B.E. Eilts

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.

B-mode ultrasonography of the bull testicle

The scrotum and testicles from 20 bulls aged 6 mo to 10 yr were obtained from a slaughterhouse and ultrasonically scanned to determine the normal echographic anatomy Ultrasonically, the normal bull testicle was homogeneous and moderately echogenic. The mediastinum testis was a linear structure in the center of the testicle and was slightly more echogenic than the parenchyma. The head and tail of the epididymis were easily identified on all testicles, but the epididymal body and ductus deferens were difficult to identify consistently. Ultrasound scanning of the testicles may prove to be a valuable noninvasive diagnostic technique for evaluating testicular diseases in bulls.

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.

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.

B-mode ultrasound observations of bull testes during breeding soundness examinations

The testes of 78 adult Beefmaster bulls (13 to > 31 mo old) were scanned with a 5 MHz, linear array portable ultrasound unit during routine breeding soundness examinations. Videotape recordings were used to measure mediastinum testis width and fluid width between visceral and parietal tunics (fluid width), and to count the number of fibrotic foci. Ultrasound results were compared to breeding soundness score parameters, which included total score, age classification, and percentage of primary and secondary sperm abnormalities. The overall mean mediastinum testis width and fluid width were 0.33 cm +/- 0.133 and 0.10 cm +/- 0.13, respectively; 15 testes had 1-8 fibrotic foci. There were no significant differences in mediastinum testis width and fluid width between left and right testes when compared by breeding soundness score classification or by age classification. There were no significant differences in mediastinum testis width, fluid width, breeding soundness score, or primary or secondary sperm abnormalities in bulls with fibrotic foci compared to bulls without fibrotic foci. There appears to be no significant information that routine testicular ultrasound examination adds to accepted breeding soundness evaluations performed on the same day.

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.

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.

CHARACTERIZATION AND COOLED STORAGE OF SEMEN FROM CORN SNAKES (ELAPHE GUTTATA)

Abstract The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4°C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (x̄ = 852 × 106 ± 585 × 106 spermatozoa/ml). Morphologically, a mean of 75.7 ± 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (x̄ = 1,859 × 106 ± 1,008 × 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (x̄ = 601 × 106 ± 439 × 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4°C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.

The effect of reducing hindquarter elevation time after artificial insemination in bitches

Hindquarter elevation time after artificial insemination in dogs was reduced from the common and arbitrarily used 10 min to only 1 min after insemination. Artificial insemination with fresh undiluted semen was conducted in 32 breedings using 15 hound bitches. The overall pregnancy rate was 91% (29/32), with an average litter size of 7.35 puppies per pregnancy. The pregnancy rate was not altered by reducing the 10-min (n = 14) hindquarter elevation time to 1 min (n = 18; P = 0.30). Similarly, the litter size was not different between groups (P = 0.40).

Fish Handling and Ultrasound Procedures for Viewing the Ovary of Submersed, Nonanesthetized, Unrestrained Channel Catfish

Abstract This study addressed the development of rapid, straightforward, and minimally stressful procedures for the ultrasound imaging of ovaries of channel catfish Ictalurus punctatus in a commercial hatchery setting. The objectives were to (1) describe the ultrasound imaging equipment and settings used, (2) describe the fish handling procedures during imaging, and (3) illustrate image orientation with respect to the physical positioning of the probe and the catfish. Ultrasound images of the ovaries of channel catfish were recorded as digital video recordings (audio video interleave format) and as still images (ultrasound image format files). This study integrated the use of nonanesthetized, submersed fish within a recirculating tank system or portable container and a submersed waterproof probe, which enabled us to use water as a transmission medium for ultrasound. This allowed us to image the fish in ventral recumbency (upright swimming position) without using a physical restraint in the tank system, or...