SCINTIGRAPHIC MEASUREMENT OF UTERINE CLEARANCE IN MARES

Uterine clearance of technetium 99m-albumin colloid (^99mTc-μAA) was qualitatively and quantitatively measured in 5 reproductively normal mares and 5 mares susceptible to endometritis (infertile). The percentage of 370 MBq ^99mTc-μAA cleared from the uterine lumen within 2 hr of intrauterine infusion was measured in 10 mares on day 3 of estrus and 48 hr after ovulation. The procedure was repeated 3 times on day 3 of estrus in 6 mares to determine repeatability. Six mares were infused with 1110 MBq ^99mTc-μAA on day 3 of estrus to evaluate the effect of increasing the dose to reduce the imaging time. There was no statistically significant difference in the mean percentage of radiocolloid cleared from the uterus during day 3 of estrus or 48 hr after ovulation or in the percent cleared when the studies were repeated in individual mares. There was no statistically significant difference in uterine clearance between the 370 and 1110 MBq dose studies in each mare from 15 to 120 min. Reproductively normal mares cleared approximately 50% of the radiocolloid from the uterus by 120 min while susceptible mares cleared less than 15%.     

Consequences of Intrauterine Enrofloxacin Infusion on Mare Endometrium

Endometritis is an important cause of infertility in mares. Enrofloxacin is a broad-spectrum antibiotic to which most equine endometritis pathogens are not resistant. The objective of this study was to determine whether enrofloxacin is safe to use as a conventional intrauterine infusion treatment. Nine healthy mares received intrauterine infusions of enrofloxacin (Baytril 100, 100 mg/mL, Bayer Health Care LLC, Animal Health Division) at 2.5 mg/kg daily for 3 days. Ultrasonographic examination and vaginal examinations were performed during the study. Endometrial biopsies were performed before treatment (S0) and 24 hours after the last treatment (S1) to evaluate acute effects. For evaluating chronic effects, biopsies were performed at 14 days (S2) and 60 days posttreatment (S3). Biopsies were graded histologically by the Kenny and Doig category scale. Difference in histological biopsy grade before and after treatment was compared between biopsies by using a repeated-measures one-way analysis of variance. and significant changes in grades were used to assess treatment effects. The vaginal and ultrasonographic examination after intrauterine infusion of enrofloxacin showed that all mares had severe purulent vaginitis and uterine fluid accumulation of ≥2 cm, with ≥1.5-cm thickening of the endometrial wall which persisted in most mares until the end of the study. Histologically, there was acute endometrial ulceration, necrosis, and hemorrhage in biopsy S1 in all mares, categorized as grade III. In biopsy S2, most mares developed fibrosis and inflammation graded as IIb (four of nine mares) or III (four of nine mares). In biopsy S3, fibrosis was extensive and had variable inflammation, graded as IIb (two of nine mares) or III (five of nine mares), with some mares healing to grade IIa (two of nine mares). There was an overall worsening of endometrial biopsy grade from I to III at S3 compared with S0 (P < .001). These results confirm that enrofloxacin is not suitable for conventional intrauterine infusion treatment in mares.     

Endometrial and serum gentamicin concentrations in pony mares given repeated intrauterine infusions

Endometrial tissue and blood serum gentamicin (GT) concentrations were determined in 6 ovariectomized pony mares given intrauterine infusions (50 ml of a 5% commercial aqueous solution of GT) each day for 5 consecutive days. The mares were subjected to the following 3 treatments: (1) GT infusion only (trial A, control); (2) progesterone plus GT (trial B, P + G); and (3) estradiol plus GT (trial C, E + G). Endometrial tissue concentrations of GT (micrograms/g) at 24 and 120 hours were significantly higher (P less than 0.05) in trials B (65.54 +/- 15.57 and 100.33 +/- 19.27) and C (73.33 +/- 22.53 and 74.09 +/- 8.60) than in trial A (4.23 +/- 0.70). Endometrial concentration for trial A at 120 hours was also significantly higher than trial A at 24 hours. There was no significant difference (P greater than 0.05) in endometrial concentrations among trials A, B, and C at 120 hours. Serum GT concentrations were significantly lower than endometrial tissue concentrations. The highest serum concentrations of GT found in every trial occurred at 6 hours after each intrauterine infusion of GT. The highest overall serum concentration of GT (micrograms/ml) determined occurred in trial B (8.30 +/- 1.28) at 78 hours. There was no significant difference in serum concentrations of GT between days of treatment, except for trial A at 78 and 102 hours, respectively. Serum concentrations of GT were significantly higher (P less than 0.05) than trial A at 30, 54, 78, and 102 hours in trial B, and at 78 and 102 hours in trial C. There was no significant difference in serum concentrations of GT between trials B and C.     

