Watson, E.D., Stokes, C.R. and Boume, F.J., 1987b. Cellular and humoral defence mechanisms in mares susceptible and resistant to persistent endometritis. Vet. Immmunol. Immunopathol., 16: 107- 121. Watson, E.D., Stokes, C.R. and Boume, F.J., 1987c. Influence of administration of ovarian steroids on the function on neutrophils isolated from the blood and uterus of ovariectomized mares. J. Endocrinol., 112: 443-448. Watson, E.D., Stokes, C.R., David, J.S.E. and Boume, F.J., 1987d. Concentrations of uterine luminal prostaglandins in mares with acute and persistent endometritis. Equine Vet. J., 19: 31-37. Watson, E.D., Stokes, C.R. and Boume, F.J., 1988. Influence of ovarian steroids on adherence (in vitro) of Streptococcus zooepidemicus to endometrial epithelial cells. Equine Vet. J., 20: 37 l-372. Widders, P.R., Stokes, C.R., David, J.S.E. and Boume, F.J., 1985. lmmunohistological studies of the local immune system in the reproductive tract of the mare. Res. Vet. Sci., 38: 88-95. Williamson, P., Munya, S., Martin, R. and Penhale, J., 1987. Dynamics of the acute uterine response to infection, endotoxin infusion and physical manipulation of the reproductive tract in the mare. J. Reprod. Fertil., Suppl., 35: 317-325.
Uterine defence mechanisms in the mare T. Katila Animal Reproduction, Faculty of Veterinary Medicine, University of Helsinki, 04840 Hautjiirvi, Finland
Abstract Breeding
or insemination
of mares is followed
by a transient
infection
and inflammation.
Normal mares eliminate bacteria and clear inflammatory by-products rapidly. Some mares are not capable of doing this. Studies on immunoglobulins, opsonins, chemotaxis and migration and phagocytosis of neutrophils have failed to show good grounds for susceptibility to uterine infections. The latest studies indicate that differences between these two kinds of mares are best explained by factors involved in uterine clearance: cervical dilation, myometrial activity and lymphatic drainage. Keywords: Mare; Endometritis; Uterine defence; Uterine drainage
1. Introduction infection and inflammation of the endometrium is an inevitable sequel to of mares. It has been believed that the uterus is grossly contaminated with bacteria during coitus (Bryans, 1962) and that bacteria elicit the acute inflammatory reaction in the uterus (Peterson et al., 1969). A recent study has shown that inflammation related to breeding is induced sooner by spermatozoa than by bacteria (Kotilainen et al., 1994). The quantity of bacteria after insemination (AI) or natural cover of normal mares was relatively low and quickly eliminated (Kotilainen et al., 1994; Katila, 1995). However, the intensity of the inflammation depended on the concentration of semen: frozen and centrifuged fresh semen caused the most intensive inflammation (Kotilainen et al., 1994). The defence mechanisms of the uterus against foreign invaders comprise complex interactions between different elements. The anatomical barriers have to be intact. Pneumovagina, urine pooling and cervical incompetency are severe predisposing factors to endometritis (Van Camp, 1986). The major barrier to ascending bacterial contamination may be the vulvovaginal fold (Himichs et al., 1988). Cellular components, immunoglobulins, bactericidal substances and mechanical factors all have their own important role to fulfil. Many of these factors are modified by steroid hormones. Transient
breeding
0378-4320/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved. PI1 SO378-4320(96)01507-2
198
2. Susceptibility
T. Karila/Animal Reproduction Science 4.2 (1996) 197-204
to uterine infections
Although the concept of a susceptible versus a resistant mare has been known for decades, it has not been clear what fails in the uterine defence of these mares. Numerous studies have approached this problem from various angles: immunoglobulins, chemotaxis, migration and phagocytosis of neutrophils, opsonins, components of uterine fluid, mechanical drainage, etc. In these studies, intrauterine inoculations of bacteria or some irritating agents, like oyster glycogen, have been commonly used. The vast majority of mares are fully capable of rapidly eliminating bacteria that have gained entrance to the uterus following breeding, parturition or intrauterine manipulations, but some other mares are not. Hughes and Loy (1969) and Peterson et al. (1969) showed that young mares were highly resistant to bacterial inoculations, whereas older or barren mares remained chronically infected. Mares prone to permanent infection are usually older, have a history of being barren or show clinical signs of endometritis and tend to belong to biopsy categories IIB or III (Kenney and Doig, 1986), but they cannot be classified as susceptible or resistant for these reasons only. The ultimate test is to infuse the mare’s uterus with bacteria. If she is able to clear the infection within 96 h on her own, she should be considered resistant to uterine infections (Troedsson, 1991). LeBlanc (1994) has suggested the infusion of charcoal into the uterus during oestrus. No charcoal is recovered in lavage done 48 h later in resistant mares, but those susceptible have retained charcoal in the uterus (LeBlanc, 1994).
