Altered gene expression in embryos and endometrium collected on day 8 of induced aluteal cycles in mares.

Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1 ng/mL) when compared with control cycles during diestrus (high progesterone; > 4 ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those related to prostaglandin synthesis and normal pregnancy establishment. Seven cyclic mares with a median age of 6.5 years (range 3-16) were utilized in a crossover design. Mares in estrus were artificially inseminated to a fertile stallion and randomly assigned to control or AL groups. Mares received either saline solution (control mares) or PGF2α (AL mares), twice daily on days 0, 1, and 2 and once daily on days 3 and 4. Serial blood samples were collected daily from day 0 (ovulation) until the day of embryo collection and endometrial biopsy on day 8. Mares were monitored until they returned to estrus, and artificially inseminated. Mares were switched to the opposite treatment group only after a successful embryo collection occurred during the previous cycle and only cycles that produced embryos were used for analyses. The study design resulted in paired samples from each mare for analyses. No significant rise in progesterone was observed in the AL group with mean concentrations of plasma progesterone remaining <1.0 ng/mL from ovulation until embryo collection on day 8. This is in sharp contrast to the control (luteal) cycle where a post-ovulatory rise in plasma progesterone was observed. Real-time RT-PCR was utilized to evaluate the expression of ESR1, PGR, CYP19A1, P19, SLC35A1, OCD, APOB, AQP3, NEU2 transcripts in the embryos and PTGS2, P19, ESR1, HK2, sPLA2, PGR, CTGF, IFNE, FGF9, SLC36A2 expression in the endometrium. Four transcripts showed increased expressed in embryos developed during AL cycles ESR1, P19, APOB and PGR (p < 0.05). Four transcripts showed increased expressed in endometrium developed during AL cycles sPLA2, PGR, ESR1, FGF9 (p < 0.05) and four transcripts showed decreased expression P19, CTGF, IFNE, HK2 (p < 0.05). Additionally, staining differences were present in endometrial staining for both ERα and PR receptor during AL cycles compared with control cycles. Embryos and endometrium developed in a progesterone-deprived environment during induced aluteal cycles demonstrated altered transcript expression. These results indicate that adequate progesterone levels may be a key mediator of the appropriate embryo-maternal environment during early preimplantation embryo development.

Morphological evaluation of Day 8 embryos developed during induced aluteal cycles in the mare.

A novel in vivo model utilizing serial administrations of PGF2α to induce aluteal cycles in the mare was used to evaluate the effects of progesterone-deprivation on the morphology of in vivo preimplantation embryos. We hypothesized that equine embryos produced during induced aluteal cycles (AL) would be developmentally affected, characterized by earlier embryo stage at collection, smaller embryo diameter, and lower quality grade, compared with those collected on the same day post-ovulation from control cycles during diestrus (high progesterone; > 4 ng/mL). Seven cyclic mares with a median age of 6.5 years (range 3-16) were utilized in a crossover design. Mares in estrus were artificially inseminated to a fertile stallion and randomly assigned to control or AL groups. Mares received either saline solution (control mares) or PGF2α (AL mares), twice daily on days 0, 1, and 2 and once daily on days 3 and 4. Serial blood samples were collected daily during estrus and until the day of embryo collection 8 days after ovulation. Mares were monitored until they returned to estrus, and artificially inseminated. Mares were switched to the opposite treatment group only after a successful embryo collection occurred during the previous cycle. Only cycles that produced embryos were used for analyses. No significant rise in progesterone was observed in the AL group with mean concentrations of plasma progesterone remaining <1.0 ng/mL from ovulation until embryo collection on Day 8. This is in sharp contrast to the control (luteal) cycle where a post-ovulatory rise in plasma progesterone was observed. The mean daily concentrations of plasma progesterone were significantly higher in control vs. AL group beginning at Day 3 and remained so until Day 8. The mean (±SEM) embryo diameter of AL embryos was 171 ± 5 µm compared to 756 ± 99 µm for control embryos. The majority of the Day 8 AL embryos were classified as morulas (3/9) or early blastocysts (5/9) with only 2 embryos of quality grade 1 compared to the Day 8 control embryos that were mostly expanded blastocysts (6/7) with 5 of 6 being of quality grade 1. This study shows that serial administrations of PGF2α were able to prevent significant rises in plasma progesterone, thus inducing aluteal cycles characterized by a progesterone-deprived environment for developing embryos. Embryos collected from induced aluteal cycles were adversely affected as demonstrated by a lower quality grade, smaller diameter and earlier embryo stage at collection when compared to control embryos.

