Cytological and bacteriological examination of the Mare's endometrium

CYTOLOGICAL A N D B A C T E R I O L O G I C A L E X A M I N A T I O N OF THE MARE'S E N D O M E T R I U M

Derek Brook, BVMS, MS, DipABVP, FRCVS

INTRODUCTION

The diagnosis of equine endometritis has been the subject of considerable debate and discussion over the past few decades. Many of the earlier workers thought that the isolation of bacteria from any part of the reproductive tract was cause for concern.13 2728Gradually this idea began to change as evidence emerged that the posterior part of the tract could contain bacteria which need not have a n y effect on fertility. 3 19 31 34 41 It was acknowledged that bacteria could be present in the uterus without there being any evidence of a distruption of normal fertility. 34 Many of the earlier studies were performed using unguarded culture devices, and it is therefore possible that cervical contaminants were obtained during.the culturing procedure. 4 10 14 16 The necessity to distinguish between uterine and cervical isolates has been stressed, 23 31 and it has been shown that the cervical microflora is not necessarily the same as that of the uterus. 5 30 34 A recent paper on the relationship of various factors on broodmare fertility showed no positive correlation between the isolation of pathogens from the uterus and subsequent foaling rates. 36 The method of collection of the samples was by guarded culture instrument,abut the paper did not indicate if the "infected mares" were treated prior to conception occurring. The existence of a state of unnocuous superficial contamination of the e n d o m e t r i u m , with pathogenic bacteria, has been documented,15 21 a n d this p r e s u m a b l y o c c u r r e d commonly in the mares studied by Schideler. 36 In order not to prescribe unnecessary treatment to mares with only superficial contamination, it would be beneficial to Author's address: 20515 Covina Hills Rd., Covina, CA 91724 Acknowledgement: 1 am grateful for financial assistance from the California Thoroughbred Breeders Foundation. aSquires E. Personal communication 16

be able to distinguish between bacteria causing only a contamination and bacteria causing a significant endometritis. In an attempt to do this several studies have been c o n d u c t e d on the application of exfoliative endometrial cytology. 7 23 32 37 39 40 It has been established that there is usually a good correlation between the presence of bacteria and the presence of neutrophils, 8 23 39 thereby indicating, in these studies at least, that superficial contamination was relatively rare. This latter statement is made with the assumption that neutrophils are not normally present in the uterine lumen of a healthy mare, and that their presence is indicative of a significant inflammatory process. In the hope of further clarifying these points, a study was instigated whereby a group of mares were repeatedly swabbed during various phases of their r e p r o d u c t i v e life, a n d the s w a b s s u b j e c t e d to simultaneous bacteriological and cytological examinations. This report describes how the swabs were obtained, processed and evaluated. It endeavors to interpret the findings in the light of subsequent reproductive performance. MATERIAL

AND

METHODS

Mares Sampled: One hundred and thirty-four Thoroughbred mares, comprising all the mares on two breeding farms, were used in this study. Mares were swabbed at different stages of the estrus cycle, as well as post-foaling and postnatural service. Many samples were taken in attempt to elucidate points of interest and not because of necessity as far as achieving conception, e.g. many maiden mares were sampled. A total of four hundred and ninety samples were obtained. EQUINE VETERINARY SCIENCE

Figure 1. Kalayjian guarded culture rod with swab enclosed (a) and exposed (b), Rocket (London) biopsy punch (c).

Figure 2. Smear from a maiden mare in estrus showing large number of epithelial cells and mucous. (Diff-Quick stain x 100).

Collection Procedure:

The mares were positioned in an examination chute, situated in a clean dry dustfree area. Their tails were bandaged and the vulva and surrounding areas washed with mild soap and water. The area was then thoroughly dried with disposable paper towels. A guarded culture instrument b (Figures 1, 1A) was introduced into the vagina by means of a lubricated c gloved hand. The index finger was inserted into the cervix and the tip of the culture roR guided into the uterus. The central plastic rod was advanced, displacing the plastic guard cap (Figure lb) thus exposing the swab within the uterus. T h e i n n e r plastic rod was twisted back and forth several times with the swab in contact with the endometrium. The rod was then pulled back into the outer plastic tube and the entire device withdrawn from the reproductive tract. Bacteriological E x a m i n a t i o n