Disposition of gentamicin in the genital tract of cows

The distribution of gentamicin (G) in plasma and uterine lumen was studied following intramuscular (i.m.) and intrauterine (i.u.) treatment. A Foley catheter was inserted into one uterine horn and retained in place by inflation of the cuff. This provided a closed system for collection of uterine lumen samples and analysis of the concentration of gentamicin for 6 h following treatment. Four normal cycling and healthy cows in dioestrus were given i.m. injections of 4 mg gentamicin/kg BW and another two were given i.m. injections of 2 mg gentamicin/kg BW gentamicin. The uteri were infused with 50 ml saline containing phenolsulphonphthalein (PSP) indicator. Blood and infused solution (IS) samples were periodically collected during the 6-h period following i.m. administration. Six hours after injection, approximately 183.7 micrograms gentamicin and 39.4 micrograms gentamicin were accumulated in the uterine lumen of cows receiving 4 mg gentamicin/kg BW and 2 mg gentamicin/kg BW, respectively. The amount of gentamicin reaching the blood stream after i.m. administration of 4 mg gentamicin/kg BW was 2.89 times that reached after administration of 2 mg gentamicin/kg BW based on the area under the curve of plots of plasma concentration of gentamicin versus time. Four normal-cycling and healthy cows in dioestrus were given i.u. infusions of gentamicin (225-275 mg) diluted in 50 ml saline containing PSP indicator using a Foley catheter in a closed system. Samples from the IS and blood were collected at various intervals for 6 h after infusion. Following i.u. infusion of gentamicin, an average of 29.4% of the dose was absorbed into the bloodstream. The majority of the dose of gentamicin (70.6%) remained in the uterine lumen throughout the 6-h period.     

Effect of surgical manipulation, placental fluid, and flunixin meglumine on fetal viability and prostaglandin F2 alpha release in the gravid uterus of mares

Twenty-one pregnant mares with single or twin conceptuses between 41 and 65 days of gestational age were allotted to 5 treatment groups. A ventral median celiotomy was performed in all mares. In group-1 mares (3 mares, single conceptus), the uterus and fetus were palpated for 5 minutes. In group-2 mares (3 mares, single conceptus, flunixin meglumine), 250 ml of sterile placental fluid was injected into the nongravid uterine horn. In group-3 mares (4 mares, unicornuate twin conceptuses), group-4 mares (3 mares, unicornuate twin conceptuses, flunixin meglumine), and group-5 mares (8 mares, bicornuate twin conceptuses, flunixin meglumine), 1 conceptus was removed from the uterus via hysterotomy. All mares received progesterone prophylactically until day 100 of gestation or until the fetus died. The 3 mares in group 1 delivered clinically normal, live foals. The mean prostaglandin F2 alpha metabolite (PGFM) plasma concentration peaked at 180 +/- 5.2 pg/ml during uterine manipulation and fetal palpation, then declined to baseline by 1 hour. Free placental fluid (group 2) undermined the chorioallantois ventrally and resulted in fetal death within 3 hours after surgery. The mean PGFM plasma concentration peaked at 39 +/- 4 pg/ml following injection of placental fluid. None of the remaining fetuses in the 7 mares with unicornuate twin conceptuses (groups 3 and 4) survived. Five mares with unicornuate twin conceptuses (group 5) delivered single viable foals. In another mare in group 5, the fetus was alive 4 days after surgery, when the mare was euthanatized for a fractured femur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma concentrations of 13,14-dihydro-15-ketoprostaglandin F2 alpha in mares during uterine involution.

Twelve mares were allowed to foal naturally, after which they were monitored to study uterine involution. Starting on day 3 after parturition, the internal genital tract was examined per rectum manually and ultrasonographically every other day for changes in uterine characteristics and ovarian activity. By day 5, gravid and nongravid uterine horns were similar in size, and by day 7, uterine fluid was absent. On day 7 after parturition, endometrial biopsy samples were obtained for histologic evaluation, and uterine swab specimens were obtained for microbiologic culture. Uterine swab specimens from 10 of 12 mares had slight bacterial growth. The uteri of 8 of the 12 mares were histologically involuted by day 7. All mares ovulated 7 to 12 days after parturition. Concentrations of 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were measured in jugular plasma samples obtained daily for 21 days after parturition. Concentrations of PGFM were low by the day after parturition, and there was no significant correlation between uterine involution and PGFM concentrations in these mares. All 12 mares were bred at the first estrus after parturition, and 9 became pregnant.     

