Epidemiologic aspects of Taylorella equigenitalis

Contagious equine metritis (CEM) is a sexually transmissible disease in mares. Although the disease is commonly diagnosed by culturing the causative bacterium Taylorella equigenitalis (T. equigenitalis) . false negative results do occur. A recently developed Polymerase Chain Reaction (PCR) assay, however, appeared to be much more sensitive, with initial results indicating an unexpected high incidence of the agent in selected horses. In this study, samples from 107 randomly selected mares with no clinical signs of CEM submitted for conventional culture were all negative for T. equigenitalis . but in the PCR-assay 54 (49%) were positive for Taylorella -DNA. Positives in the PCR-assay were found in all breeds tested, even in horses imported from the isolated population in Iceland. These findings suggest that T. equigenitalis was present long before it was first isolated in 1977, The high incidence of Taylorella in horse populations without apparent clinical signs of CEM, the occurrence of incidental clinical case and the known variability between strains, all indicate that Taylorella is endemic in the horse population. In order to explore whether the organism is present in species other than the horse, we also used the PCR-assay on clinically health donkeys (n = 14), zebras (n = 15), Przewalski horses (n = 2) and cows (n = 21). All the animals showed negative results except one of the Przewalski horses, and one cow that was repeatedly found to give positive reaction. We also found that the fertility of 7 stallions with cultures positive for Taylorella (6 used in an AI-program and 1 by natural breeding) was not affected, as shown by the normal range of foaling rates in mares inseminated or bred by these stallions. The overall results may be interpreted to mean that Taylorella is of limited significance in horse breeding.     

Contagious equine metritis--outbreak of the disease in Kentucky and laboratory methods for diagnosing the disease.

Contagious Equine Metritis (CEM) was initially reported during the 1977 breeding season in England (Crowhurst, 1977) and Ireland (Timoney, Ward & Kelly, 1977. The disease has also been diagnosed in France and Australia (Huges, Bryden & MacDonald, 1978). The first occurrence of CEM in the United States followed the importation or 2 stallions from France late in 1977 which resulted in an outbreak early in the 1978 breeding season (Swerczek, 1978). Mares usually develop clinical signs of CEM 8--10 days after being covered by an infected stallion, when a copious, greyish discharge is seen. Other mares may not show any outward signs of disease, but may have a shortened dioestrous period. Many mares recover spontaneously from the disease, but a small proportion become carriers of the CEM organism. The stallion does not show any clinical signs of disease, but remains a carrier. In this paper we recommend various laboratory procedures for the diagnosis of CEM in mares and stallions.     

The role of international transport of equine semen on disease transmission.

Despite the numerous benefits of having the capability to transport semen internationally, there are serious potential ramifications if that semen is contaminated with a communicable disease. BACTERIA: Many commensal bacteria colonize the exterior of the stallion penis and are not regarded as pathogenic. They may be cultured from an ejaculate. Alterations of the normal bacterial flora on the exterior genitalia may cause the growth of opportunistic bacteria such as Klebsiella pneumonia, Pseudomonas aeruginosa, Streptococcus zooepidemicus, which, if inseminated, may cause infertility in susceptible mares. Contagious equine metritis (CEM), a highly transmissible, true venereal disease of horses, is caused by the gram-negative coccobacillis, Taylorella equigenitalis. Even with the use of rigorous testing protocols, the current techniques used may not ensure accuracy of results. VIRUSES: Equine coital exanthema (equine herpes virus type 3; EHV-3) is a highly contagious virus that causes painful lesions on the stallion's penis and mare's vulva. Although it is primarily transmitted through coitus, infected fomites have also been implicated in its spread. Therefore, it is possible that the virus can potentially be transmitted to the ejaculate through penile contact with an artificial vagina or sleeve. Equine arteritis virus appears to be becoming more prevalent in recent years. The most common method of transmission is through respiratory disease, but the organism can also be shed in the semen of asymptomatic stallions. Equine infectious anemia virus has also been found to be present in the semen of an infected stallion, although no evidence exists at this time that there is venereal transmission of this disease. PROTOZOA: Dourine, caused by Trympanosoma equiperidum, is a venereal disease found only in Africa, South and Central America and the Middle East. Serological testing using complement fixation is recommended for diagnosis. Piroplasmosis, a disease caused by Babesia equi or by a less severe strain, Babesia caballi, has received a great deal of attention in recent years due to the increased transfer of horses between countries. It is considered to be enzootic in many areas of the southern US, and is found throughout the world. The protozoal agent is most often spread by ticks, but mechanical transmission has also been documented; therefore, there is concern for venereal transmission if blood from an infected horse contaminates the semen.     