Within one hour of collection the swabs were stroked onto blood and MacConkey agar plates, labeled and incubated aerobically at a p p r o x i m a t e 37°C. They were examined for evidence of bacterial growth at twelve hour intervals, note being made of the a p p r o x i m a t e number of colonies present and time of their appearance. Organisms n o t r e c o g n i z a b l e by g r o w t h c h a r a c t e r i s t i c s were submitted to a specialized laboratory for identification. No attempt was made to classify organisms as either pathogenic or non-pathogenic. The number of colonies on the plates were counted after forty-eight hours and at that stage the cultures were considered negati~ve if there were 0-5 colonies, doubtful if there were 5-10 colonies, and positive if there were m o r e than 10 colonies on the plate. Cytological E x a m i n a t i o n

A clean dry microscope slide was held in one hand and the s a m e s w a b as h a d b e e n used to m a k e the bacteriological examination was rolled back and forth several times on the center of the slide. It was then labeled, air dried, fixed with methyl alcohol and stained bKalayjian Industries, Long Beach, CA c KY Jelly, Johnson and Johnson, New Brunswick, NJ Volume 5, Number 1

Figure3. Higher magnification of Figure2"Taiis"of mucus are apparent, originating at each epithelial cell. (Diff-Quik x

400).

by the "Wright's Dip Stat" stain, d The slide was examined and note made of the different cell types present, there is a p p r o x i m a t e numbers and the presence or absence of mucous and bacteria. The following cell types were observed: a. Epithelial cells. These were the most c o m m o n cells seen and represented exfoliation from the lining of the endometrium. There was considerable distortion and disruption of m a n y of these cells and wide variation in the number of epithelial cells seen. Some smears contained large sheets of cells (Figures 2 & 3) and others had virtually none. Maiden and younger mares tended to produce smears containing m o r e epithelial cells than older mares. Smears f r o m mares in anestrus contained cells which were usually either cuboidal or low columnar (Figure 4). Diestrus samples generally showed slightly taller epithelial cells (Figure 6). The tell columnar cells (up to 50 lzm) often exhibited basal c y t o p l a s m i c vacuolation (Figure 6). Cilia were apparent on the luminal borders of only very few cells (Figure 4). On dMedi-Chem Inc., Santa Monica, California 17

Figure 4. Cubiodal epithelial cells in a smear from an anestrus mare. (Diff-Quik x 100).

Figure 5. Columnar epithelial cells from a mare in diestrus. Arrow shows ciliated boarder. (Diff-Quik x 1000).

Figure 6. Smear from a mare in mid-estrus showing tall columnar cells with basal cytoplasmic vacuolation, (arrow). ~(Diff-Quik x 1000).

Figure 7. Smear from a reproductively normal mare showing degenerate epithelial cell nuclei. (Diff-Quik x 1000).

Figure 8. Cornified epithelial cells in an endometrial smear, indicative of vaginal contamination. This was obtained from a mare 10 days post-partum.

Figure 9. Endometrial smear from an infected mare showing (a) e r y t h r o c y t e s , (b) large m a c r o p h a g e s , (c) i m m a t u r e neutrophils, (d) mature oeutrophils, (e) bacteria. (Diff-Quik x

400). 18

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Figure 10. Endometrial smear showing degenerate epithelial cells and an eosinophil (arrow). (Diff-Quik x 400).

Figure 11. Smear from a mare 6 days post-partum showing multinucleate and macrophage (arrow). (Diff-Quik x 400).

Figure 12. P o s t - f o a l i n g e n d o m e t r i a l smear s h o w i n g erythrocytes and vacuolated macrophages (arrow). (Diff-Quik x 1000),

Figure 13. Smear from a mare infected with P s e u d o m o n a s spp., showing debris and clumps of bacteria (arrow). (Diff-Quik

several occasions degenerate epithelial cell nuclei were numerous on the smears (Figure 7). Cornified epithelial cells (Figure 8) were seen rarely, usually in post-foaling samples. degenerate. Sometimes it was difficult to distinguish between degenerate eipthelial cells and degenerate neutrophils. Occasionally bacteria could be seen either overlying the neutrophils or actually within them (Figure 9). An a p p r o x i m a t e quantitative estimation was made as follows: No neutrophils per l0 fields x 400 magnification was considered negative; one to five in 10 fields was considered doubtful and more than five per 10 fields was considered postitive. c. Eosinophils. These cells were a very rare finding, only being apparent in two smears (Figure 10). One smear with only scattered eosinophils, was from a mare five days post-foaling and the other one, which produced large numbers, was f r o m a mare who developed a vaginal discharge ten days after being covered. d. Lymphocytes. Again these were rare and sometimes very difficult to distinguish f r o m i m m a t u r e neutrophils.