Measurement of total volume and protein concentration of intrauterine secretion after intrauterine inoculation of bacteria in mares that were either resistant or susceptible to chronic uterine infection.

Undiluted uterine secretion was used to determine the concentration of total protein and the accumulated volume of uterine secretion after a bacterial inoculation in mares susceptible and resistant to chronic uterine infection (CUI). The uterus of 6 susceptible and 5 resistant mares was inoculated with 5 x 10(6) Streptococcus zooepidemicus on the third day of estrus. Using a tampon inserted in the uterus, secretions were sampled at 5, 12, 24, and 36 hours after inoculation, followed by intrauterine lavage with phosphate buffered saline solution. The concentration of protein was determined in the undiluted secretion as well as in the uterine washing and the total amount of accumulated uterine secretion was calculated. Protein concentrations in plasma were compared before and after absorption by the tampon. Protein concentration of plasma before and after absorption by the tampon did not differ. Mares susceptible to CUI accumulated significantly (P less than 0.001) more fluid in the uterus than mares resistant to CUI, and uterine washings from the resistant mares were significantly (P less than 0.05) more dilute than those from the susceptible mares. Significant differences in protein concentrations between susceptible and resistant mares were not found. It was concluded from this study that the described method to sample undiluted uterine secretion was practical and reliable for the analysis of protein concentration. Various concentrations of uterine secretions in washings from susceptible and resistant mares emphasizes the importance in using undiluted uterine secretions or dilution markers in washings when intrauterine products are analyzed.     

Ultrasonographic uterine changes and vaginal discharges following intrauterine infusion of liquid paraffin in cows

This study was conducted to examine uterine changes and uterocervical discharges following intrauterine infusion with liquid paraffin (LP) during the luteal phase by ultrasonic and vaginoscopic examinations in cows. Multiparous dairy cows (n=10) were infused with 50 ml physiological saline (PS group; n=5) or liquid paraffin (LP group; n=5) on day 10 or 11 after ovulation (day 0: ovulation). Vaginoscopic, rectal and ultrasonogaphic examinations were carried out at 0.25, 1, 2, 3, 4, 6, 8, 12 and 24 h after the LP and PS infusion and then at daily intervals until subsequent ovulation after the infusions. The mean volumes of recovered discharges from the vagina within 6 h after infusion were significantly greater (P<0.05) in the LP group than in the PS group (33.0 +/- 9.9 vs.14.0 +/- 13.9 ml). Yellowish-white discharge was first observed at 3.2 +/- 0.5 and 3.6 +/- 0.6 h after infusion and lasted for 12.2 +/- 2.9 and 2.1 +/- 1.5 days for the LP and PS groups, respectively, showing a significant difference (P<0.05) in duration. Subsequently, transparent discharge appeared again 2-3 days before the subsequent ovulation after the treatments in both groups and disappeared on the day prior to or the day of ovulation. During the immediate examination after the infusion, the cavity of the uterine horn appeared anechoic and dilated in the images of both groups. The anechoic images changed to echoic images at 2.2 +/- 0.8 and 2.6 +/- 0.9 h after the infusion in the LP and PS groups, respectively, and the echoic images lasted for 12.2 +/- 2.9 and 2.1 +/- 1.5 days in the LP and PS groups, respectively. These results suggest that the appearance and disappearance of intrauterine anechoic and echoic images reflect the appearance and disappearance of the characteristics of the recovered LP/PS-like liquid and yellowish-white and transparent discharges from the vagina.     

Investigation of diagnostic importance of platelet closure times measured by Platelet Function Analyzer--PFA 100 in dogs with endotoxemia

