Immunology of infective preterm delivery in the mare.

Placentitis is reported to be the cause of 9.8-33.5% of abortions, stillbirths and perinatal losses in horses. Bacterial infections are responsible for 53% of placentitis cases with Streptococcus equi ssp. zooepidemicus being isolated in 28% of these cases. Clinically, mares may have a vaginal discharge, show udder development, lactate prenatally and deliver a premature or dead foal. Major aspects of the pathogenesis of infectious preterm delivery that may require more effective therapeutic targeting are myometrial contraction, immunological aspects of preterm delivery, and the effects of proinflammatory cytokine signalling on activation of the fetal hypothalamic-pituitary-adrenal axis. This article focuses on current knowledge of inflammatory signalling secondary to equine placentitis, and the interplay among inflammation, loss of myometrial quiescence and activation of the fetal hypothalamic-pituitary-adrenal axis.

Cushioned versus noncushioned centrifugation: sperm recovery rate and integrity.

It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10(6) sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), and 1800NC (96.7 ± 2.6) groups was significantly higher than the 900C (68.7 ± 4.6) and 1800C (79.6 ± 3.5) groups. The D0 TM and PM were not different between the CON, 900NC, 900C, and 1800C, but were lower for the 1800NC group. The D1 TM and PM of the 900NC (75.2 ± 3.8 and 71.1 ± 4.1) and 900C (76.2 ± 3.7 and 72.4 ± 4.0) groups were significantly higher than the 1800NC (71.7 ± 4.1 and 67.3 ± 4.4) and 1800C (71.6 ± 4.1 and 67.2 ± 4.4) groups, and the CON (66.2 ± 4.5 and 60.0 ± 4.8) group was significantly lower than the other groups. The D1 PLM of the CON, 900NC, 900C, 1800NC, and 1800C groups were not different. The ACR on D1 was significantly lower for the CON (93.0 ± 2.4) group compared with all other groups. Optimal RR preserving sperm integrity was obtained in the 900NC group.

Factors affecting sperm recovery rates and survival after centrifugation of equine semen.

Conventional centrifugation protocols result in important sperm losses during removal of the supernatant. In this study, the effect of centrifugation force (400 or 900 × g), duration (5 or 10 min), and column height (20 or 40 mL; Experiment 1); sperm concentration (25, 50, and 100 × 10(6)/mL; Experiment 2), and centrifugation medium (EZ-Mixin CST [Animal Reproduction Systems, Chino, CA, USA], INRA96 [IMV Technologies, Maple Grove, MN, USA], or VMDZ [Partnar Animal Health, Port Huron, MI, USA]; Experiment 3) on sperm recovery and survival after centrifugation and cooling and storage were evaluated. Overall, sperm survival was not affected by the combination of centrifugation protocol and cooling. Total sperm yield was highest after centrifugation for 10 min at 400 × g in 20-mL columns (95.6 ± 5%, mean ± SD) or 900 × g in 20-mL (99.2 ± 0.8%) or 40-mL (91.4 ± 4.5%) columns, and at 900 × g for 5 min in 20-mL columns (93.8 ± 8.9%; P < 0.0001). Total (TMY) and progressively motile sperm yield followed a similar pattern (P < 0.0001). Sperm yields were not significantly different among samples centrifuged at various sperm concentrations. However, centrifugation at 100 × 10(6)/mL resulted in significantly lower total sperm yield (83.8 ± 10.7%) and TMY (81.7 ± 6.8%) compared with noncentrifuged semen. Centrifugation in VMDZ resulted in significantly lower TMY (69.3 ± 22.6%), progressively motile sperm yield (63.5 ± 18.2%), viable yield (60.9 ± 36.5%), and survival of progressively motile sperm after cooling (21 ± 10.8%) compared with noncentrifuged semen. In conclusion, centrifuging volumes of ≤ 20 mL minimized sperm losses with conventional protocols. With 40-mL columns, it may be recommended to increase the centrifugal force to 900 × g for 10 min and dilute the semen to a sperm concentration of 25 to 50 × 10(6)/mL in a milk- or fractionated milk-based medium. The semen extender VMDZ did not seem well suited for centrifugation of equine semen.

Cytogenetic and molecular characterization of Y isochromosome in a 63XO/64Xi(Yq) mosaic karyotype of an intersex horse.