The epidemiology of contagious equine metritis (CEM) in England 1977--1978).

Following an outbreak of CEM in England during 1977 a Code of Practice was introduced to control the disease in 1978. The Code recommended a bacteriological screening programme for Thoroughbred mares and stallions and improved standards of hygiene on the stud farm. As a result of the implementation of the Code a number of asymptomatic carrier mares was detected. Stallions which had transmitted CEM in 2977 and were treated did not transmit the disease during 1978. Two small outbreaks of CEM were reported during the 1978 breeding season.     

Equine arteritis virus

Equine arteritis virus (EAV) is a small, enveloped, positive-stranded RNA virus, in the family Arteriviridae , W.H.ich can infect both horses and donkeys. While the majority of EAV infections are asymptomatic, acutely infected animals may develop a wide range of clinical signs, including pyrexia, limb and ventral edema, depression, rhinitis, and conjunctivitis. The virus may cause abortion and has caused mortality in neonates. After natural EAV infection, most horses develop a solid, long-term immunity to the disease. Marzz and geldings eliminate the virus within 60 days, but 30 to 60% of acutely infected stallions will become persistently infected. These persistently infected animals maintain EAV within the reproductive tract, shed virus continuously in the semen, and can transmit the virus venereally. Mares infected venereally may not have clinical signs, but they shed large amounts of virus in nasopharyngeal secretions and in urine, which may result in lateral spread of the infection by an aerosol route. The consequences of venereally acquired infection are minimal, with no known effects on conception rate, but mares infected at a late stages of gestation may abort. Identification of carrier stallions is crucial to control the dissemination of EAV. The stallions can be identified by serological screening using a virus neutralization (VN) test. If positive at a titer of >/= 1:4, the stallion should be tested for persistent infection by virus isolation from the sperm-rich fraction of the ejaculate, or by test mating Shedding stallions should not be used for breeding, or should be bred only to mares seropositive from a natural infection or from vaccination, the mares should be subsequently isolated from seronegative horses for three weeks after natural or artificial insemination. A live attenuated (ARVAC) and a formalin-inactivated (ARTERVAC) vaccine are available. Both vaccines induce virus-neutralizing antibodies, the presence of which correlates with protection from disease, abortion, and the development of a persistent infection. Serological investigations indicate that EAV has a worldwide distribution and that its prevalence is increasing. As a consequence, an increasing number of equine viral arteritis (EVA) outbreaks is being reported. This trend is likely to continue unless action is taken to slow or halt the transmission of this agent through semen.     

Contagious equine metritis: a review

Contagious Equine Metritis (CEM) is a highly contagious venereal disease of horses caused by a fastidious, Gram-negative coccobacillus which grows best on chocolate agar under microaerophilic conditions (5-10% CO2). Clinically, the disease is characterized by a copious watery-to-mucopurulent, vaginal discharge two to ten days after breeding by an infected stallion (11, 13). Shortened estrous cycle lengths are common and may be the only indication of endometritis in some instances (7). Inapparent carriers of the disease in both the mare and stallion make control of the disease more difficult. Outbreaks of CEM have been reported in England, Ireland, France, Australia and the United States. The current information concerning CEM is reviewed.     