3. Effect of ovarian steroids It is commonly agreed that mares are more susceptible to infectious endometritis during progesterone dominance than when under the influence of oestrogen or with no hormonal influence at all. The modifying effects of steroids on various uterine functions have been studied in intact cycling mares or in hormone-treated ovariectomised mares. In many instances, the results are conflicting. One explanation is the different techniques used in the collection of uterine fluid. The most common method has been the lavage technique, which does not take into account how much fluid there is in the uterine lumen before saline injection. Concentrations of various components of uterine fluid can be accurately measured by absorbing uterine fluid into tampons (Katila et al., 19901, but with this method the total uterine content of a substance remains obscure, because the volume of uterine fluid is not measured. Troedsson (1991) combined these two techniques to allow a more accurate estimation of the total amount of a certain substance in the uterine fluid. Sampling techniques may also be a good explanation for discrepancies between the studies that have compared normal mares and mares with chronic endometritis. Susceptible mares accumulated six times more fluid in the uterus after bacterial challenge than did normal mares (Troedsson and Liu, 1992). Administmtion of hormones did not affect the phagocytic capacity of circulating neutrophils (Washburn et al., 1982). After bacterial challenge, the situation changed. At 2 days after experimental infection, circulating polymorphonuclear leukocytes (PMN) from oestrogen-treated mares showed better phagocytic response and streptocidal activ-
T. Katila / Animal
Reproduction
Science 42 (1996)
197-204
199
ity than progesterone-treated or control mares (Washburn et al., 1982). Random migration of blood PMNs and phagocytosis by uterine neutrophils were reduced in progesterone-treated mares 18 h after intrauterine infusion of Streptococcus zooepidemicus (Watson et al., 1987~). It is not known whether progesterone is a primary cause of this apparent neutrophil deficiency. Neutrophils may also be adversely affected by the persistence of bacteria in the progesterone-dominated uterus (Watson et al., 1987~). When looking at physical drainage of the uterus, the effects of hormones become obvious. Under the influence of progesterone the cervix is closed, whereas under oestrogen dominance it stays open. Myometrial activity of the uterus changes with the stage of oestrous cycle (Troedsson, 1991). Uterine epithelial cells from mares treated with oestradiol demonstrate significantly lower bacterial adherence than cells collected from mares treated with progesterone (Watson et al., 1988). It is probable that all these factors contribute to the marked difference between mares treated with progesterone and those treated with oestrogen in the clearance of bacteria and non-antigenic markers after intra-uterine inoculation (Evans et al., 1986).
4. Immunoglobulins Immunoglobulins were detected in equine uterine flushings for the first time by Kemrey and Khaleel (1975), who isolated six classes of immunoglobulins: IgG,, IgG,, IgG,, IgT, IgA, and IgM. The equine endometrium is considered part of the mucosal immune system, which has the potential for local immunoglobulin synthesis (Widders et al., 1985). The early assumption that problem mares did not have enough IgA, led to the use of colostrum as a source of immunoglobulins in the treatment of endometritis. However, Asbury et al. (1980) had previously reported that mares, who were not able to clear bacteria after intrauterine inoculation, had, in fact, higher concentrations of antibodies in uterine secretions than resistant mares. Although immunoglobulins are an essential component of defence, the susceptibility of some mares to uterine infections does not result from immunoglobulin deficiency.
