The intersection of gender and drug use-related stigma: A mixed methods systematic review and synthesis of the literature.

BACKGROUND: Substance use-related stigma is a significant barrier to care among persons who use drugs (PWUD). Less is known regarding how intersectional identities, like gender, shape experiences of substance use-related stigma. We sought to answer the following question: Do men or women PWUD experience more drug use stigma? METHODS: Data were drawn from a systematic review of the global, peer-reviewed scientific literature on substance use-related stigma conducted through 2017 and guided by the Stigma and Substance Use Process Model and PRISMA guidelines. Articles were included in the present analysis if they either qualitatively illustrated themes related to the gendered nature of drug use-related stigma, or quantitatively tested the moderating effect of gender on drug use-related stigma. RESULTS: Of the 75 studies included, 40 (53 %) were quantitative and 35 (47 %) were qualitative. Of the quantitative articles, 22 (55 %) found no association between gender and drug use-related stigma, 4 (10 %) identified women who use drugs (WWUD) were more stigmatized, and 2 (5 %) determined men who use drugs (MWUD) were more stigmatized. In contrast, nearly all (34; 97 %) of the qualitative articles demonstrated WWUD experienced greater levels of drug use-related stigma. CONCLUSION: The quantitative literature is equivocal regarding the influence of gender on drug use-related stigma, but the qualitative literature more clearly demonstrates WWUD experience greater levels of stigma. The use of validated drug use-related stigma measures and the tailoring of stigma scales to WWUD are needed to understand the role of stigma in heightening the disproportionate harms experienced by WWUD.

Biophysics of zebrafish (Danio rerio) sperm.

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37+/-0.02 (SEM), an isosmotic cell volume (V(o)) of 12.1+/-0.2 microm(3) (SEM), a water permeability (L(p)) in 10% dimethyl sulfoxide of 0.021+/-0.001(SEM)microm/min/atm, and a cryoprotectant permeability (P(s)) of 0.10+/-0.01 (SEM)x10(-3)cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24h at 0 degrees C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.

Capacitation-like changes in equine spermatozoa following cryopreservation.

The primary objective of this study was to assess plasma membrane characteristics and activation of signal transduction pathways in equine spermatozoa during both in vitro capacitation and cryopreservation. Significant plasma membrane restructuring, as assessed by measurement of plasma membrane lipid disorder and phospholipid scrambling, was not observed until after cryopreservation and subsequent thawing (P < 0.05). Although in vitro capacitated cells also displayed increased plasma membrane lipid disorder and phospholipid scrambling (P < 0.05), it appeared that regulation of these events in in vitro capacitated versus cryopreserved equine spermatozoa was not identical. Addition of 5 microM staurosporine to the capacitation media reduced plasma membrane phospholipid scrambling (P < 0.05), but supplementation to the freezing extender prior to cryopreservation did not. Furthermore, progesterone was able to induce a greater degree of acrosomal exocytosis in in vitro capacitated versus frozen/thawed spermatozoa. Expression of phospholipid scramblase, a protein thought to be important in plasma membrane phospholipid scrambling, did not differ between treatments. Comparison of protein tyrosine phosphorylation patterns between in vitro capacitated and cryopreserved cells demonstrated a divergence in signal transduction. Cellular signaling in in vitro capacitated equine spermatozoa appeared to be in part dependent on activation of the cAMP/PKA pathway, whereas signaling in cryopreserved cells seemed to proceed predominantly through alternative pathways. Taken together, these data support the idea that capacitation and "cryocapacitation" are not equivalent processes.

Spermatozoal response to osmotic stress.

The process of sperm cryopreservation imparts on sperm cells the stress of low-temperature and drastic osmotic change. Damage to the cell plasma membrane results in cell injury in a number of cellular structures and associated functions. Studies in the author's laboratory have focused upon the various mechanisms of osmotic and thermal injury including plasma membrane lipid structure, mitochondrial membrane potential, and intracellular signaling. We have determined that cryoinjury to sperm, as for somatic cells, is a multi-factorial event and some of these events are reversible while some are not.

Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides.