This study was performed to evaluate the diagnostic importance of the platelet closure times measured by the Platelet Function Analyzer (PFA-100) in dogs with endotoxemia. E. coli endotoxin was given intravenously once, at the dose of 0.02 mg/kg or 1 mg/kg in groups I (n=9) and II (n=8), respectively. Normal saline (0.1 ml/kg) was injected in group III (n=8).The dogs were monitored for 48 h, and venous blood samples were collected prior to (baseline) and at intervals of 0.5, 1, 2, 4, 6, 8, 12, 24 and 48 h subsequent to the treatments. The white blood cell (WBC), platelet counts, and hematocrit (Hct) values were recorded. Platelet closure times were determined, using collagen/epinephrine (CEPI) and collagen/adenosine diphosphate (CADP) cartridges. Within 0.5 h after the endotoxin application baseline WBC and platelet counts (mean +/-SD) decreased significantly (p<0.001) to 2000 +/- 500 and 1850 +/- 200 cells/microl or 69.000 +/- 12.500 and 27.000 +/- 6.400 cells/microl in groups I and II, respectively. Platelet counts remained low during the first 1-48 h, but the WBC count was high at the 8th-48th h, in groups I and II, compared with baselines (p<0.001). After the application of the endotoxin, Hct values increased from baseline values of 37 +/- 3 or 39 +/- 2% to 48 +/- 2 or 51 +/- 3%, within 1 h (p<0.001), in groups I and II, respectively. Hct values in group II were notably higher (p<0.001) than those of group I, during the 2nd-48th h. Hematological parameters and closure times did not differ significantly throughout the study in group III. Baseline closure time ranged from 79 +/- 5 seconds (s) to 86 +/- 5 s for CADP and 144 +/- 13 s to 159 +/- 14 s for CEPI in all dogs (n=25). At 0.5 h after the endotoxin, the closure times of CADP as well as CEPI declined to 62 +/- 6 s and 76 +/- 8 s in group I (p<0.001) and 57 +/- 5 s and 75 +/- 6 s in group II (p<0.001). Afterwards, closure time prolonged to the levels of 280 +/- 8 s (CADP) and 294 +/- 5 s (CEPI) by 48 h (p<0.001) in group II, but returned to the baseline limit in group I. In conclusion, our results show that the shortened closure times may serve as a very early diagnostic sign of endotoxemia, prolonged closure times however may be used as an index for the severity of endotoxemia.     

Comparative effects and safety of ivermectin in pregnant mares

Mares (n=20) approaching 3 months of pregnancy were assigned randomly to 1 of 4 treatment groups. Treatments were (a) an IM injection of ivermectin at 600 mcg/kg, (b) various conventional anthelmintic drugs at the manufacturers' recommended dose, (c) and IM injection of the ivermectin vehicle (placebo) and (d) no anthelmintic treatment during the trial. All anthelmintic treatments were administered at 60-day intervals up to and including the date of parturition. Fecal egg counts, arginase, hemoglobin and packed cell volume values were determined at bi-weekly intervals during the trial and there were no statistically significant differences determined between the treatment groups for these parameters. None of the mares showed any adverse clinical signs during the course of this study and all 20 mares delivered live foals which remained on the research farm until they were sold as year-lings. Mares treated with ivermectin had significantly (P<0.01) lower egg per gram counts than mares in the conventional treatment group. Multiple hematological and clinical chemistry values were determined for all mares and resulting foals within 12 hours post-parturition. A one-way analysis of variance showed no clinically relevant statistically significant differences between treatment groups in either mares or foals at 12 hours post-parturition. This study suggests that ivermectin at 600 mcg/kg is safe and highly efficacious when administered to pregnant mares.     

Factors affecting the composition of mare uterine fluid

The influx of protein and polymorphonuclear leucocytes (PMN) into the uterine lumen was examined at different intervals after intrauterine infusion of fluids. The intrauterine infusion of both phosphate buffered saline (PBS) and a solution of lipopolysaccharide (LPS) derived from Escherichia coli resulted in a biphasic influx of protein in the uterine flushings peaking three and six hours after infusion. LPS infusion caused an additional influx of protein at 24 hours. The initial influx of protein preceded a biphasic influx of PMN which peaked six and 24 hours after both infusions. Uterine flushings obtained following PBS and LPS infusion contained both serum-derived and uterine-specific proteins. To investigate whether the influxes were a general response to reproductive mucosal stimulation, several regions of the reproductive tract were subjected to physical manipulation. Results suggested that these influxes were initiated chiefly by stimulation of the cervical and, or, uterine region.     

Evaluation of peritoneal fluid following intestinal resection and anastomosis in horses.

