Proteins associated with the early intrauterine equine conceptus.

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

Changes in major proteins in the embryonic capsule during immobilization (fixation) of the conceptus in the third week of pregnancy in the mare.

During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulation = day 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (n = 55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a major capsule-associated protein that migrated at approximately 10 kDa was identified by N-terminal sequencing as equine beta2 microglobulin (beta2M). During fixation, beta2M in the capsule underwent limited proteolysis to an approximately 8 kDa form lacking nine amino acids from the N terminus, and was subsequently degraded. Expression of beta2M mRNA was detected in the yolk-sac wall tissues and endometrium between days 13.5 and 17.5. During this period, beta2M in the capsule was evidently not part of a complex with major histocompatibility complex class 1 heavy alpha chain bands because these were undetectable in the capsule and uterine lavage. Uterocalin (p19) was detected in uterine lavage and capsule throughout fixation, but in yolk-sac fluid only before fixation. These studies indicate that intact beta2M is a major protein associated with the embryonic capsule before fixation, after which it undergoes limited proteolysis to a truncated approximately 8 kDa form that remains in the capsule after the conceptus is immobilized.

Production of capsular material by equine trophoblast transplanted into immunodeficient mice.

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

Estimation of sodium and potassium concentrations in the uterine fluid of mares by microdialysis and ion chromatography.

Equine blastocyst fluid has a lower [Na+], a higher [K+] and a lower osmolality than does normal blood serum. Based on the assumptions that the sodium pump is primarily responsible for fluid accumulation and that ions transported actively into the blastocyst increase blastocyst osmolality above that of the external medium, we hypothesized that the [Na+] and the osmolality of mare uterine fluid are lower than those of blastocyst fluid. Microdialysis and ion chromatography were used to estimate [Na+] and [K+] of uterine fluid. Mares (n=10) were used for in vivo measurements at different stages of the oestrous cycle and during early pregnancy. On the basis of the results of studies in vitro, the mean +/- SD intrauterine [Na+] and [K+] were 110.0 +/- 15.1 and 12.3 +/- 6.8 mmol l(-1), respectively. These results indicate that uterine fluid of mares has a lower [Na+] and a higher [K+] than does normal serum. However, the [Na+] in uterine fluid is considerably higher than that of blastocyst fluid. Thus, these results do not support our hypothesis and the mechanism of production of hypotonic blastocyst fluid in horses remains unclear.

Evidence for the presence of sodium- and potassium-dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus.

The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium- and potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse transcriptase polymerase chain reaction was conducted on two sets of five conceptuses recovered between 12 and 28 days (+/- 1 day) after ovulation. Messenger RNAs encoding the alpha1 and beta1 subunit isoforms of Na+,K+-ATPase were detected in all embryonic tissues examined. Western blot analysis showed that alpha1 and beta1 subunits are both present in Day 15 conceptuses. Trophoblast tissues from 19 conceptuses between 8 and 31 days after ovulation were stained immunohistochemically using primary antibodies against the alpha1 and beta1 subunit isoforms of the Na+,K+-ATPase. Both isoforms were detected in all sections. Trophoblastic vesicles, prepared from 6 conceptuses between 12 and 14 days after ovulation, were used to investigate the inhibition of blastocyst expansion with ouabain after collapse induced with cytochalasin D. In normal medium there was a mean 3-fold increase, and in ouabain (10(-6) M) a mean 3-fold decrease, in the volume of vesicles that had been partially collapsed with cytochalasin D. We therefore conclude that, despite the hypotonicity of the blastocyst fluid in the early horse conceptus, the Na+,K+-ATPase plays a role in its accumulation, as in other species.

Osmolality of equine blastocyst fluid from day 11 to day 25 of pregnancy.