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.

Relationship between infection, inflammation and premature parturition in mares with experimentally induced placentitis.

REASONS FOR PERFORMING THE STUDY: Ascending placentitis results in premature birth and high foal mortality. By understanding how placentitis induces premature delivery, it may be possible to develop diagnostic markers and to delay premature delivery pharmacologically, thereby decreasing perinatal foal mortality. OBJECTIVE: To identify relationships between bacterial infection, inflammation and premature parturition in mares with experimentally induced placentitis. MATERIALS AND METHODS: Experiment 1: Concentrations of allantoic fluid prostaglandins (PGs) F2alpha and E2 were measured in 8 mares after intracervical inoculation with Streptococcus equi ssp. zooepidemicus (at Days 285-291 of gestation) until parturition and compared with controls (n = 4). Experiment 2: mRNA expression of interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF)-alpha and IL-8 in the chorioallantois from inoculated mares in Experiment 1 were compared with 7 mares that foaled normally. RESULTS: Bacterial inoculation resulted in 7 aborted fetuses and birth of one premature, viable foal. Infection was associated with inflammation of the chorioallantois in the region of the cervical star, isolation of bacteria and high concentrations of PGE2 and PGF2alpha in allantoic fluid obtained within 48 h of delivery (P = 0.04). Chorioallantois from all mares expressed mRNA for IL-8, TNF-alpha, IL-6 and IL-1beta. Experimentally infected mares expressed more mRNA for IL-6 (P = 0.003) and IL-8 (P = 0.009) in the cervical star region and more mRNA for IL-6 (P = 0.004) in tissues from placental horns than control mares. CONCLUSIONS AND CLINICAL RELEVANCE: Bacterial placentitis may result in liberation of cytokines from the chorioallantois and prostaglandin formation leading to abortion or birth of a precociously mature foal.

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.

Cefquinome concentrations in endometrium after intrauterine treatment of cobactan 4.5% in mares and inflammatory response of the endometrium to this treatment.

This study was conducted to measure the concentration of cefquinome in the endometrium of mares after intrauterine treatment and to evaluate associated inflammation. Mares (n = 14) were randomly assigned to one of the following groups: (i) control (n = 4) were either not treated (n = 2) or received (n = 2) lactated Ringer's intrauterine for 1 or 3 days; (ii) treated mares (n = 10) received intrauterine cefquinome for 1 or 3 days. After at least 10 days had passed following the last treatment and ovulation, mares were given Prostaglandin F2alpha (PGF2alpha) and were randomly assigned to an alternate treatment. Endometrial biopsy samples were taken at 2, 8, 24 and 48 h, or at 4, 12 and 36 h, after the last treatment. Biopsy samples were taken at the same time points from control mares (n = 2) and lactated Ringer-treated mares (n = 2). Cefquinome concentrations were quantified using a high-performance liquid chromatography (HPLC) assay and inflammation was assessed using haematoxylin and eosin (H&E)-stained sections. Concentrations of cefquinome [559 (1 day) and 595 microg/g (3 days) at 2 h, and 403 (1 day) and 370 microg/g (3 days) at 4 h] were similar between treatment groups at 2 and 4 h after treatment (p > 0.05). At 8 h, as well as at 24 and 48 h, concentrations were greater in the 3-day group (17 vs 301 microg/g, 3 vs 80 microg/g and 0.1 vs 0.2 microg/g, respectively) (p < 0.05). No significant differences (p > 0.05) in the inflammatory response at 2-48 h after treatment were found between groups.

Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin.

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.

Effect of nitric oxide synthase inhabitors on ovulation in hCG-stimulated mares.

Recent studies suggest that nitric oxide (NO) may have a role in regulating ovarian physiology. To investigate the role of NO during ovulation in mares, inhibitors of the nitric oxide synthase (NOS) were administered to estrous mares. Forty cycling mares (20 horses and 20 pony mares) were allotted to one of the three treatment groups. Once a follicle was at least 27 mm in diameter, but smaller than 35 mm, mares were given one of the following treatments: saline solution 0.9% (n = 20, w/v, i.v., every 12 h), Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME; n = 10, 148 micromol/kg, i.v., every 12 h), or aminoguanidine hemisulfate (AG; n = 10, 406 micromol/kg, i.v., every 12 h). When a follicle >30 mm was present on one of the ovaries, ovulation was induced with hCG (2,500 IU, i.v.). The median time of ovulation (+/-6 h) after hCG administration for the treatment groups was 42, 84 and 54 h for mares treated with saline solution, L-NAME and AG, respectively. There was no significant difference between the groups treated with AG or L-NAME (P = 0.06); however, these groups were different from the control group (P < 0.05). The delayed ovulation caused by the administration of NOS inhibitors suggests a role for NO in follicular growth and ovulation in horses.

Osmotic sensitivity of canine spermatozoa.

The objective of this study was to determine osmotic tolerance of canine spermatozoa. The study comprised three experiments: (1) spermatozoa suspended either in an egg yolk-citrate (EYC) extender or in Kenney skim milk extender were exposed to NaCl solutions ranging from 290 to 1500 mOsm; (2) spermatozoa suspended in EYC were exposed to 550 to 1500 mOsm solutions of glucose, galactose, or fructose; and (3) spermatozoa suspended in EYC or glucose-bovine serum albumin (G-BSA) were exposed to 0.6 M (approximately 900 mOsm) or 1.2 M (approximately 1600 mOsm) solutions of glycerol, ethylene glycol (EG), or dimethyl sulfoxide (Me(2)SO). In all experiments, motility and membrane integrity of spermatozoa were assessed after they were diluted into isotonic medium at 37 degrees C. Exposure of canine spermatozoa to solutions of either NaCl or monosaccharides at osmolalities >500 mOsm caused a significant reduction of motility (P<0.01). Motility of spermatozoa was more affected by osmotic stress than their membrane integrity. Osmotic sensitivity of canine spermatozoa was dependent on the type of extender; spermatozoa suspended in the Kenney extender were more resistant to osmotic stress than those suspended in the EYC extender. Despite their sensitivity to exposure to high concentrations of nonpermeating agents, canine spermatozoa were rather resistant to exposure to glycerol and EG. However, Me(2)SO was toxic to canine spermatozoa; motility was substantially reduced after spermatozoa were exposed to 0.6 M Me(2)SO. The type of extender also affected the sensitivity of canine spermatozoa to Me(2)SO; spermatozoa suspended in the EYC extender were more resistant than those suspended in G-BSA. It was concluded that canine spermatozoa are sensitive to osmotic stress, but are tolerant to shrinking and swelling caused by exposure to permeating cryoprotectants.

The effects of ph, osmolarity and urine contamination on equine spermatozoal motility.

Urospermia has been reported as a cause of infertility in numerous species. The detrimental effects of urine on spermatozoa are due, at least in part, to changes in pH and osmolarity. Semen was collected and subjected to conditions of varying pH (Experiment 1), of varying osmolarity (Experiment 2), and various quantities and concentrations of urine (Experiment 3) and effects on motility were recorded. Finally, semen was contaminated with urine and then either of 2 semen extenders was added, with or without centrifugation, in an attempt to alleviate the detrimental effect of urine on motility (Experiment 4). The results of these experiments showed that alterations in pH and osmolarity negatively affected stallion sperm motility. Optimal pH and osmolarity appeared to be approximately 7.7 and 315, respectively. Contamination of the ejaculate with urine significantly decreased sperm motility. Smaller quantities of dilute urine were less detrimental than larger quantities of dilute urine, and dilute urine was less detrimental than more concentrated urine. The addition of semen extender restored the motility of urine contaminated semen to that of the uncontaminated control, however centrifugation to remove urine provided no significant advantage.