e. Macrophages. These were very c o m m o n in postfoaling smears, especially from mares producing an excessive discharge. They often appeared large, multinucleated and vacuolated (Figures II, 12 & 13). f. Erythrocytes. These were again not ¢ J m m o n but usually occurred in smears taken in the post-foaling period (Figures 9, 1 I and 12). They were occasionally also seen in cases of severe acute endometritis. g. Bacteria and Yeasts. These were again an infrequent finding, even in smears from infected mares. Their appearance varied f r o m large numbers of organisms to only a few sporadic ones (Figures 9, 13 & 14). Yeasts were seen occasionally and were usually a c c o m p a n i e d by neutrophils (Figure t5). h. Mucous. The a m o u n t visible on the smear varied greatly and was most obvious in young mares in estrus (Figure 2). M a r e s s h o w i n g c h r o n i c p r o b l e m s were u s u a l l y biopsied using a Rocket biopsy bunch (Figure IC). The technique and method of interpretation is described elsewhere 2~. Treatment of infected mares followed

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x 400).

19

Figure 14. Gram positive anaerobic rod from a smear o f a post-partum mare. (Diff-Quik x 1000).

Figure 15. Endometrial smear from a mare infected with (7. albicans showing fungal elements. (Diff-Quik x 400).

standard procedures as outlined in other reports ~2. Minimum contamination technique breeding was used on some mares and followed published methods2L RESULTS A total of 490 swabs were taken from 134 mares. After being subjected to cytological and bacteriological examination, the samples were divided into the following categories: t. 290 (59%) were negative both bacteriologically and cytologically. 2. 96 (20%) were positive both bacteriologically and cytologically. 3.24 (5%) were positive bacteriologically and negative cytologically. 4. 39 (8%) were negative bacteriologically and positive cytologically. 5. 41 (8%) were doubtful. 20

As would be expected a fairly large proportion of the mares producing both negative results conceived without any problem. False negatives occurred in 15% of the samples and all these were from mares not fully in heat at the time of collection. A pregnant mare was sampled when she showed signs of estrus 18 days post-covering. T w e n t y p e r c e n t of the swabs p r o d u c e d b o t h neutrophils and bacteria. Quite a high percentage of these were from mares that had either recently foaled or recently been covered. The remainder, except one, fell into two categories, those with endometritis which responded to treatment and those with the condition which did not respond. The treatment was often extensive and c o m p r i s e d i n t r a - u t e r i n e i r r i g a t i o n s through several heat periods. Several mares were biopsied and the presence of endometritis confirmed histologically. One mare conceived without treatment despite having a moderate growth of E. coli and numerous neutrophils on a smear; she was covered and left the breeding farm before any treatment could be given. The majority of samples producing bacteria but no neutrophils came from mares not fully in estrus. The bacteria most commonly isolated in this group was E. coli. Samples producing neutrophils but no bacteria came from three categories of mares, those having either foaled recently, been covered recently or undergone recent antibiotic treatment. There were also several samples in this group which came from mares not fully in heat, but which when re-sampled showed presence of bacteria. Of the samples that were doubtful categories are worthy of mention. One was a group of mares, usually younger ones, producing a slight growth of E. coli, which subsequently rid themselves of infection and conceived. The other was a group of mares, usually older, which subsequently produced more positive results and often. had a history of chronic endometritis. Post.foaling

samples

Sixty-seven samples were taken from eighteen mares at times ranging from nine hours to eleven days post-partum Even though all foals were born normally and no retained afterbirths occurred, there was tremendous variation in the amount of apparent affection that occurred. The severity of this did not seem to be age related and often the clinical appearance of the mare gave no indication of the severe r e a c t i o n (as d e t e c t e d by c y t o l o g i c a l examination) occurring within her uterus. Mares five days post-foaling produced smears containing large n u m b e r s o f red b l o o d cells, m a c r o p h a g e s a n d neutrophils, but were completely free of inflammatory cells at nine days. Other mares revealed hardly any reaction through this entire period. As an arbitrary guide, mares with no bacteria and either none or only a few neutrophils at seven to ten days or earlier were usually covered on the foal heat. Those with positive samples at ten days or more were usually either short cycled or a l l o w e d to c o m e b a c k i n t o h e a t n a t u r a l l y at approximately thirty days post-partum, lfthey were then free of bacteria and neutrophils they were covered, if not they were either allowed one more heat period to clear up EQUINE VETERINARY SCIENCE

or they were treated. All the mares in this group conceived.