5. Migration of neutrophils into the uterus The influx of PMNs into tissues is the hallmark of the acute inflammatory response. In normal mares, the first neutrophils enter the uterus within 1 h after AI (Katila, 1995) or experimental infection (Watson et al., 1987d). After AI (Katila, 1995) or bacterial inoculation (Williamson et al., 1987; Katila et al., 1990), the highest numbers of neutrophils are found around 6-12 h. Peak neutrophil numbers do not differ between susceptible and resistant mares (Liu et al., 1986; Williamson et al., 1987), but in persistently infected mares, PMNs stay at a higher level for a longer period of time. In healthy mares, neutrophils gradually diminish in numbers, and 48 h after AI they have disappeared (Katila, 1995).
200
T. Katila/Animal Reproduction Science 42 (19%) 197-204
6. Chemotaxis and neutrophil migration in vitro Chemotactic agents, which enhance the migration of peripheral neutrophils, include serum (Blue et al., 1984; Strzemienski et al., 19841, uterine fluid (Strzemienski et al., 1984) and especially serum (Watson et al., 1987b) and uterine fluid (Blue et al., 1984) from infected mares. This is obvious, since uterine fluid after bacterial infusion contains many chemoattractive components, e.g. certain metabolites of arachidonic acid (Watson et al., 1987a). The first studies using uterine neutrophils and chemotactic chambers indicated that PMNs collected 12 h after bacterial infusion from susceptible mares did not respond to chemotactic agents and were nondeformable (Liu et al., 1985). A later study suggested that a premature migration dysfunction occurred 12 h after infection in mares belonging to biopsy category III (Liu et al., 1986). The same study also showed that in susceptible mares, there is a continued recruitment of PMNs from the peripheral circulation in response to the presence of bacteria. When these mares were sampled later than 12 h post inoculation, different and new populations of neutrophils were obtained (Liu et al., 1986). Watson et al. (1987c) discovered that uterine neutrophils did not migrate under agarose. Neutrophils that have already phagocytosed may run out of plasma membrane for locomotion (Watson et al., 1987c.j. It can be concluded from the above studies that there are no real differences between susceptible and resistant mares in the migration of PMNs.
7. Opsonisation and phagocytosis The first papers on opsonisation in the mare’s uterus demonstrated that the addition of serum to uterine flushings significantly enhanced opsonisation of bacteria (Asbury et al., 1982) and that this was a complement-dependent event (Asbury et al., 1984). These results, and the initial finding that susceptible mares had a deficiency in the release of opsonins, initiated the use of intra-uterine plasma therapy (Asbury, 1984). Later studies did not support the early hypothesis that susceptible mares are not effective in opsonisation (Brown et al., 1985). In fact, haemolytic complement activity was significantly greater in uterine washings from susceptible than resistant mares (Watson et al., 1987b). This is understandable because of the persistent nature of inflammation in these mares. No opsonins were detected prior to uterine infection, but after bacterial inoculation they accumulated in the uterine lumen (Brown et al., 1985). Opsonic activity in the uterus was approximately of the same magnitude as that in the circulation (Magnusson and Jonsson, 19911, and it involved both complement and antibody (Brown et al., 1985; Watson, 1988). Release of opsonins is an important component of uterine defence, but susceptibility to uterine infections does not depend on defective opsonisation. Neutrophils derived from the uterus are more effective in phagocytosis than those derived from blood, perhaps because they have already been activated in the inflamed uterus (Watson et al., 1987c). Studies on the phagocytosing ability of uterine PMNs comparing resistant and susceptible mares have produced conflicting results. One has to bear in mind that uterine neutrophils are not a homogeneous population of cells, and
T. Katila/Animal Reproduction Science 42 (19%) 197-204
201
thus, not as easy to study as blood PMNs. They have already responded to chemotactic signals by the time they have migrated into the uterus. They come from an environment which is full of bacteria and various inflammatory by-products. Some of them have already shown phagocytic activity, which has changed the cell. The ability of PMNs to phagocytose and kill yeast blastospores-a test used to measure phagocytosis-decreases after bactericidal activity (Watson et al., 1987b). The ability to migrate may have been lost because of plasma membrane changes. The lifetime of a neutrophil is only 12-15 h. In the continued presence of bacteria, new PMNs are recruited from the circulation (Liu et al., 1986). When sampling mares with endometritis, old and dying PMNs and new ones are obtained in the same collection.