Horse conceptuses collected between Day 11 and Day 18 of pregnancy float in isotonic media. To investigate this phenomenon, blastocyst fluids from 30 conceptuses from 13 mares were analysed for osmolality and for concentrations of Na+, Cl-, K+, glucose, urea and creatinine. In conceptuses from Group A, samples from Day 11 to Day 16 yielded the following results (mean +/- s.e.m.): osmolality, 121.4 +/- 1.5 mOsm kg-1; Na+, 11.0 +/- 2.2 mM; Cl-, 29.3 +/- 2.5 mM; K+, 26.2 +/- 2.6 mM; glucose, 0.6 +/- 0.1 mM; urea, 6.0 +/- 0.6 mM; creatinine, 9.6 +/- 1.1 microM. Between Day 16 and Day 25, the osmolalities and Na+ concentrations increased gradually with age but the former never exceeded 255 mOsm kg-1. Fluids from Group B were obtained from eight conceptuses exposed to saline solutions of different osmolalities for various periods of time. An increased perivitelline space of 1-3 mm became evident at the lower pole of the floating conceptus after 45 min of exposure to solutions with osmolalities of > or = 300 mOsm kg-1, suggesting that Na+ and Cl- diffuse freely through the capsule but not through the trophoblast. The significance of the hypo-osmolality of equine blastocyst fluid is discussed but remains unclear.

[Hematological reference values for foals in the first two months of life]. / Hämatologische Referenzwerte beim Fohlen in den ersten zwei Lebensmonaten.

Hematologic reference values were established in 18 healthy foals in the first two months of life. Blood samples were collected prior to colostrum consumption and at 30 hours, 1 week, 3, 5, 7 and 9 weeks of age. PCV, Hb and RBC decreased during the first week and RBC, but not PCV and Hb, increased toward the end of the second month. With the exception of the sample at 1 week, the foals had mean RBC values significantly higher than those of controls. Mean MCV and MCH did not change during the first week, but decreased slightly thereafter; all means were smaller than in controls. The numbers of neutrophils increased mainly during the first week, and the numbers of lymphocytes increased gradually during the first two months. The mean N:L-ratio was highest at 1 week and lowest at 5 weeks.

The relationship between cycle stage and results of uterine culture in the mare.

A total of 368 uterine swabs were taken from mares in heat (n = 202) and from mares that were not in heat (n = 166). From 72 of the mares, two swabs were taken; one during either seasonal anoestrus or dioestrus, and one during oestrus. Swabs were taken during anoestrus/dioestrus in 94 other mares and during oestrus in the remaining 130 mares. Bacteriological cultures were done aerobically and classified as negative, insignificant or significant. There was a trend for more positive cytological specimens during oestrus than during anoestrus/dioestrus. The proportions of significant cultures were 12 of 202 (5.9%) in oestrous mares and 14 of 166 (8.4%) in anoestrous/dioestrus mares. Generally more insignificant than negative cultures were obtained during oestrus compared to anoestrus/dioestrus. Seven of 27 cultures that were classified as significant were associated with positive cytological specimens, and only 2 of 16 specimens that yielded pure cultures of E. coli contained neutrophils. Although the culture results did not differ significantly between the cycle stages, oestrus should be the preferred time for uterine examination.

A clinical trial on the effect of prostaglandin F2 alpha on placental expulsion in dairy cattle after caesarean operation.

One hundred and thirty dairy cattle that had undergone a caesarean operation because of dystocia were assigned either to a treatment group, receiving 25 mg dinoprost (synthetic prostaglandin F2 alpha) or to a control group, receiving 5 ml saline solution, administered intramuscularly after removal of the calf and reposition of the closed uterus into the abdominal cavity. In 52 of 65 (80.0 per cent) prostaglandin-treated animals and in 38 of 65 (58.5 per cent) control animals the complete placenta was expelled within 12 hours. This difference was statistically significant (P < 0.05).

Streptomycin-resistant Escherichia coli as a marker of vulvovestibular contamination of endometrial culture swabs in the mare.