Because of its location on the sperm surface and its multiple functions during fertilization, the PH-20 protein is a potential target for contraceptive vaccines. Cynomolgus macaques were immunized using four different adjuvants together with synthesized peptides or recombinant proteins representing selected regions of macaque PH-20. The synthesized peptide (amino acids 387-412, designated Peptide 4) was used as a linear molecule in a 1:1 ratio with a peptide sequence of tetanus toxoid, as well as a multiple antigenic peptide (MAP) matrix held together by scaffolding lysine residues. In the MAP construct, the ratio of Peptide 4 to tetanus peptide was 4:1. To circumvent the poor production of recombinant PH-20 in bacterial cells, two truncated forms of the molecule were expressed in Escherichia coli, G18 (encoding amino acids 143-510) and E10 (encoding amino acids 291-510). The adjuvants were Montanide ISA 51, Titermax Gold, Syntex adjuvant formulation (SAF), and QS-21. All of the antigen/adjuvant combinations produced significant immune responses as measured by ELISA. The circulating antibodies from immunized animals recognized macaque sperm surface PH-20 on Western blots and were shown by indirect immunofluorescence to bind to the surface of macaque sperm. Montanide and Titermax were associated with higher titers of anti-PH-20 antibodies than QS-21 and SAF adjuvants. Immunization with Titermax, however, resulted in sterile abscesses in 4 of 8 animals injected. We conclude that antigens derived from synthesized peptides and recombinant proteins representing selected regions of the PH-20 molecule can be used as vaccine components in combination with the adjuvant Montanide to elicit a significant sperm-directed antibody response in immunized macaques.

Effect of hyaluronic acid/chondroitin sulfate on healing of full-thickness tendon lacerations in rabbits.

Viscoat, a high-molecular-weight, highly purified hyaluronic acid (HA) and chondroitin sulfate (CS) compound, was instilled around rabbit plantaris tendon following full-thickness laceration and surgical repair. After 3 weeks of immobilization, no significant difference in adhesion strength or tensile strength of the healing tendons existed between Viscoat-treated tendons and controls. This contradicts previous studies which suggest that hyaluronic acid reduces postoperative tendon adhesions. Further studies examining tendon adhesions after less severe degrees of tendon injury and using direct, quantitative measurement techniques are warranted to demonstrate whether HA has a beneficial effect on tendon healing.

The effect of exogenous follicle stimulating hormone treatment on anterior pituitary and testicular function in the prepubertal bull.

The effect of exogenous follicle stimulating hormone (FSH) on testicular function was studied in twenty 4-month-old Holstein bulls during two seasons (ten in late winter and ten in summer). Five mg FSH or saline were given subcutaneously at 12-hour intervals (10 mg/day) for ten days, after which time the testes were removed, weighted, and incubated in vitro with 0, 15, or 150 ng luteinizing hormone (LH) for 3 hours. FSH treatment resulted in significantly heavier testes (38%) in the summer (39.3 +/- 1.6 vs. 28.5 +/- 1.5 g) but not in late winter. Epididymal weight was not affected. Across seasons, serum LH concentrations were higher (P = 0.06) in FSH-treated bulls (2.39 +/- 0.13 vs 1.80 +/- 0.13 ng/ml); the largest increase occurred in the summer. FSH treatment did not change serum testosterone levels in late winter, whereas in the summer serum testosterone concentrations were three-fold higher (0.90 +/- 0.06 vs 0.29 +/- 0.02 ng/ml; P less than 0.05) in the FSH-treated bulls. In addition, serum testosterone concentrations increased with time of FSH treatment. FSH treatment stimulated a 2.4-fold increase (P less than 0.05) in testosterone content of the testes across seasons and a three-fold increase (P less than 0.05) in testosterone synthesis in vitro in the summer. There was no dose effect of LH on vitro in the summer. There was no dose effect of LH on in vitro testosterone synthesis. We conclude that administration of exogenous FSH to intact prepubertal bulls is capable of altering testicular function, but that seasonal influences can modify this function.

Capacitation in vitro of stallion spermatozoa: comparison of progesterone-induced acrosome reactions in fertile and subfertile males.

Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of biological and chemical stimuli. In the present report, we have investigated the ability of progesterone to stimulate acrosome reactions of stallion sperm capacitated in vitro. Motile sperm were selected by a two-layer Percoll gradient centrifugation and were incubated in TALP medium modified by the 1:1 (v/v) addition of TEST-yolk medium for 5 hours at 39 degrees C, under 5% CO2 in humidified air. Sperm incubated in vitro in TALP-TEST medium had a higher percentage of acrosome reactions following the addition of progesterone (3.18 mumol/L) compared to controls (P < 0.05). Furthermore, sperm from stallions classified as fertile on the basis of breeding history had higher percentages of progesterone-induced acrosome reactions in comparison with stallions classified as subfertile (P < 0.05). Acrosome reactions were assessed routinely by fluoresceinated lectin binding, but the physiological appearance of induced acrosome reactions was confirmed at the ultrastructural level by transmission electron microscopy. We conclude that 1) TALP-TEST medium supports stallion sperm capacitation in vitro, 2) progesterone-induced acrosome reactions are physiological, and 3) sperm from fertile stallions may be more responsive to progesterone-induced acrosome reactions than those of subfertile stallions. This is the first report in a nonhuman species that differences exist between the sperm of fertile and subfertile males in the ability to capacitate and acrosome react in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Equine sperm-oocyte interaction: the role of sperm surface hyaluronidase.

The plasma membrane over the sperm head of several mammalian species has been shown to express a glycerolphosphatidylinositol-linked hyaluronidase known as PH-20. This protein has been associated with the sperm's interaction with the oocyte cumulus matrix and zona pellucida. The characteristics of PH-20 in equine sperm have not been clearly defined. In this study, ejaculated gel-free semen from five stallions and epididymal sperm from isolated epididymis from 10 stallions was used to characterize the PH-20 activity in equine sperm. Affinity purified anti-equine PH-20 polyclonal antibody was used to immunodetect sperm surface-associated PH-20 and immunolabel whole sperm. The intracellular calcium indicator, Fluo-3, was used to assess sperm intracellular calcium. Stallion sperm express a surface-associated hyaluronidase localized to the posterior sperm head region in ejaculated sperm. Following in vitro capacitation and acrosomal exocytosis, the inner acrosomal membrane (IAM) displays intense hyaluronidase fluorescence suggesting that the IAM and hyaluronidase plays a significant role in zona penetration by sperm. Sperm incubated in hyaluronan (HA)-containing capacitation medium display an elevated intracellular calcium concentration (P<0.01) that is associated with translocation of PH-20 antigenic sites on the sperm surface in addition to increases in protein tyrosine phosphorylation. Caput- and cauda-derived sperm display developmentally unique PH-20 immunofluorescence expression patterns. These data suggest that the differential expression of PH-20 in ejaculated and epididymal sperm could be involved in cumulus penetration, sperm-egg recognition, and oolemmal fusion in this species.

Zona pellucida binding and zona-induced acrosome reactions in horse spermatozoa: comparisons between fertile and subfertile stallions.

Diagnostic tests that probe sperm function are needed to determine the potential etiologies of subfertility and to explore treatments of subfertility in stallions. Using epifluorescence and phase contrast microscopy, a comparison was made between ejaculates from 3 fertile and 3 subfertile stallions in which sperm-zona pellucida binding and acrosomal status were measured. Motile spermatozoa were selected by Percoll gradient centrifugation and were capacitated in vitro using TEST:TALP capacitation medium at 39 degrees C under humidified air containing 5% CO2. Concentration of motile spermatozoa was held constant during co-incubation with oocytes for fertile and subfertile ejaculates. The total number of zona pellucida-bound spermatozoa was higher for fertile stallions than for subfertile stallions (P < 0.05). Similarly, the percentage of acrosome reactions in zona pellucida-bound spermatozoa was higher for the 3 fertile stallions than for the 3 subfertile stallions (P < 0.05). These results indicate that spermatozoa from fertile stallions may interact with female gametes differently from that of subfertile stallions and suggest that sperm functions are measurable and may vary with fertility.

The effect of postbreeding uterine lavage on pregnancy rate in mares.