Postoperative abdominal fluid changes were compared in 2 groups of horses; those undergoing double small-colon resection and anastomosis (n = 10) and those undergoing exploratory celiotomy alone (n = 5). Peritoneal fluid was collected before surgery and on postoperative days 1, 3, 5, and 7. Total and differential nucleated cell counts, RBC numbers, and total protein and fibrinogen concentrations were evaluated. In both groups, all values were significantly higher than normal on the first postoperative day (after small-colon resection and anastomoses, WBC = 130,350 +/- 23,310 cells/microliters, RBC = 7,389,000 +/- 6,234,000 cells/microliters, total protein = 3.63 +/- 0.16 g/dl; after exploratory celiotomy alone, WBC = 166,620 +/- 34,340 cells/microliters, RBC = 295,000 +/- 86,070 cells/microliters, total protein 4.38 +/- 0.54 g/dl). The number of total peritoneal nucleated cells and RBC significantly decreased after the first postoperative day, whereas total protein and fibrinogen concentrations, percent neutrophils, and percent mononuclear cells remained unchanged. None of the values had returned to normal by postoperative day 7 (after small-colon resection and anastomoses, WBC = 45,600 +/- 8,765 cells/microliters, RBC = 95,390 +/- 53,380 cells/microliters, total protein = 4.39 +/- 0.23 g/dl; after exploratory celiotomy alone, WBC = 43,340 +/- 7,746 cells/microliters, RBC = 12,860 +/- 11,790 cells/microliters, total protein = 3.92 +/- 2.20 g/dl.) The resection and anastomosis group had a significantly lower total protein concentration on the first postoperative day and a significantly higher mean total RBC count over the entire 7-day postoperative evaluation than did horses that underwent celiotomy alone. Other values in the 2 groups of horses did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of abortion during midgestation in mares

Four standardbred mares, 99 to 153 days pregnant, were treated with a synthetic prostaglandin analogue, prostalene, in an attempt to induce abortion. The mares received subcutaneous injection of either 2 mg prostalene (recommended luteolytic dose); 4 mg prostalene (double luteolytic dose) or 4 mg twice at intervals of 12 hours or 24 hours. The prostalene treatment resulted in cervical relaxation, increased tone of the uterus and decreased plasma concentrations of progesterone. None of the pregnant mares aborted within seven days after the first prostalene treatment. Abortions were subsequently induced by single or multiple intrauterine infusions using warm hypertonic saline. There were no postabortion complications.     

Supplemental l-Arginine Shortens Gestation Length and Increases Mare Uterine Blood Flow before and after Parturition

Supplementing diets with l-Arginine (Arg) improves female reproductive performance and reproductive blood flow in other species. The objectives of this study were to investigate uterine artery blood flow changes before and after parturition, and evaluate blood flow in Arg supplemented and control mares by Doppler ultrasonography. Sixteen light-horse mares began Doppler ultrasonography evaluation, 21 days before expected foaling date (EFD) and continued until day 7 postparturition. The mares under treatment (n = 8) were supplemented with 100 g Arg, once daily, beginning with 21 days before EFD. Blood flow measurements were calculated as pulsatility index (PI) and resistance index (RI) for both uterine arteries, either ipsilateral or contralateral; to uterine horn of established pregnancy; defined gravid uterine artery (GUA) and non-gravid uterine artery (NGUA), respectively. The mares under treatment had a shorter gestation length (337 ± 1.7 days) as compared to control (345 ± 2.1 days; P ≤ .05). No differences in gestation length were observed between groups when examined by age, parity, EFD, or sex of foal. Both GUA and NGUA uterine artery diameter decreased from the day before parturition to day 7 after parturition (P ≤ .001). During this time period, both PI and RI increased (P ≤ .01); indicating less blood flow. A treatment effect was observed with Arginine-treated mares having greater blood flow prepartum in the NGUA (P ≤ .001) and postpartum in the GUA (P ≤ .05), for both indices. The data demonstrated that supplementing mares with Arg shortened gestation length and increased uterine arterial blood flow before and after parturition.     

Effects of aerosolized feedyard dust that contains natural endotoxins on adult sheep

OBJECTIVE: To determine the clinical, clinicopathologic, and histologic effects of aerosolized feedyard dust that contains natural endotoxins on adult sheep. ANIMALS: Eighteen 3-year-old Saint Croix sheep. PROCEDURE: A prospective randomized controlled study was conducted. There were 2 treatment groups (dust-endotoxin group, n = 9; control group, 9). Aerosolized feedyard dust was provided continuously during a 4-hour period for each application (once in week 1, 3 times in week 2, and 7 times in week 3) to sheep in a semiairtight tent. All sheep were euthanatized and necropsied 8 hours after the treatment group received the last dust treatment. Variables measured before and after each dust treatment were rectal temperature, total WBC count, and concentrations of fibrinogen and haptoglobin. RESULTS: Mean amount of dust administered during each treatment was 451 g/4 h. Filter collection indicated 51 mg of dust/m3 and 7,423 ng of endotoxin. Mean rectal temperature at 8 hours (40.4 C) and mean WBC counts 12 and 24 hours after dust treatment were significantly higher for the treated group than the means of the respective variables for the control group. Similar responses were observed with repeated dust-endotoxin treatments; however, with each subsequent treatment, there was a diminished response. Sheep in the treatment group had generalized alveolar septal thickening and hypercellularity. CONCLUSIONS AND CLINICAL RELEVANCE: Feedyard dust induced a temporary febrile response and leukocytosis in sheep in the treatment group. Exposure to dust that contains endotoxins may be a stressor preceding acute infectious respiratory tract disease of marketed sheep.     