8. Mechanical clearance During recent years, increasing attention has focused on mechanical drainage through the cervix. The most common pathogen in equine endometritis, Streptococcus zooepidemicus has the ability to adhere to endometrial epithelial cells, which may help the bacteria to combat physical clearance mechanisms (Watson et al., 1988). Evans et al. (1986) showed that physical clearance mechanisms are an important factor in the persistence of infections. Young mares cleared inoculated bacteria, charcoal, and microspheres more rapidly than older mares (Evans et al., 1987). Progesterone treatment made previously resistant mares susceptible to bacterial challenge (Evans et al., 1987). Using scintigraphy, LeBlanc et al. (1994) showed that susceptible mares were more likely to retain radiocolloid in the uterus than resistant mares during oestrus or 48 h after ovulation. However, some nulliparous mares did not clear radiocolloid, nor did normal mares during dioestrus. These workers concluded that poor cervical dilation may delay uterine clearance. Pycock (cited in Allen, 1993) also considered the functioning of the cervix to be critical in the drainage of the uterus: mares with intrauterine fluid collections often had an abnormal cervix. Manual dilation of the cervix together with oxytocin and intrauterine treatments started early in oestrus helped these mares to conceive (Allen, 1993). Troedsson and his group studied the role of myometrial activity in physical clearance. Troedsson et al. (1993) showed that in all mares there was a visible increase in myometrial activity following bacterial infusion, but resistant mares demonstrated a greater myometrial activity in frequency, duration and intensity than susceptible mares. The researchers concluded that susceptible mares had impaired electrical myometrial activity. Troedsson et al. (1993) also assumed that prostaglandins, released from activated PMNs, may be responsible for increased myometrial activity during infection. In a subsequent study, the same group showed that PGF,,, PGE, and oxytocin induced myometrial activity (Troedsson et al., 1995). When uterine clearance was measured in mares with scintigraphy, both oxytocin and PGF*, stimulated clearance of intrauterine radiocolloid (Cadario et al., 1995). The postbreeding treatment of problem mares with oxytocin has become a commonly used therapeutic regimen in the stud farm practice (LeBlanc, 1994).
202
T. Katila /Animal
Reproduction
Science 42 (1996) 197-204
Besides cervical dilation and myometrial contractions, lymphatic drainage may be important in uterine clearance. Lymphatic vessels and lymph nodes drain the equine uterine submucosa and uterine lumen of excess fluid. The efficacy and rate of lymphatic drainage were decreased in susceptible mares compared with resistant mares (LeBlanc et al., 1995). Many susceptible mares have lymphatic lacunae in endometrial biopsies (LeBlanc, 1994). LeBlanc (1994) proposed the following hypothesis for the pathogenesis of endometritis in mares. If the uterus is not cleared before the cervix is closed, lymphatics drain the uterine contents in normal mares. In susceptible mares, inflammatory by-products remain in the uterine lumen, producing more inflammation and irritation of the endometrium. Removing intrauterine fluid containing inflammatory by-products by saline lavage and inducing uterine contractions with oxytocin improved conception rates considerably in a treatment trial, which included mares with a history or evidence of endometritis (LeBlanc, 1994). In conclusion, all aspects of neutrophil function are vital in the defence against bacterial invaders: migration in sufficient numbers into the desired site in response to chemotaxis, coating of bacteria by opsonins (complement, IgG and others), phagocytosis and intracellular killing. There is no conclusive evidence, however, that any of these are deficient in mares susceptible to endometritis. Increasing evidence shows that differences in the mechanical drainage of the uterus may provide the best explanation for susceptibility to uterine infections. Adequate cervical dilation, myometrial contractions and intact lymphatic drainage are required for the maintenance of uterine health.