Genome sequence of Taylorella equigenitalis MCE9, the causative agent of contagious equine metritis

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We herein report the genome sequence of T. equigenitalis strain MCE9, isolated in 2005 from the urethral fossa of a 4-year-old stallion in France.     

Coital exanthema in stallions.

Equine coital exanthema can be produced experimentally in stallions by inoculation with an equine herpesvirus (strain 65/61) and be transmitted during coitus with an infected mare. Serological responses to this infection include the production of complement-fixing and serum-neutralizing antibodies which reach maximum levels 14 to 21 days after infection. Complement-fixing antibodies decline rapidly and are usually not detectable by 60 days after infection, whereas serum-neutralizing antibody activity is maintained for at least 1 year. This disparity provides a useful method for the diagnosis of recent infections and estimation of the temporal incidence of infection in groups of mares. It seems likely that clinically normal carrier mares spread the virus on studs where the disease has previously not occurred.     

Epidemiological observations on contagious equine metritis in Kentucky, 1978.

Contagious equine metritis, introduced by importation of 2 comtaminated stallions from France, affected 54 Thoroughbred brood mares during the 1978 breeding season in Kentucky. The infection was diagnosed bacteriologically and by the use of a complement fixation test. Although lateral spread to stallions, and probably to a few mares, occurred through human agency in the breeding sheds of 2 stud farms, control measures instituted early in the epidemic confined the disease to brood mares bred by stallion on only these farms.     

Tests for cooperative behaviour between stallions

Breeding groups with multiple stallions occur sympatrically with single-stallion breeding groups in feral horse, Equus caballus, populations. Mutualism and reciprocal altruism between stallions have been proposed to explain the origin and functioning of multistallion bands. However, empirical support for these hypotheses is contradictory and incomplete. Furthermore, there are no explicit tests of the predictions that each hypothesis makes about stallion behaviour and social structure. We compared nine multistallion and 18 single-stallion bands in the Kaimanawa Ranges, New Zealand. Compared with agonistic behaviours, affiliative behaviours were relatively unimportant in the relationships between stallions within bands. The number of stallions in the band did not have a positive influence on mare group size, stability, home range quality or reproductive success in bands. Furthermore, there was a positive relationship between aggression ('intolerance') by the dominant towards subordinate stallions and the subordinates' effort in mare group defence ('helping') but a negative relationship between helping effort by subordinates and their proximity to, and mating with, the bands' mares. Therefore, the predictions of the mutualism and reciprocal altruism hypotheses were not supported. Indeed, for some of the predictions we found the opposite outcomes to be true. Multistallion bands had significantly poorer reproductive success, and dominant stallions were less tolerant of subordinates that helped most and reduced their access to mares. Nevertheless, in all other respects Kaimanawa stallions in multistallion bands behaved like those described elsewhere. Thus, we reject cooperative hypotheses for multimale breeding groups in horses and discuss the mate parasitism and consort hypotheses as better alternatives. Copyright 2000 The Association for the Study of Animal Behaviour.     

Effects of racing on equine fertility

Racing and fertility are connected with each other in many ways. Stress and increased body temperature induced by racing may have negative effects on fertility, but on the other hand, high quality nutrition and management of racing horses may have positive effects. Fertility may also be genetically associated with racing performance. The analysed data consisted of Finnish mating records of Standardbreds (n=33,679) and Finnhorses (n=32,731), from 1991 to 2005, and the harness racing records of both mares and stallions. Fertility was measured by foaling outcome, and racing performance was measured by best time and number of races. We used racing results from the mating year and from the entire career, to study both short-term and long-term effects of racing on fertility. The analyses were conducted with a linear mixed model, where racing was fitted as a fixed factor. In a separate bivariate analysis we measured the genetic correlation of racing and fertility, applying a threshold model for the fertility trait. For mares, racing after the first mating or more than 10 times during the mating year diminished the foaling outcome. However, racing only before the first mating or 1-5 times during the mating year had positive effects on mare fertility. Stallion fertility did not suffer from racing during the mating year. The mares with the best career racing records had the highest foaling rates, but this was probably due to preferential treatment. The genetic correlation between best racing record and fertility was favourable but weak in the Finnhorse (-0.24±0.08), and negligible in the Standardbred (-0.15±0.11).     