Effect of dose and day of treatment on uterine response to oxytocin in mares.

To determine the effect of dose and day of oxytocin treatment on intrauterine pressure, 6 normal mares were treated with 10 or 25 IU oxytocin 2 days before ovulation, on the day of ovulation and 2 days after ovulation. Intrauterine pressure (IUP) was measured using micro-tip-catheters (one placed intrauterine, a second and third serving as reference sensors in the vagina and external to the mare) and transmitted by telemetry for 30 min to establish a baseline before saline was administered, iv, and for an additional 30 min after saline administration. Oxytocin was then given, iv, and IUP was recorded for 60 min. No change in IUP was observed after saline injection. The administration of both 10 (n=16) and 25 (n=10) IU oxytocin induced a response (P<0.01). The intensity of response depended on the day of administration (P<0.01) and the dose of oxytocin (P<0.001). The variation of response was significantly greater after 10 IU oxytocin (CV 15.78%) compared with 25 IU oxytocin (CV 6.42%). The uterine response was greatest on Day 2 prior to ovulation and lowest on Day 2 after ovulation. The response was negatively correlated to increasing plasma progesterone (10 IU oxytocin: r = -0.435, 25 IU oxytocin: r = -0.265). There was no correlation between the uterine response and plasma estradiol-17beta concentration (P<0.01). In conclusion the results of this study show that oxytocin administration to mares before ovulation provides a greater response than after ovulation. A decline in the intensity of response after ovulation can be compensated for with a higher dose of oxytocin. Furthermore, the use of the multiple catheter technique is an effective method for assessing changes in uterine pressure.

Anti-sperm antibodies and seminal characteristics after testicular biopsy or epididymal aspiration in dogs.

A study was performed to determine if performing testicular biopsies or epididymal aspirates in dogs would induce sperm-bound anti-sperm antibodies (ASA), affect long-term sperm production or semen quality. Semen was collected from 8 mature dogs 3 times a week before and after hemicastration and then 3 times a week after testicular biopsy (n=3 and 1 control) or epididymal aspiration (n=3 and 1 control). Detection of anti-sperm IgG (ASA) on sperm cells was performed by flow cytometry analysis using a flow cytometer. Two dogs with testicular biopsies became positive for ASA 16 d after testicular biopsy and remained positive for 7 and 9 d, respectively. One dog that had an epididymal aspirate became positive 13 d after epididymal aspiration and remained positive for 35 d. One dog became positive 21 d after hemicastration and remained positive for 28 d. Sperm output declined significantly in 7 of 8 dogs after hemicastration. A control epididymal aspirate treatment dog had decreased sperm output, and a testicular biopsy treatment dog had increased sperm output. None of the dogs with ASA had significant changes in sperm output after treatment. Sperm motility declined significantly in 3 dogs after hemicastration. An epididymal aspiration treatment dog had a decrease in sperm motility, a control epididymal aspirate treatment dog and a control testicular biopsy treatment dog each had increases in sperm motility. None of the dogs with ASA had significant changes in motility. The percentage of normal spermatozoa significantly decreased in 3 dogs and significantly increased in 1 dog after hemicastration. Two dogs that had testicular biopsies and 1 dog that had an epididymal aspiration had decreases in percent normal sperm. Two of 3 dogs with decreases in percent normal sperm after treatment had ASA, but 2 dogs with ASA had no change in motility. Hemicastration, epididymal aspiration, and testicular biopsy can induce ASA production within 2 wk of the procedure, but ASA are transient and do not have a predictably negative effect on total sperm output or motility. Testicular biopsy and epididymal aspiration are safe diagnostic procedures, but further work investigating post-treatment fertility must be done before final conclusions can be made.