References Allen, W.R., 1993. Proceedings of the John P. Hughes International Workshop on Equine Endometritis. Equine Vet. J.. 25: 184-193. Asbury, A.C., 1984. Uterine defense mechanisms in the mare: The use of intrauterine plasma in the management of endometritis. Theriogenology, 21: 387-393. Asbury, A.C., Halliwell, R.E.W., Foster, G.W. and Longino, S.J., 1980. Immunoglobulins in uterine secretions of mares with differing resistance to endometritis. Theriogenology, 4: 299-308. Asbury, AC., Schultz, K.T., Klesius, P.H., Foster, G.W. and Washburn, SM., 1982. Factors affecting phagocytosis of bacteria by neutrophils in the mare’s uterus. J. Reprcd. Fertil., Suppl., 32: 151-159. Asbury, A.C., Gorman, N.T. and Foster, G.W., 1984. Uterine defense mechanisms in the mare: Serum opsonins affecting phagccytosis of Streptococcus zooepidemicus by equine neutrophils. Theriogenology, 21: 375-385. Blue, H.B., Blue, M.G., Kemrey, R.M. and Merritt, T.L., 1984. Chemotactic properties and protein of equine uterine fluid. Am. J. Vet. Res., 45: 1205-1208. Brown, A.E., Hansen, P.J. and Asbury, A.C., 1985. Opsonization of bacteria by uterine secretions of cyclic mares. Am. J. Reprod. Immunol., 9: 119-123. Bryans, J.T., 1962. Research on bacterial diseases of horses. Lectures for Stud Managers’ Course, Lexington, Kentucky, pp. 155- 156. Cadario, M.E., Thatcher, M.J.D. and LeBlanc, M.M., 1995. Relationship between prostaglandin and uterine clearance of radiocolloid in the mare. Biol. Reprod. Mono, 1: 495-500. Evans, M.J., Hamer, J.M., Gason, L.M., Graham, C.S., Asbury, A.C. and Irvine, C.H.G., 1986. Clearance of bacteria and non-antigenic markers following intra-uterine inoculation into maiden mares: effect of steroid hormone environment. ‘lheriogenology, 26: 37-50. Evans, M.J., Hamer, J.M., Gason, L.M. and Irvine, C.H.G., 1987. Factors affecting uterine clearance of inoculated materials in mares. J. Reprod. Fertil., Suppl., 35: 327-334.
T. Katila /Animal
Reproduction Science 42 (1996) 197-204
203
Hinrichs, K., Cummings, M.R., Sertich, P.L. and Kenney, R.M., 1988. Clinical significance of aerobic bacterial flora of the uterus, vagina, vestibule, and clitoral fossa of clinically normal mares. J. Am. Vet. Med. Assoc., 193: 72-75. Hughes, J.P. and Loy, R.G., 1969. Investigations on the effect of intrauterine inoculations of Streptococcus zooepidemicus in the mare. Proc. 15th Annual Convention of the American Association of Equine Practitioners, pp. 289-292. Katila, T., 1995. Onset and duration of uterine inflammatory response of mares after insemination with fresh semen. Biol. Reprod. Mono, 1: 515-5 17. Katila, T., Lock, T.F., Hoffman, W.E. and Smith, A.R., 1990. Lysozyme, alkaline phosphatase, and neutrophils in uterine secretions of mares with differing resistance to endometritis. Tberiogenology, 33: 723-732. Kenney, R.M. and Doig, P.A., 1986. Equine endometrial biopsy. In: D.A. Morrow (Editor), Current Therapy in Theriogenology. W.B. Saunders, Philadelphia, PA, pp. 723-729. Kenney, R.M. and Khaleel, S.A., 1975. Bacteriostatic activity of the mare uterus: A progress report on immuno-globulins. J. Reprod. Fertil., Suppl., 23: 357-358. Kotilainen, T., Huhtinen, M. and Katila, T., 1994. Sperm-induced leukocytosis in the equine uterus. Theriogenology, 41: 629-636. LeBlanc, M., 1994. Breakdown in uterine defense mechanisms in the mare: Is a delay in physical clearance the culprit? Proc. of the Annual Meeting of the Society of Theriogenology, Kansas City, MO, pp. 121-129. LeBlanc, M.M., Neuwirth, L., Asbury, A.C., Tran, T., Mauragis, D. and Klapstein, E., 1994. Scintigraphic measurement of uterine clearance in normal mares and mares with recurrent endometritis. Equine Vet. J., 26: 109-l 13. LeBlanc, M.M., Johnson, R.D., Caldenvood Mays, M.B. and Valderrama, C., 1995. Lymphatic clearance of India ink in reproductively normal mares and mares susceptible to endometritis. Biol. Reprod. Mono, I: 