Sexual behavior following introduction of a stallion into a group of mares

A rested stallion was introduced daily for 30 days into each of three herds of 20 mares. Observations of sexual and mating behavior were made for one hour. The stallion remained with a herd until another stallion was introduced the following day. Other mares were isolated from stallions and were bred by artificial insemination. The diameter or growth rate of the preovulatory follicle for the six days preceding ovulation and the length of the interovulatory interval for mares which did not become pregnant were not affected significantly by the presence of a stallion. The number of breedings per hour of observation (2.4 ±0.2) and the length of the interval from introduction of a stallion into the herd of mares to first breeding (12 ±1 min) were significantly different among stallions, but the length of the interval between breedings (17 ±2 min) was not. The mean number of breedings per stallion per hour was not affected significantly by the number of posturing (estrous) mares. The number of times that the stallion rebred the same mare when more than one mare postured during the observation hour (49%) was greater (P<0.01) than what would be expected to occur by chance (30%). The hypothesis that breeding occurs preferentially in those estrous mares that are closest to ovulation was not supported, except for significantly lower breeding activity in posturing mares on days 8 and 7 before ovulation and on days 0 (day of ovulation) and ?1 (26% bred) than on days 6 to 1 (52%).     

Fertility of frozen-thawed stallion semen cannot be predicted by the currently used laboratory methods

The aim of the project was to use current simple and practical laboratory tests and compare results with the foaling rates of mares inseminated with commercially produced frozen semen. In Exp. 1, semen was tested from 27 and in Exp. 2 from 23 stallions; 19 stallions participated in both experiments. The mean number of mares per stallion in both experiments was 37 (min. 7, max. 121). Sperm morphology was assessed and bacterial culture performed once per stallion. In Exp. 1, progressive motility after 0, 1, 2, 3, and 4 h of incubation using light microscopy, motility characteristics measured with an automatic sperm analyzer, plasma membrane integrity using carboxyfluorescein diacetate/propidium iodide (CFDA/PI) staining and light microscopy, plasma membrane integrity using PI staining and a fluorometer, plasma membrane integrity using a resazurin reduction test, and sperm concentration were evaluated. In Exp. 2, the same tests as in Exp. 1 and a hypo-osmotic swelling test (HOST) using both light microscopy and a fluorometer were performed immediately after thawing and after a 3-h incubation. Statistical analysis was done separately to all stallions and to those having ≥ 20 mares; in addition, stallions with foaling rates < 60 or ≥ 60% were compared. In Exp. 1, progressive motility for all stallions after a 2 – 4-h incubation correlated with the foaling rate (correlation coefficients 0.39 – 0.51), (p < 0.05). In stallions with > 20 mares, the artificial insemination dose showed a correlation coefficient of -0.58 (p < 0.05). In Exp. 2, the HOST immediately after thawing showed a negative correlation with foaling rate (p < 0.05). No single test was consistently reliable for predicting the fertilizing capacity of semen, since the 2 experiments yielded conflicting results, although the same stallions sometimes participated in both. This shows the difficulty of frozen semen quality control in commercially produced stallion semen, and on the other hand, the difficulty of conducting fertility trials in horses.     

Management and fertility of mares bred with frozen semen.