Sex chromosome aberrations commonly lead to abnormal sexual development. Here we cytogenetically and molecularly characterized Y isochromosome in an intersex horse. Blood lymphocyte analysis showed a mosaic karyotype with 96% 63,XO and 4% 64,Xi(Y) cells. Molecular analysis of the isochromosome was carried out by fluorescence in situ hybridization and polymerase chain reaction with male-specific and pseudoautosomal markers from the horse Y chromosome. We found that the isochromosome was monocentric, composed of 2 long arms, carrying 2 sets of genes of the pseudoautosomal region (PAR) and the male-specific region of the Y (MSY), including the SRY - thus being genetically equivalent to Y disomy. Sequence analysis of a 1,955-bp region including the SRY exon, the promoter and the UTRs, revealed no mutations in the aberrant Y. The presence of an intact SRY in a small proportion of cells is the proposed cause for the intersex phenotype. Given that the i(Yq) was present in a mosaic form, both post-zygotic and meiotic mechanisms of its origin were proposed. We speculated that nonmosaic 64,Xi(Yq) karyotypes might be rare or absent because of the likely instability of the i(Yq) during cell division. Genetic and phenotypic implications of Y isochromosome formation in other mammals are discussed in the light of the diversity of Y chromosome organization between species.

Persistent breeding-induced endometritis after hysteroscopic insemination in the mare.

Low-dose insemination has been proposed to reduce persistent breeding-induced endometritis (PBIE) in mares with delayed uterine clearance (DUC). Others proposed that hysteroscopic insemination induces an exaggerated inflammatory response and should be avoided in DUC mares. The objectives here were to evaluate presence and severity of PBIE in normal and DUC mares after hysteroscopic insemination with fresh semen, and to determine if hysteroscopy could be used in DUC mares without inducing excessive inflammation. Reproductively normal (n = 4) and DUC (n = 5) mares received four treatments in random order: uterine body insemination (UB, 1 × 10(9) spermatozoa, 20 ml), hysteroscopic insemination (HYST, 5 × 10(6) spermatozoa, 0.5 ml), sham hysteroscopic insemination (SHAM, semen extender, 0.5 ml) and hysteroscopic infusion of seminal plasma (SP, 0.5 ml). Significantly more DUC (50%) mares than normal (14%) mares accumulated intrauterine fluid 24 h post-treatment. The difference in fluid accumulation between DUC (40%) mares and normal (7%) mares was also significant 48 h post-treatment. Fluid scores were not significantly different between treatments in normal mares. However, treatments HYST and SHAM resulted in significantly higher fluid scores 24 h but not 48 h post-treatment in DUC mares. There was no effect of treatment or mare group on the percentage and total number of neutrophils in uterine fluid 48 h post-treatment. Percentage of neutrophils was correlated with duration of hysteroscopy in normal mares, with procedures lasting ≥ 9 min associated with PBIE. There was no effect of mare group, treatment or duration of hysteroscopy on pregnancy rate. Hysteroscopy induces a transient inflammation that is not more severe than that after conventional artificial insemination, suggesting no contraindication to its use in DUC mares.

Immediate and delayed (after cooling) effects of centrifugation on equine sperm.

The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility was assessed by computer-assisted semen analysis, and samples were stained with SYBR-14/propidium iodide (PI) for viability and with PI/fluorescein isothiocyanate (FITC)-Peanut aglutinin (PNA) (Arachis hypogaea) for acrosomal integrity. The effect of treatment and day on motility, viability, and acrosomal integrity was determined using a mixed linear model. Compared with the other treatments, centrifugation at 4500xg reduced all end points measured (P<0.05). Both 400xg and 900xg yielded lower recovery rates than that of 4500xg (NC=100.0+/-0.0%; 400xg=54.4+/-8.6%; 900xg=75.0+/-7.1%; 4500xg=97.9+/-2.8%; P<0.05). Centrifugation at 400xg or 900xg did not damage equine sperm. Based on these findings, further studies of centrifugal forces between 900xg and 4500xg are warranted to determine the optimal force that maximizes recovery rate, minimizes sperm damage, and does not affect fertility.

Disorders of sexual development in the dog and cat.

Normal sexual differentiation occurs in three sequential steps--establishment of chromosomal (genetic) sex, development of gonadal sex, and development of phenotypic sex. Errors in the establishment of chromosomal, gonadal, or phenotypic sex cause abnormal sexual differentiation. Affected individuals are identified with a wide variety of patterns from ambiguous genitalia, to apparently normal genitalia with sterility or infertility. When a patient is suspected of having a disorder of sexual development, analysis of the chromosomal constitution and complete gross and histopathologic description of the gonads, internal and external genitalia are required to correctly categorize the type of disorder.

Low-dose insemination--why, when and how.