Opsonins of Streptococcus in uterine flushings of mares susceptible and resistant to endometritis: control of secretion and partial characterization

The release of opsonins into the uterine lumen of mares susceptible or resistant to endometritis was examined after intrauterine inoculation of a filtrate of Streptococcus culture fluid or vehicle. Uterine flushings were collected at 0.5 hour before and 2, 4, 6, 8, and 24 hours after inoculation on day 2 or 3 of estrus and on day 7 or 8 after ovulation. Amounts of opsonins in flushings were quantified as the H2O2 produced by leukocytes incubated with flushings-opsonized bacteria, compared with H2O2 produced by leukocytes incubated with nonopsonized bacteria. Opsonin values in flushings increased (P less than 0.025) in all mares after inoculation of filtrate or vehicle. For mares resistant to endometritis, opsonin values were greater at diestrus than at estrus. The opposite was true for mares susceptible to endometritis, resulting in a status (susceptible vs resistant) X stage of cycle interaction (P less than 0.025). Overall, opsonins were higher (P less than 0.05) in flushings of mares susceptible to endometritis than in flushings of mares resistant to endometritis, but this difference was only apparent at estrus. Preliminary characterization of opsonins in uterine secretions by ammonium sulfate fractionation and gel filtration indicated that opsonins were mainly associated with an ammonium sulfate-soluble fraction of high molecular weight (greater than 4 X 10(6] and an ammonium sulfate-precipitable fraction that was associated with immunoglobulin G.     

Retrospective study on the incidence of postinsemination uterine fluid in mares inseminated with frozen/thawed semen

Uterine fluid accumulation has been reported after insemination or natural breeding of mares. This retrospective study examined the factors affecting the incidence of uterine fluid after insemination of frozen semen. Specifically, this study determined the association between mare age, reproductive status, fluid accumulation, and pregnancy rates in mares. Records were available from 283 warmblood mares throughout 496 cycles. Mares were divided into maiden, foaling, and barren and age groups of 3 to 9, 10 to 16, and more than 16 years. Mares were inseminated only once with frozen semen within 4 to 8 hours before or after ovulation. Ultrasound examinations were performed 12 to 18 hours after insemination. A depth of at least 20 mm of fluid was considered significant. Mares with less than 20 mm were treated with oxytocin, and those with more than 20mm of fluid were given oxytocin and uterine lavage. Pregnancy determination was performed at 14 to 16 and 30 to 50 days after ovulation. Fluid level of more than 20 mm was recorded in 25% of the cycles. Barren mares and aged mares (10-16 and > 16 years) had a higher incidence of uterine fluid accumulations. Per-cycle pregnancy rate was lower (45%) in mares with uterine fluid than in mares without uterine fluid (51%). This difference was primarily due to the reduction in fertility of mares who were older than 16 years and retained fluid after insemination. Apparently, oxytocin and lavage treatments provided acceptable fertility in the other groups of mares that had uterine fluid.

Introduction

Use of equine frozen semen is accepted by the majority of horse registries. According to several field studies,[1, 2, 3, 4 and 5] insemination of frozen semen has resulted in acceptable pregnancy rates. Postbreeding fluid accumulation is a physiologic inflammation that clears the uterus of foreign material such as excess spermatozoa, seminal plasma, bacteria, and extenders. [6, 7, 8, 9 and 10] Uterine fluid can be easily diagnosed with ultrasonography. [10, 11 and 12] Persistent postbreeding uterine fluid has been associated with a decrease in fertility after natural mating or artificial insemination (AI) of fresh semen. [11, 12 and 13] Predisposing factors to persistent fluid accumulations are reduced myometrial contractions, poor lymphatic drainage, large overstretched uterus, and cervical incompetence. [7, 14 and 15] Normal mares are able to expel uterine fluid quickly after inseminations, whereas susceptible mares accumulate fluid in their uterine lumen for more than 12 hours after breeding or insemination. [10]It is commonly stated that insemination with frozen semen leads to greater post-AI fluid accumulation than insemination with fresh or cooled semen or after natural mating. Apparently, there is only 1 controlled study on this comparison.[7] The authors reported that infusion of frozen semen resulted in a greater inflammatory response than natural breeding. In a field study, [16] 16% of mares naturally mated had persistent postbreeding fluid accumulations compared with a 30% rate reported for mares inseminated with frozen semen. [1 and 2] More recently, Watson et al. [17] reported a postbreeding fluid accumulation rate of 16%, which is identical to that reported for natural mating. [16] It is difficult to compare studies because details of mare selection and insemination or breeding frequencies are not always reported. Obviously, a higher proportion of barren and aged mares in a study would increase the incidence of postbreeding fluid accumulation. [1 and 2]The study presented herein was a retrospective study designed to determine the incidence of postbreeding fluid accumulation in a large number of mares inseminated with frozen semen. Associations were determined between mare age, reproductive status and fluid accumulation, and pregnancy rate in mares with and without uterine fluid accumulation.

