D.L. Paccamonti

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.

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.

The effects of parturition and peripartum complications on the peritoneal fluid composition of mares

Abnormalities in peritoneal fluid are diagnostically useful for managing equine colic; however, their significance in post-dystocia mares is not known. This study was to determine what changes, if any, occurred following obstetrical manipulations. Peritoneal fluid samples were collected from 2 groups of foaling mares to establish control values, and from a third group that had developed clinical abnormalities (CAb,n = 14) or had made an uneventful recovery (CN,n = 36) following fetal extraction. In Group 1 mares, samples were collected before and after induced parturitions (n = 7), and although the total nucleated cell count was increased (P < 0.02) the median values for peritoneal fluid composition remained within the normal reference range. In Group 2 mares, samples were collected after unassisted foalings (n = 10) on postpartum Days 1, 3, 5 and 7, and the peritoneal fluid values remained within the normal reference range. In the Group 3 (CN) mares neither assisted vaginal delivery or fetotomy caused median peritoneal fluid values to rise above the normal reference range. Although remaining within normal limits, the total nucleated cell count was increased (P < 0.01) on Day 2. The median peritoneal fluid total protein value for Group 3 (CAb) mares was greater than the median value for Group 3 (CN) mares on Day 1 (P < 0.05) and Day 2 (P < 0.001). The peritoneal fluid total nucleated cell count in Group 3 (CAb) mares with a uterine tear, vaginal laceration involving the peritoneal cavity, or a ruptured mesocolon was greater than in Group 3 (CN) mares (P < 0.02). The median peritoneal fluid percentage of neutrophils value for Group 3 (CAb) mares was higher than for Group 3 (CN) mares on both Days 1 and 2 (P < 0.02). Elevation of a single peritoneal fluid value in the postpartum mare may be incidental; however, increases in 2 or more of these (total protein > 3.0 g/dl; total nucleated cell count > 15,000 cells/microl; percentage of neutrophils > 80%) is clinically significant.

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).

Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification

Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts.

Day of cycle affects changes in equine intrauterine pressure in response to teasing

Oxytocin is released in response to teasing during both estrus and diestrus in mares, and at least during estrus, teasing results in an increase in electromyographic activity in the uterus. Exogenous oxytocin causes an increase in intrauterine pressure and prior studies have shown that this response is correlated to the day of the estrous cycle. To determine if teasing causes an increase in intrauterine pressure and if this response varies by day of the cycle, intrauterine pressure was measured while mares were teased with a stallion 2 days before ovulation, on the day ovulation was detected and 2 days after ovulation. A significant increase in intrauterine pressure was observed in response to teasing both 2 days before ovulation and on the day of ovulation, when plasma concentrations of progesterone were low. No significant increase in intrauterine pressure was observed in response to teasing 2 days after ovulation when progesterone concentrations were elevated. Management practices that include teasing or stallion exposure may be beneficial in stimulating uterine clearance mechanisms in mares during the preovulatory period.