501-506. Liu, I.K.M., Cheung, A.T.W., Walsh, E.M., Miller, M.E. and Lindenberg, P.M., 1985. Comparison of peripheral blood and uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis: Chemotactic and cell elastimetry analysis. Am. J. Vet. Res., 46: 917-920. Liu, I.K.M., Cheung, A.T.W., Walsh, E.M. and Ayin, S., 1986. The functional competence of uterine-derived polymorpho-nuclear neutrophils (PMN) from mares resistant and susceptible to chronic uterine infection: A sequential migration analysis. Biol. Reprod., 35: 1168-l 174. Magnusson, U. and Jonsson, K., 1991. A method for the accurate measurement of opsonic activity in uterine secretions of the mate. Theriogenology, 36: 737-747. Peterson, F.B., McFeely, R.A. and David, J.S.E., 1969. Studies on the pathogenesis of endometritis in the mare. hoc. 15th Annual Convention of the American Association of Equine Practitioners, pp. 279-287. Strzemienski, P.J., Do, D. and Kenney, R.M., 1984. Anti-bacterial activity of mare uterine fluid. Biol. Reprod., 31: 303-311. Troedsson, M.H.T., 1991. Uterine defense mechanisms in the mare. Ph.D. Thesis, University of California, Davis, USA. Troedsson, M.H.T. and Liu, I.K.M., 1992. Measurement of total volume and protein concentration of intrauterine secretion after intrauterine inoculation of bacteria in mares that were either resistant or susceptible to chronic infection. Am. J. Vet. Res., 53: 1641- 1644. Troedsson, M.H.T., Liu, I.K.M., Ing, M., Pascoe, J. and Thurmond, M., 1993. Multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. J. Reprod. Fertil., 99: 307-313. Troedsson, M.H.T., Liu, I.K.M., Ing, M. and Pascoe, J., 1995. Smooth muscle electrical activity in the oviduct and the effect of oxytocin, prostaglandin F,, and Prostaglandin E, on the myometrium and the oviduct of the cycling mare. Biol. Reprod. Mono, 1: 475-488. Van Camp, S., 1986. Breeding soundness examination of the mare and common genital abnormalities encountered. In: D.A. Morrow (Editor), Current Therapy in Theriogenology. W.B. Saunders, Philadelphia, PA, pp. 654-661. Washburn, S.M., Klesius, P.H., Ganjam, V.K. and Brown, B.G., 1982. Effect of estrogen and progesterone on the phagocytic response of ovariectomized mares infected in utero with P-hemolytic streptococci. Am. J. Vet. Res., 43: 1367-1370.
204
T. Katila/Animal Reproduction Science 42 (1996) 197-204
Watson, E.D.,‘1988. Opsonins in uterine washings influencing in vitro activity of equine neutrophils. Equine Vet. J., 20: 435-437. Watson, E.D., Stokes, C.R. and Boume, F.J., 1987a. Influence of arachidonic acid metabolites in vitro and in uterine washings on migration of equine neutrophils under agarose. Res. Vet. Sci., 43: 203-207. Watson, E.D., Stokes, C.R. and Boume, F.J., 1987b. Cellular and humoral defence mechanisms in mares susceptible and resistant to persistent endometritis. Vet. Immmunol. Immunopathol., 16: 107- 121. Watson, E.D., Stokes, C.R. and Boume, F.J., 1987c. Influence of administration of ovarian steroids on the function on neutrophils isolated from the blood and uterus of ovariectomized mares. J. Endocrinol., 112: 443-448. Watson, E.D., Stokes, C.R., David, J.S.E. and Boume, F.J., 1987d. Concentrations of uterine luminal prostaglandins in mares with acute and persistent endometritis. Equine Vet. J., 19: 31-37. Watson, E.D., Stokes, C.R. and Boume, F.J., 1988. Influence of ovarian steroids on adherence (in vitro) of Streptococcus zooepidemicus to endometrial epithelial cells. Equine Vet. J., 20: 37 l-372. Widders, P.R., Stokes, C.R., David, J.S.E. and Boume, F.J., 1985. lmmunohistological studies of the local immune system in the reproductive tract of the mare. Res. Vet. Sci., 38: 88-95. Williamson, P., Munya, S., Martin, R. and Penhale, J., 1987. Dynamics of the acute uterine response to infection, endotoxin infusion and physical manipulation of the reproductive tract in the mare. J. Reprod. Fertil., Suppl., 35: 317-325.