Samples taken post-coitus F i f t y - f o u r s a m p l e s were t a k e n f r o m t h i r t y - t w o different mares at periods up to five days post-coitus. Fifteen mares conceived in the estrus that the samples were taken and none of them were given any treatment. All fifteen had a negative culture twenty-four hours postservice, but seven still had neutrophils present at that time. Of the remaining mares, six were given a twentyfour hour post-breeding treatment because of their previous histories. Five of these six had both bacteria and neutrophils present at this time. All six were re-examined at forty-eight hours and all but one were free of bacteria at this stage. The remaining one was re-examined at seventy-two hours and found to be clear. These six mares conceived. The remaining eleven mares did not conceive in the estrus that the sample was taken. Six of the eleven had, as had the previous group, bacteria still present at twenty-four hours post-coitus. Despite minimum contamination technique breedings in subsequent heats only five of these mares eventually conceived. One mare, who had bacteria present at four days post-covering, c o n c e i v e d b u t s u b s e q u e n t l y r e s o r b e d the fetus. Consequently she was proved to be infected. DISCUSSION The collection of material for the diagnosis of equine endometritis has been the subject of recent review articles,l 9 and the methods used in this study are discussed in those articles. The variation in the size of fhe eipthelial cells seen, from cuboidal in anestrus to tall columnar in estrus, has been noted by others.6, 17 21 23 35 Basal cytoplasmic vacuolation in the tall columnar cells of the estrus smears has also been noted on biposy samples. 2~ The presence of degenerate epithelial cells has been prevously reported to be associated with poor reproductive performance 37 but in this study that relationship could not be confirmed. Cornified epithelial cells were thought to be an indication of vaginal contamination and were usually only present in post-foaling smears. The great variation in the number of epithelial cells seen is difficult to explain. It may be due to collection procedures and a curettage device might be more beneficial in obtaining larger numbers of cells. 24 29 Generally the younger healthier mares produced the greatest number of epithelial cells. Cilia were apparent on the luminal borders of very few epithelial cells, which may be due to collection and processing procedures. Neutrophils were usually present in large numbers, if at all, but on rare occasions only a few were seen and m o s t of these samples were from mares not fully in heat. Neutrophils undergoing pyknosis, kariolysis, etc., were usually associated with more severe infections. This has also been noted by others. 29 Lymphocytes were rarely seen and it has been proposed that many of them undergo degeneration during transepithelial migration. 21 Eosinophils were also very rare, being seen in only two smears, it has been stated that eosinophils are usually associated with yeast and Volume 5, Number 1

fungal infections, but this was not apparent in this series. 29 One m a r e p r o d u c e d a l a r g e n u m b e r o f eosinophils approximately ten days after being covered and a biopsy taken at that stage also showed heavy eosinophil infiltration of sub-epithelial tissue. The possibility of an allergic reaction to the semen was considered, but the mare subsequently conceived t o t h e same stallion. The presence of large macrophages appeared to be confined to smears taken during the postfoaling period and this would agree with work done on endometrial biopsies. 21 The fact that bacteria were visible on only a few of the smears from mares producing infected swabs is difficult to understand. This finding has been noted by others 39 and it may be that different staining techniques would be helpful. As would be expected, a high percentage of mares whose uteri were free of bacteria and neutroohils, conceived easily and produced live foals. It is important to note that there were a number of negative swabs taken from mares in partial estrus which were subsequently positive when repeated on the same mares in full estrus. False negatives have been reported previously, 3 8 10 162633 and it may be due to the dryness of the endometrium in anestrus, diestrus or even early estrus, The fact that none of the mares conceived who produced negative results when in partial estrus and positive swabs in full estrus, is thought to possibly indicate a compromised defense system. Healthy mares subjected to e x p e r i m e n t a l infection will show a rapid reaction to the infection regardless of whether they are in estrus or diestrus. ~9 Several of the chronically infected mares in this series would show repeated negative culture and smear in diestrus and then positive culture and smear in estrus and because of this it is thought advisable to only sample mares in strong estrus. The finding of samples containing aerobic bacteria but no neutrophils, was quite rare and in agreement with published literature, 39 It is thought that this is indicative of either a "carrier state" or that the tissue is not able to react to bacteria. Contamination from the posteria tract during the collection procedure is another possibility 9 31 but should be minimal using the particular method here. Yeasts have been shown to be often associated with surface contamination, 2~ but i n this series they were usually accompanied by neutrophils. Observing neutrophils in the absence of aerobic bacteria is thought to arise in a variety of situations 3 39 e.g. the presence of antimicrobial preparations in the uterus, the existence of bacteria other than aerobes, or non-infectious irritation. Results of this study confirm this view in that 75% of the samples showing neutrophils but no bacteria came from mares that had either just foaled, recently been covered or were undergoing treatment. If anaerobes had been a significant problem one would have expected more cases showing positive cytology but negative culture results. The possibility of anaerobic and aerobic bacteria being present at the same time should not be excluded. 9 Samples taken in the post-foaling period showed that there was an enormous variation in the reaction recurring. Some young apparently healthy mares would become severely infected and have an active pronounced 21