PGFM response to exogenous oxytocin and determination of the half-life of oxytocin in nonpregnant mares.

We investigated the half-life of oxytocin in reproductively normal mares and the prostaglandin response after oxytocin administrations. Mares were given oxytocin, 10 or 25 iu, i.v., on the day of, or 2 days after, ovulation, and frequent jugular blood samples were collected for analysis of oxytocin and Prostaglandin F metabolite (PGFM) by RIA. Neither dose of oxytocin nor day of treatment affected the half-life of the exogenous oxytocin, which was determined to be 6.8 min. A significant increase in PGFM was observed within 6 min of oxytocin administration and peak values were observed within 10 min. PGFM response after oxytocin administration on the day of ovulation appeared elevated compared to the response 2 days after ovulation.

Ultrasound as an aid for diagnosis of ovarian abscesses in two mares.

This report describes two mares presented for evaluation of anorexia, fever of unknown origin, and weight loss. Clinical examination, laboratory findings, and transrectal ultrasonographic images suggested ovarian abscessation. One mare was successfully treated medically. Because of financial considerations, the second mare was euthanatized and a postmortem examination was performed. At necropsy, there was an enlarged right ovary with an adhesion to the large colon. Microscopic findings were characteristic of an ovarian abscess.

Reproductive parameters of miniature stallions.

Breeding soundness evaluation (BSE) of stallions is a routine component of stud farm practice. Guidelines for assessing satisfactory breeding potential have been developed using data derived from stallions of full-size breeds. In view of the increasing popularity of miniature stallions, knowledge of normal semen parameters of these stallions is important. Therefore, testicular measurements and semen parameters from 216 sexually rested miniature stallions were obtained. Semen was collected twice, 1.5 to 3 h apart, using an artificial vagina. Values were averaged over the 2 collections because of the sexual inexperience of the stallions. The smaller stallions (Group A, 72 to 86 cm; Group B, 87 to 96 cm) had smaller testicles (P<0.05), and Group A stallions had the lowest ejaculate volume (P<0.05) compared with Group C (97 to 104 cm) stallions. Thus, although there was no difference in the concentration of spermatozoa per milliliter between groups of stallions, Group A stallions had fewer total spermatozoa in their ejaculate than Group C stallions (4.31+/-0.47x10(9) vs. 5.41+/-0.30x10(9), P<0.05). Moreover, miniature stallions had smaller testicles and fewer total spermatozoa in their ejaculate than is commonly accepted as normal in full-size stallions. Average total scrotal width of miniature stallions was found to be 7.13, 7.38 and 7.95 cm for Groups A, B and C, respectively. The average total number of spermatozoa in the ejaculates of miniature stallions in this study was 4.94+/-0.22x10(9) cells, with 1.75+/-0.09x10(9) total normal, motile spermatozoa. When only stallions <96.5 cm in height were considered (conforming to requirements of the American Miniature Horse Association Registry), the average total number of spermatozoa in the ejaculates was 4.59+/-0.30x10(9) cells, with 1.70+/-0.11x 10(9) total normal, motile spermatozoa. Based on these findings, different criteria should be used to evaluate the potential breeding soundness of miniature stallions than are commonly applied to full-size stallions.

Fertility in bitches artificially inseminated with extended, chilled semen.

Sixteen bitches were artificially inseminated with either fresh, 24 h-chilled or 48 h-chilled extended semen over 38 estrous cycles. A commercial system for extending, chilling and transporting semen commonly used in the equine industry was used Pregnancy rates and litter sizes of the bitches inseminated with extended, chilled semen (19/20, 95%; litter size = 7.1) were not significantly different from those observed in bitches inseminated with fresh semen (17/18, 94%; litter size = 7.2; P > or = 0.89). These results show that a commercial system for extending, chilling and transporting equine semen is an attractive and efficient method of shipping canine extended chilled semen.