To investigate the vulvovestibular contamination of endometrial culture swabs in the mare, a liquid culture of a streptomycin-resistant strain of Escherichia coli was applied to the vulvovestibular area of mares and used as a marker of contamination of endometrial culture swabs. Prior to taking endometrial swabs, the perineal area was washed with soap, rinsed with water, and dried. Endometrial culture swabs were taken from mares that were in anestrus or diestrus and from mares that were in estrus. When a manual transvaginal swabbing technique was used, 22 of 24 endometrial swab specimens from 12 mares were contaminated with the experimental bacterial strain; culture of only one endometrial swab yielded more than nine colonies. When a speculum approach was employed, three of 12 swab specimens from 12 mares yielded between one and three colonies. The stage of cycle had no effect on the extent of contamination, but the proportion of positive cultures was significantly smaller when swabs were taken via a vaginal speculum approach, compared to a manual transvaginal approach. Complete preclusion of vulvovestibular contamination of endometrial swab specimens was not achieved; however, fewer than ten colonies can be expected even in mares in which the vulvovestibular area has been thoroughly contaminated with a broth culture, provided that the perineal area is adequately cleaned prior to swabbing.

[Clinico-chemical blood parameters in foals in the first two months of life]. / Klinisch-chemische Blutparameter beim Fohlen in den ersten zwei Lebensmonaten.

Eighteen healthy foals were studied from birth until 2 months of age. Blood samples were obtained at the following times: presuckle, 30 hours, 1, 3, 5, 7 and 9 weeks of age. Changes in serum P, Mg, Ca, Na, K, Cl, iron, AP, ASAT, ALAT, GGT, GLDH, CK, lipase, urea, creatinine, cholesterol, triglyceride, uric acid, protein and fibrinogen and in plasma total solids were examined and the values compared to reference values of adult horses. There were characteristic age related changes in several parameters. Single measurements should be interpreted cautiously to allow for individual variations.

Mononuclear cell infiltration of the equine endometrium: immunohistochemical studies.

Endometrial sections from mares with varying degrees of mononuclear cell infiltration were examined for immunoglobulin (Ig)A-, IgM-, IgG(T)- and IgG(Fc)-containing cells, luminal and glandular epithelial cell Ig-staining and free interstitial Ig-staining, using a peroxidase anti-peroxidase technique. Mares with mild to moderate (Group 2) and mares with severe diffuse mononuclear cell infiltration, superimposed by acute endometritis (Group 3), had significantly higher numbers of Ig-containing cells than genitally-normal mares (Group 1). The differences between Groups 1 and 3 were significant for all four isotypes. In Groups 1 and 2, numbers of IgA-containing cells were significantly larger than numbers of IgM- and IgG(T)-containing cells. Generally, more glandular epithelial cells stained for IgA and IgM than for IgG(T) and IgG(Fc), and Ig-staining for all isotypes increased from Group 1 to Group 3. Free interstitial staining did not appear to differ among the three groups, but IgG(Fc)- and IgG(T)-staining generally was more intense than IgA- and IgM-staining. The efficiency of uterine defence in the mare does not seem to depend solely on humoral factors, and defects involving other components of the defence system may contribute to failure of the uterus to clear infection.

[Postcoital uterine microbe colonization and endometritis in the mare]. / Die postkoitale uterine Keimbesiedlung und Endometritis bei der Stute.

In the mare, natural breeding is associated with bacterial contamination of the reproductive tract. The purpose of this study was to examine postcoital bacterial contamination and the resulting inflammatory response of the uterus. Uterine swabs for bacteriological and cytological examination were obtained from 80 mares. Each mare was sampled once between 4 and 69 hours postbreeding. In mares which did not conceive, sampling was repeated at the following estrus. The findings were compared with those obtained prior to breeding and correlated with the breeding outcome. Bacteria were cultured from 72.5% of the postcoital swabs. There was a wide spectrum of organisms which included species known as potential causes of endometritis. Neutrophilic granulocytes were found in varying concentrations in 48.8% of cases. In 16.3% of mares both bacteriological and cytological examinations were negative. Mares with positive bacteriological and/or cytological results at the postcoital examination had better foaling rates compared to the remaining mares (p less than 0.05). The postcoital findings did not correlate with those of the prebreeding examination, or with the interval between breeding and sampling, or with the different stallions.