c e l l u l a r r e a c t i o n d e s p i t e an a p p a r e n t l y n o r m a l parturition. Alternatively, others appeared to have a very minor neutrophil response which lasted twenty-four hours or less. It was impossible in the large majority of these mares to correlate the clinical signs with the cellular response occurring e.g. some mares would have an intense large macrophage infiltration with no external evidence of a discharge. It may eventually be possible to base the decision on whether or not to cover a mare at foal heat on a cytological examination conducted on a certain day post-foaling. In this very limited study, it appeared that mares that were free of bacteria and neutrophils by the seventh day generally conceived if covered during foal heat. Swabs taken post-natural service showed a remarkable variation in the degree of reaction occurring in the endometrium. Young healthy mares which conceived easily were free of bacteria by twenty-four hours postservice, but 40% still had neutrophils present; whereas at forty-eight hours the neutrophils had largely disappeared. Problem mares usually had bacteria present for longer than twenty-four hours and some benefit was gained by detecting these mares and subsequently using minimum contamination technique breeding. It is probable that mares with compromised endometrial defense systems can be found by cytological and bacteriological examination at twenty-four and fortyeight hours post-breeding thereby allowing one to instigate methods to assist the mare in combating infection.

REFERENCES 1 Allen WE and Newcombe JR: Aspects of genital infection and swabbing techniques in the mare. Vet Rec 104:228-231, 1979. 2. Asbury AC: Equine Medicine and Surgery. 3rd Edition, 13051308, 1982. American Veterinary Publications Inc., Santa Barbara. 3. Asbury AC: Current Therapy in Equine Medicine, 410-414, 1983. WB Saunders and Co., Philadelphia. 4. Bain AM: The role of infection in infertility in the Thoroughbred mare. Vet Rec 78:168-173, 1966. 5. Blanchard TL, Garcia MC, Hurtgen JP and Kenney RM: C o m p a r i s o n of two t e c h n i q u e s for o b t a i n i n g e n d o m e t r i a l bacteriological cultures in the mare. Theriogen. 16:85-91, 1981. 6. Brandt GW: The significance and interpretation of uterine biopsy in the mare. Prec. 16th Ann Conv A A E P 279-294, 1970. 7. Brook D: Diagnosis of equine endometrial Candidiasis by direct s m e a r and successful t r e a t m e n t with a m p h o t e r i e i n B and oxytetracyeline. JS Aft Vet Assoc) 53:261-263, 1982. 8. Brook D: Exfoliative Endometrial Cytology in the Mare. M Sc Thesis, University of Stellenboseh, Stellenboseh, South Africa, 1983. 9. Brook D: The diagnosis of equine bacterial endometritis. J Compend C Ed 6:5 $200, 1984. 10. Bruner DW: Notes on genital infection in the mare. Cornell Vet 41:65-68, 1951. 11. Collins SM: A study of the incidence of cervical and uterine infection in Thoroughbred mares in Ireland. Vet Rec 78:673-676, 1964. 12. Conboy HS: Diagnosis and therapy of equine endometritis. Proc 24th Ann Conv A A E P St. Louis, Missouri. 165-174, 1978. 13. Day FT: The veterinary clinicians approach to breeding problems in mares. Vet Rec 69:125%1267, 1957. 14. Dimock WW and Edward PR: Pathology and bacteriology of the reproductive organs of mares in relation to sterility. Res Bull Ky Agric Exp Stn 186, 1928.