Materials and methods

Mares

Records were available from 283 warmblood mares inseminated with frozen semen at the Cristella Veterinary Clinic in Italy during 1998 to 2001. Mares ranging in age from 3 to 20 years were inseminated with semen that was frozen in 10 centers and was from 34 stallions. The broodmare population was subdivided into 3 reproductive groups: 89 maiden mares (mean age, 7.2 years), 106 foaling mares (mean age, 9.4 years), and 87 barren mares (mean age, 11.9 years). Maiden mares older than 7 years were selected with biopsy scores of 1 or 2 only. Barren mares were open for no more than 2 consecutive seasons and had negative cytology and bacteriology scores. Age groups were divided as follows: 3 to 9 years (n = 132), 10 to 16 years (n = 137) and older than 16 years (n = 14). Data from 496 cycles were used. Distribution of the estrous cycles was 172, 157, and 167 in the maiden, foaling, and barren groups, respectively; and 224, 244, and 28 in the youngest, intermediate, and oldest groups, respectively.

Mare reproductive management and artificial insemination protocol

During estrus, all mares underwent a daily ultrasound examination with a 5-mHz transrectal probe (SA 600 Vet; Medison Inc., Seoul, South Korea) until 1 or more 35-mm ovarian follicles were detected. Ovulation was then induced by the intravenous administration of 2000 IU of human chorionic gonadotropin (hCG). Ultrasound examination was performed 12 hours after hCG treatment and then every 4 to 8 hours until ovulation occurred. Mares were inseminated only once within a period of 4 to 8 hours before or after ovulation. The semen used was thawed according to the distribution center's instructions and had the following minimum post-thaw quality requirements: not less than 200 × 10⁶ progressively motile spermatozoa per dose and a minimum of 30% progressive spermatozoal motility. Foaling mares were not inseminated at their first postpartum (“foal heat”) estrous period, because pregnancy rates are recognized to be lower than during the subsequent estrous periods.[18] During the first postpartum estrus, ovarian ultrasound scan examinations were performed every 2 to 3 days until an ovulation was detected. A prostaglandin F₂α injection was given 5 days later to short-cycle the mare.

Postinsemination monitoring

An ultrasound examination of the reproductive tract was performed 12 to 18 hours after insemination to detect any intrauterine fluid accumulation. The presence and depth of intrauterine fluid was recorded. Twenty millimeters or more of grade II or III intrauterine fluid[19] was recorded as a significant amount of fluid. Mares with less than 20 mm of fluid were treated with an intravenous injection of 20 IU oxytocin. For mares with more than 20 mm of fluid, oxytocin was administered, and the uterus was flushed daily with buffered saline solution: 1-L aliquots were infused and recovered until the recovered fluid was clear. In these mares, oxytocin treatment was repeated up to 3 times daily. Post insemination treatments were performed for no more than 4 days after ovulation had occurred.Pregnancy diagnosis was performed with ultrasound at 14 to 16 days after ovulation. Scans were then repeated at 30 and 50 days of gestation to confirm the presence in the uterus of an apparently healthy developing conceptus.

Statistical analysis

χ² Analysis was used to determine the effect of reproductive status and age on the incidence of fluid accumulation. In addition, the influence of persistent uterine fluid accumulation on pregnancy rates per cycle was determined for each reproductive class and age by using χ² analysis.