Semen quality, mare status and mare management during estrus will have the greatest impact on pregnancy rates when breeding mares with frozen semen. If semen quality is not optimal, mare selection and reproductive management are crucial in determining the outcome. In addition to mare selection, client communication is a key factor in a frozen semen program. Old maiden mares and problem mares should be monitored for normal cyclicity and all, except young maidens, should have at least a uterine culture and cytology performed. Mares with positive bacterial cultures and cytologies should be treated at least three consecutive days when in estrus with the proper antibiotic. With frozen semen, timing the ovulation is highly desirable in order to reduce the interval between breeding and ovulation. The use of ovulation inducing agents such as human chorionic gonadotropin (hCG) or the GnRH analogue, deslorelin, are critical components to accurately time the insemination with frozen semen. Most hCG treated mares ovulate 48h post-treatment (12-72h) while most deslorelin (Ovuplant) treated mares ovulate 36-42h post-treatment. However, mares bred more than once during the breeding cycle appear to have a slight but consistent increase in pregnancy rate compared to mares bred only once pre- or post-ovulation. In addition, the "capacitation-like" changes inflicted on the sperm during the process of freezing and thawing appear to be responsible for the shorter longevity of cryopreserved sperm. Therefore, breeding closer to ovulation should increase the fertility for most stallions with frozen semen. Recent evidence would suggest that breeding close to the uterotubal junction increases the sperm numbers in the oviduct increasing the chances of pregnancy. Post-breeding examinations aid in determining ovulation and uterine fluid accumulations so that post-breeding therapies can be instituted if needed. Average pregnancy rates per cycle of mares bred with frozen semen are between 30 and 40% with a wide range between sires. Stallion and mare status are major factors in determining the success of frozen semen inseminations. Pregnancy rates are lower for barren and old maiden mares as well as those mares treated for uterine infections during the same cycle of the insemination. To maximize fertility with frozen semen, a careful selection of the stallions and mares, with proper client communication is critical. Dedication and commitment of mare owner and inseminator will have the most significant impact on the pregnancy rates.     

Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions

It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10).     

Factors influencing conception rates of Arab mares in Tunisia

In order to investigate the factors affecting the reproduction efficiency of Arab breeding mares in Tunisia, breeding data corresponding to 2340 mated cycles, over 5 years (from 2000 to 2004), from 555 mares offspring of 50 sires and bred with 17 stallions were analysed using a multivariate logistic regression. We chose logit link function and binomial distribution and we used log-likelihood-ratio (LL) and Wald tests (X² Wald) to test the mean values. The factors of variation included in the model were the year, the stallion, the age of the mare at mating, the sire and the category of the mare. Logistic regression results showed that age and sire affected both the overall conception rate and the first-cycle conception rate of the mares whilst category and stallion influenced only the overall conception rate. To our knowledge, this is the first study showing that the sire of the mare can affect both the first-cycle conception rate and the overall conception rate of the mares.     

A survey of the fertility of Icelandic stallions

Very limited information is available on the breeding performance of Icelandic stallions, let alone the effect that management practices may have had on such performance. As an extensively kept, largely genetically isolated breed of horse it provides a good model for the study of factors that affect reproductive performance without the additional complication of selective breeding, infectious infertility and breed effect. A survey was conducted using 27 Icelandic stallions covering 1590 mares within the normal Icelandic breeding system (May to September). During the season, stallions cover mares within three periods of time, each period being of a similar length (average 35.5 days). During period 1, mares are covered in hand and at pasture. During periods 2 and 3, all mares are covered at pasture. The overall fertility rate for Icelandic stallions was calculated. The effect of a range of variables on fertility was investigated statistically using a number of models in an attempt to minimise the effect of confounding factors. An overall adjusted fertility rate for Icelandic stallions of 67.7% was obtained. The following factors were shown to have a significant effect on fertility: age of mare (P<0.001), training level of stallion (P<0.05) and method of breeding (P<0.05). For some individual stallions reproductive status of the mare also had a significant (P<0.001) effect. Many of these factors have been observed to effect FR in other more intensively managed equine populations. However, the less dramatic detrimental effect of age and the lack of a significant effect of mare reproductive status in most stallions suggests that infertility problems are less evident in Icelandic mares, possibly due to less emphasis on selection for athletic performance and the accepted culling of subfertile stock.     