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15. Farrelly BT and Mullaney PE: Cervical and uterine infection in Thoroughbred mares. Ir Vet J 11:201-212, 1964. 16. Gadd JD: The relationship of bacterial culture, microscopic smear examination and medical treafment to surgical correction of barren mares. Proc 24th Ann Cony A A E P St. Louis, Missouri. 362369, 1975. 17. Hammond J and Wadzieki K: Anatomical and histological change during the oestrus cycle of the mare. Proc R Soc B 130:1,1941. 18. Hoppe RE Domanski A and Dobrowolska A: Cause and treatment of inflammation of the genital tract in the mare with special reference to the process caused by haemolytic cocci. Third Int Congr Animal Reprod Cambridge, England 11:83, 1956. 19. Hughes JP, Loy RG, Asbury AC and Burd HE: The occurrence of Pseudomonas in the reproductive tract of mares and its effect on fertility. Cornell Vet 56:595-609, 1966. 20. Hughes JP and Loy RG: Investigations on the effect of intrauterine inoculations of streptococcus zooepidemicus in the mare. Proc 15th Ann Cony A A E P 289-292, 1969. 21. Kenney RM: Clinical and pathological changes of the mare's endometrium as detected by biopsy, with a note on early embryonic death. JA VMA Assoc 172:241-206, 1978. 22. Kenney RM, Bergman RV, Cooper WL and Morse GW: Minimal contamination techniques for breeding mares: technique and p r e l i m i n a r y findings Proc 21st A n n Cony A A E P Boston Massachusetts, 327-335, 1975. 23. Knudsen O: Endometrial cytology as a diagnostic aid in mares. Cornell Vet 54:415-422, 1964. 24. Knudsen O: A combined cytological and bacteriological examination in the mare. Prec. 28th Ann Conv A A E P 431-434, 1982. 25. Knudsen O and Sollen P. Methods for obtaining samples from uterus i0 COw and mare. Nord Vet Med 13:449-450, 1961. 26. Lieux. P, Baker RH, DeGroot A, Laskey HH, Raynor RE, Simpson JG and Tohler E Results of a survey on bacteriological culturing of broodmares. JA UVMA) 1460-1464,1970. 27. Millar R and Francis J The relation of clinical and bacteriological findings to fertility in Thoroughbred mares. Aust Vet J 50:351-355, 1974. 28. MiUar, WC and Barnett, G Twenty years of equine research. The Veterinarian 5:51-64, 1968. 29. Neely D: Equine Reproduction, 41-51, 1983. Veterinary Learning Systems, Inc. 30. Newcombe JR: Comparison of the bacterial flora of three sites in the genital tract of the mare. Vet Rec 102:169-170, 1982. 31. Ommert W: Broodmare symposium. Proc lOth Ann Cony A A E P 99-101, 1964. 32. Peterson FB, McFeely RA and David JSE: Studies on the pathogenesis of endometritis in the mare. Proc 15th Ann Conv A A E P 279-289, 1969. 33. Rasbech NO: Effects of equine genital infections on reproduction. Nord Vet Med 17:305-317, 1965. 34. Scott P, Daley P, Gidley-Baird G, Sturgess S and Frost AJ: The aerobic bacterial flora of the reproductive tract of the mare. Vet Rec 17:58-61, 1971. 35. Seaborn E: The oestrus cycle in the mare and some associated phenomena. Anat Ree 30:22%321, 1925. 36.Schideler RK, McChesney AE, Voss JL and Squires EL: Relationship of endometrial biopsy and other management factors on fertility of broodmars. J Eq Vet Sci 2:5-10, 1982. 37. Solomon WJ, Sehultz RH and Fahning ML: A study of chronic infertility in the mare utilizing uterine biopsy, cytology cultural methods. Proc 18th Ann Conv A A E P 55-67, 1974. 38. Swerczek TW Early fetal death and infectious placental disease in tb~ mare. Proc 26th Ann Conv A A E P 173-179, 1980. 39. Wingfield-Digby NJ: The technique and clinical application of endometrial cytology in the mare. Equine Vet J 10:167-170, 1978. 40. Wingfield-Digby NJ and Ricketts SW: Results of concurrent bacteriological and cytological examinations of the endometrium of mares in routine stud farm practice J Reprod Fert Suppl 32:181-185, 1982. 41. Woolcock JB: Equine Bacterial Endometritis. Veterinary Clinics o f North America: Large Animal Practice 241-251. WB Saunders & Co., Philadelphia, 1980.

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