The typical dose for insemination into the uterine body of the mare is > 300 x 10(6) progressively motile spermatozoa (PMS) and an insemination dose of > 200 x 10(6) PMS is recommended for frozen-thawed semen. Low-dose insemination techniques allow for a drastic reduction in the numbers of spermatozoa required to achieve pregnancy. Acceptable pregnancy rates can be achieved with doses ranging from 1 to 25 x 10(6) PMS in volumes ranging from 20 to 1000 microL. Two techniques have been described: hysteroscopic insemination and transrectally guided deep horn insemination using a pipette. Similar pregnancy rates can be attained by either method when 5 x 10(6) PMS are used. Hysteroscopic insemination may provide an advantage when the dose is 1-3 x 10(6) PMS. These techniques have the potential to make more efficient use of frozen-thawed or sex-sorted semen from certain stallions. The use of low-dose insemination to improve fertility of infertile stallions warrants further investigation.

Uterine clearance mechanisms during the early postovulatory period in mares.

Uterine response to inoculation with Streptococcus zooepidemicus organisms, 51Cr-labeled 15-microns microspheres, and charcoal was evaluated in 9 mares (4 resistant and 5 susceptible to endometritis) to determine mechanical and cellular clearance rates during the early postovulatory period. Mares were inoculated at estrus prior to ovulation during estrous cycles 1, 3, and 5. Uterine swab specimens for aerobic and anaerobic bacteriologic culture and serum for progesterone determination were obtained on postovulation day 3 during estrous cycle 1, on the day of ovulation during estrous cycle 3, and on postovulation day 5 during estrous cycle 5. Immediately thereafter, the uterus was irrigated with 50 ml of sterile physiologic saline solution containing tracer amounts of 125I-labeled human serum albumin. Streptococcus zooepidemicus was isolated from 10 of 15 (67%) uterine specimens collected from susceptible mares and incubated aerobically. Escherichia coli also was isolated from 2 of the 10 specimens incubated aerobically. Anaerobic bacteriologic culture of specimens from all mares yielded no growth. Chromium-labeled microspheres were recovered twice from 2 susceptible mares, on day 0 and day 5. Charcoal was retained in 5 specimens collected from 3 susceptible mares. Bacteriologic culture of specimens from resistant mares did not yield growth. On day 0, chromium-labeled microspheres and charcoal were recovered once from 1 resistant mare. Mares susceptible to endometritis accumulated more fluid within the uterine lumen after ovulation than did resistant mares (mean +/- SEM, 52.73 +/- 15.22 ml and 7.41 +/- 1.96 ml, respectively; P less than 0.01). From this study, it appeared that uterine cellular and bactericidal mechanisms are dysfunctional during the early postovulatory period. However, there appeared to be no disruption of the mechanisms responsible for mechanical clearance of materials inoculated in the uterus.

The "horse-racing effect" and predicting decline in forced expiratory volume in one second from screening spirometry.

Longitudinal spirometric data on adults in a general population sample confirmed an overall relationship between rate of change in FEV1 (delta FEV1) and mean FEV1/Ht3. This relationship between FEV1 "slope" and "level" has been called a "horse-racing effect" and is the basis for the widely accepted concept that detecting a low FEV1 will predict a rapid decline in FEV1 and the development of clinically significant COPD. However, when both age and smoking habits were taken into account, the effect persisted only in male smokers. Even in them, it was dependent on the inclusion of part of the delta FEV1 in calculation of the mean FEV1 value. While neither a low initial FEV1/Ht3 nor percent predicted FEV1 significantly predicted a subsequent rapid fall in FEV1, presumably because of regression toward the mean, other initial spirometric variables proved better predictors, at least among male smokers. In them, a low FEV1/FVC ratio on entry was almost uniformly associated with a high rate of decline in FEV1. This was not seen in ex-smokers with similar initial spirometric findings. Also, delta FEV1 could not be predicted with any precision in women regardless of their smoking status. The findings strongly support the concept that detection of early airway disease by spirometric screening will identify those male smokers who are likely to develop clinically significant illness if they continue to smoke and that smoking cessation, even at this point, can markedly improve the prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Longitudinal changes in forced expiratory volume in one second in adults. Effects of smoking and smoking cessation.

Longitudinal changes in FEV1 (delta FEV1) in relationship to smoking habits were examined in 1,705 adults enrolled in a prospective study of a general population sample. In both men and women, there were excessive rates of decline in FEV1 in smokers, at least in those smoking more than 10 cigarettes per day. The excess decline of smokers was age dependent, particularly in men, with much of the excess loss of function occurring between 50 and 70 yr of age. Effects of smoking on delta FEV1, observed minus expected delta FEV1 and observed minus expected percent delta FEV1, were greater in men than in women even when controlled for current cigarette dose. Ex-smokers show delta FEV1 values similar to those of nonsmokers. In subjects younger than 35 yr of age, quitting smoking during follow-up was associated with an actual increase in FEV1. In men 50 to 70 yr of age, smoking cessation early in the study appeared to lead to a relatively prompt return to normal rates of functional decline. Thus, the findings suggest relatively prompt beneficial effect of smoking cessation even in late middle age.