Endometrial biopsy in mares under nonuniform breeding management conditions: Prognostic value and relationship with age.

Endometrial biopsy plays an important role in fertility examination of the mare. The purpose of this study was to examine the prognostic usefulness of endometrial biopsy under nonuniform breeding management conditions, and to clarify any relationship with age of the mare. Based on endometrial histological changes, 192 mares from different farms were classified using a four-category grading system. There were significant associations between categories and fertility. Mean pregnancy rates in the four categories were 79, 49, 33 and 0% and mean foaling rates were 70, 42, 18 and 0%, respectively. Mares older than the median age (=11 years) had, on average, higher category scores and significantly lower fertility than younger mares. Within categories, there was a tendency towards higher fertility in younger mares. Although the precise prediction of fertility of the individual mare remains difficult, it is evident from this investigation that endometrial biopsy has considerable prognostic potential even under nonuniform breeding management conditions. Furthermore, the age of a mare must be considered when estimating her fertility potential.

Relationships of total protein, specific gravity, viscosity, refractive index and latex agglutination to immunoglobulin G concentration in mare colostrum.

A colostrum sample was collected within 24 h after foaling from 27 mares and from 10 other mares a milk sample was collected several weeks post partum. Immunoglobulin G concentrations were determined quantitatively by radial immunodiffusion and semi-quantitatively using a commercial latex agglutination test. Total protein, specific gravity, viscosity and refractive index were determined and their relationships to the immunoglobulin G concentration analysed. All parameters correlated with the immunoglobulin G concentration. The latex agglutination test divided the colostrum samples into three groups with different means for immunoglobulin G and total protein concentrations. Specific gravity and the latex agglutination test were found to be the methods best suited for on-farm evaluation of colostrum quality.

Distribution of histological lesions in the equine endometrium.

The distribution of histopathological lesions in the equine endometrium was examined to investigate the representativeness of a single biopsy specimen in terms of the whole endometrium. Five sections from each of 110 uteri obtained from slaughtered mares were evaluated microscopically and classified according to a four-category grading system used for endometrial biopsies. Depending on the extent of agreement between the categories of the homologous sections, the uteri were considered to show either good agreement (81 uteri; 73.6 per cent), moderate agreement (26 uteri; 23.6 per cent) or poor agreement (three uteri; 2.7 per cent). All the homologous sections of the group showing moderate agreement were within two adjacent categories. Disagreements were more often due to variations in the distribution of fibrotic lesions than to variations in the degree of chronic infiltrative lesions. There was no seasonal effect on the apparent degree or distribution of lesions. In most cases the examination of a single biopsy, when combined with a thorough clinical examination, should provide adequate information about the condition of a mare's endometrium.

Two related cases of cerebellar abnormality in equine fetuses associated with hydrops of fetal membranes.

Hydrops allantois was diagnosed in two Haflinger mares with severe abdominal distension. Both mares were seven months pregnant. Abortion was induced with two injections of prostaglandin six hours apart followed by further manual dilation of the cervix and administration of oxytocin the next day. There were 90 and 95 litres of fluid, respectively, in the allantoic cavities which resembled extracellular fluid with regard to concentrations of urea, creatinine, sodium, potassium, calcium, magnesium, phosphate and chloride, but not total protein. Both fetuses had severe brain abnormalities which were diagnosed as cerebellar and cerebral hypoplasia associated with bilateral hydrocephalus internus and hydranencephaly and cerebellar aplasia, respectively. Both mares were pregnant by the same stallion, but a clear hereditary link was not found.