Development and characterization of a homologous radioimmunoassay for equine prolactin

A specific and sensitive homologous radioimmunoassay has been developed for equine prolactin, suitable for measuring prolactin concentrations in serum of horses. The sensitivity of the assay ranged from 0.4 to 0.6 ng/ml and the intra- and inter-assay coefficients of variation averaged 6.9 and 15.4%, respectively, for five doses of hormone. Cross-reactivity with other mammalian and nonmammalian prolactins and growth hormones was less than 20 and 0.3%, respectively. Cross-reactivity with equine growth hormone was less than 0.07%. Equine serum and pituitary extracts showed parallel dilution-response curves with equine prolactin. The percentage recovery of exogenous equine prolactin in serum was 89%. Preliminary analysis of several physiological samples (stallions, pregnant, and nonpregnant mares) yielded values from 0.6 to 12.0 ng/ml.     

Female–female competition or male mate choice? Patterns of courtship and breeding behavior among feral horses (Equus caballus) on Assateague Island

Previous research on the Assateague horses found that high-ranking females had more surviving offspring than low-ranking females. Variance in reproductive success may be the result of a variety of proximate processes that affect sexual behavior such as mate choice and mate competition. A study was done to determine whether patterns of courtship, social, and sexual behavior could be identified that would suggest mate choice and/or mate competition. Behavioral data were collected from approximately 40 sexually mature mares living in harem bands. Stallions showed more interest in the eliminations of dominant mares than subordinate mares. Males also engaged in significantly more high-intensity (e.g., mounts and copulations) sexual behavior with dominant mares than subordinate mares, and there was a trend for males to engage in more low-intensity (e.g., flehmen and ano-genital sniffing) sexual behavior with dominant mares than subordinate mares. There was no effect of mare rank on spatial relationships with the stallion; however, dominant mares did attempt to restrict reproductive access to the stallion by harassing and disrupting copulations. Higher foaling rates among dominant mares on Assateague Island could therefore be the result of rank-related mate choice by stallions and direct female competition for mating opportunities.     

Association between population structure and allele frequencies of the glycogen synthase 1 mutation in the Austrian Noriker draft horse

The aim of this study was to determine the allele frequency of the glycogen synthase 1 (GYS1) mutation associated with polysaccharide storage myopathy type 1 in the Austrian Noriker horse. Furthermore, we examined the influence of population substructures on the allele distribution. The study was based upon a comprehensive population sample (208 breeding stallions and 309 mares) and a complete cohort of unselected offspring from the year 2014 (1553 foals). The mean proportion of GYS1 carrier animals in the foal cohort was 33%, ranging from 15% to 50% according to population substructures based on coat colours. In 517 mature breeding horses the mutation carrier frequency reached 34%, ranging on a wider scale from 4% to 62% within genetic substructures. We could show that the occurrence of the mutated GYS1 allele is influenced by coat colour; genetic bottlenecks; and assortative, rotating and random mating strategies. Highest GYS1 carrier frequencies were observed in the chestnut sample comprising 50% in foals, 54% in mares and 62% in breeding stallions. The mean inbreeding of homozygous carrier animals reached 4.10%, whereas non‐carrier horses were characterized by an inbreeding coefficient of 3.48%. Lowest GYS1 carrier frequencies were observed in the leopard spotted Noriker subpopulation. Here the mean carrier frequency reached 15% in foals, 17% in mares and 4% in stallions and inbreeding decreased from 3.28% in homozygous non‐carrier horses to 2.70% in heterozygous horses and 0.94% in homozygous carriers. This study illustrates that lineage breeding and specified mating strategies result in genetic substructures, which affect the frequencies of the GYS1 gene mutation.