Results

The per-cycle pregnancy rate at 14-16 days after ovulation was 49.3% (245/496 cycles). By the end of the season, 245 of 283 mares (86.5%) were confirmed pregnant. Fluid level of at least 20 mm (grade II or III) was recorded in 126 of the 496 cycles (25.4%). Barren mares had a higher (P < .05) incidence of postbreeding fluid accumulation (64/167; 38.3%) than maiden (34/172; 19.7%) and foaling (28/157, 17.8%; Table 1) mares. The incidence of fluid accumulation was also higher in mares older than 16 years (19/28; 67.8%) than those aged 10 to 16 years (69/244; 28.2%) and 3 to 9 years (38/224; 17%). The incidence of uterine fluid was also higher (P < .05) for mares aged 10 to 16 years than those aged 3 to 9 years (Table 2). Overall, the per-cycle pregnancy rate was lower (P < .05) for mares with post-AI fluid accumulations than for those with no uterine fluid or only a small quantity of fluid (57/126, 41.9% vs 188/360, 56.2%). Pregnancy rates were similar (P > .05) for mares with or without uterine fluid when comparisons were made within maiden and barren mare groups. However, more foaling mares became pregnant when no fluid was detected after insemination. Pregnancy rate for this group (68.1%) was higher than that for maiden (44.2%) and barren (44.6%) mares (Table 3). Older mares with uterine fluid accumulations had a lower per-cycle pregnancy rate (36.8%) than mares in the same group but without fluid. Surprisingly, if no fluid was detected, the highest pregnancy rates were in mares older than 16 years ( Table 4).     

Changes in lymphocyte blastogenic response of mares during the perinatal period

A fluorometric assay was applied to evaluate blastogenesis of equine lymphocytes. Optimal culture conditions were as follows; concentrations of phytohaemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were 1 microgram/ml, 40 micrograms/ml and 10 micrograms/ml, respectively, when 5 X 10(5) lymphocytes were incubated with culture medium containing 20% pooled horse serum (PHS) for 120 hours. The relative mean stimulation index of healthy non-pregnant mares were 5.107 +/- 0.323 (M +/- SE) with PHA, 4.019 +/- 0.183 with Con A and 3.610 +/- 0.131 with PWM. Sequentially the blastogenic responses of lymphocytes from twenty mares were observed during various stages of the perinatal period. Response decreased gradually before parturition was lowest at the time of parturition (PHA: 1.923 +/- 0.174, Con A: 1.698 +/- 0.206 and PWM: 1.706 +/- 0.177), and then increased gradually after parturition towards non-pregnant levels.     

Ultrasonographic studies on the reproductive tract of mares after parturition: effect of involution and uterine fluid on pregnancy rates in mares with normal and delayed first postpartum ovulatory cycles

During breeding of mares, ultrasonographic detection of uterine fluid accumulations in the first postpartum ovulatory period was associated with significantly decreased pregnancy rates, when compared with rates in control mares (P less than 0.005). The previously gravid uterine horn was recognized as the larger horn, when assessed for size by ultrasonography, for a mean of 21 days (range, 15 to 25 days) after parturition. On the basis of similar measurements obtained during 3 ultrasonographic scans (5-day period), uterine involution was determined to be completed in a mean of 23 days (range, 13 to 29 days). Progestin treatment did not affect uterine size, fluid accumulation, or rate of involution after parturition. However, delaying the first postpartum ovulation with 8 days of progestin treatment significantly improved pregnancy rates (P less than 0.05). More (P less than 0.05) mares became pregnant (23 of 28, 82%) when ovulation occurred after day 15 in the first postpartum ovulatory period, compared with those mares that ovulated before day 15 (6 of 12, 50%). We concluded that ultrasonographic detection of uterine fluid and postpartum progestin treatment can be used to manipulate breeding strategies and to improve pregnancy rates in mares bred during the first postpartum ovulatory period.     

The Effect of Manual Removal of Placenta Immediately after Foaling on Subsequent Fertility Parameters in the Mare

Retained placenta is considered to be a common problem in postpartum mares. The incidence varies from 6% to 54% depending on the breed, with higher incidence in heavy draught mares than in light-weight mares. Retained placenta has been linked to lower postpartum oxytocin concentration, impaired uterine involution, and dystocia. The objective of this study was to assess the effect of early manual removal of placenta immediately postpartum on subsequent fertility parameters (development of free intrauterine fluid, inflammatory status of endometrium, and on pregnancy rates) and to compare them with mares with spontaneous expulsion of placenta. A total of 29 mares, mainly Irish draught, were closely monitored during foaling by closed circuit television and allocated to two groups: (1) mares that expelled the placenta spontaneously within 3 hours of foaling; and (2) mares that were cleansed manually immediately after foal delivery. All mares were examined and scanned 5 and 9 days postpartum, and free intrauterine fluid was recorded; endometrial swabs were taken 9 days postpartum for endometrial cytology and culture. None of the fertility parameters analyzed showed statistical difference between groups 1 and 2. Therefore, it can be concluded that early manual removal of placenta has no detrimental effects on subsequent fertility of mares and, therefore, can be recommended when a veterinarian attends a foaling.