One stallion and 54 mares were used in an experiment to evaluate the effect of postbreeding uterine lavage on pregnancy rate in mares. All mares were inseminated with 250 x 10(6) progressively motile sperm every other day during estrus until detection of ovulation. Mares (n = 18) were randomly assigned to one of three treatment groups: 1) no postbreeding uterine lavage (control); 2) uterine lavage at 0.5 h postbreeding; or 3) uterine lavage at 2 h postbreeding. A dilute solution of povidone-iodine (PIS; 0.05%) previously determined to render spermatozoa immotile in vitro was used to lavage the mare uteri. One liter PIS, prewarmed to 40 degrees C, was used for each lavage. Pregnancy status of mares was determined at 21 d and 36 d post ovulation, using transrectal ultrasonography. The pregnancy rate of Group 1 (66.7%) was higher than that of Group 2 (22.2%; P<0.05) or Group 3 (33.3%); P<0.10). The pregnancy rates of Groups 2 and 3 were similar (P>0.70). Evaluation of endometrial biopsies obtained from a separate set of mares (n = 3) on Day 6 post ovulation, both before and after uterine lavage, revealed no difference in the accumulation of inflammatory cells, suggesting adverse effects of lavage on fertility may have been due to excessive removal of spermatozoa from the uterus during the lavage process or damage to oviductal spermatozoa.

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C.

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.

Effects of postparturient uterine lavage on uterine involution in the mare.

Eighteen postparturient mares were used to evaluate effects of uterine lavage on uterine involution. Mares were randomly assigned to one of three treatment groups: Group 1 (seven mares), no lavage; Group 2 (five mares), lavage on Day 3 post partum; and Group 3 (six mares), lavage on Days 3, 4, and 5 post partum. Five liters sterile physiologic saline, prewarmed to 42 degrees C, were used for each lavage. Transrectal ultrasound examination of the reproductive tract was performed on Day 11 post partum to detect the presence of free fluid in the uterine lumen, to estimate the cross-sectional diameter of the uterine horns and body, and to determine if ovulation had occurred. Endometrial biopsies were also taken on Day 11 post partum to evaluate endometrial histologic characteristics. Lavage had no effect (P>0.05) on diameter of the uterine body or previously gravid uterine horn, presence of fluid in the uterine lumen, or number of mares which had ovulated by Day 11 post partum. Histologic characteristics of the endometrium (height of luminal epithelium, gland depth, relative gland vclume, and inflammatory-cell score) were not affected by treatment (P>0.05). Postpartum uterine lavage did not significantly affect uterine involution by the parameters measured in normal-foaling mares at Day 11 post partum.

Effect of dose of GnRH analog on ovulation in mares.

Proper timing of insemination for optimal conception is accomplished by frequent palpations per rectum, by ultrasonography of the preovulatory follicle and/or by treatment with hCG or GnRH. Sustained release of GnRH from implants has been shown to hasten ovulation. Therefore, 2 studies were conducted to evaluate the efficacy of a GnRH analog, deslorelin, for hastening ovulation in nonlactating cyclic mares. The GnRH implant was 2.3x3.7 mm and released deslorelin for 2 to 3 days. In Experiment 1, 60 nonlactating, cycling mares were assigned to 1 of 5 doses: 0, 1.2, 1.7, 2.2 and 2.7 mg per implant. Mares were assigned sequentially on the first day of estrus (Day 1). Ovaries were examined per rectum and with ultrasonography every 12 h until ovulation. Once the mares obtained a follicle>30 mm, they were injected subcutaneously with a GnRH implant. The mares were inseminated every other day during estrus with semen from 1 of 3 stallions. Pregnancy was determined with ultrasonography. Experiment 2, 40 nonlactating, cyclic mares were assigned to 1 of 5 treatments (same treatments as in Experiment 1). Data were obtained on interval to ovulation, duration of estrus and pregnancy rates at 12, 18 and 35 d after ovulation. Time to ovulation was shorter (P<0.05) in GnRH-treated mares than in control mares in the Experiment 1. Mean time to ovulation was 68, 49, 48, 47, 44 h in Experiment 1, and 91, 66, 58, 46, 58 h in Experiment 2 for mares given 0, 1.2, 1.7, 2.2 and 2.7 mg/mare in the 2 trials. Averaged for both experiments, the proportion of mares ovulating within 48 h of treatment was 40, 75, 85, 90 and 90% for 0, 1.2, 1.7, 2.2 and 2.7 mg/mare. For both experiments, there was no effect of GnRH on pregnancy rate. In summary, a subcutaneous implant containing GnRH analog induced ovulation in most mares by 48 h of injection, and there was no advantage of doses higher than 2.2 mg/mare.

Enterovesical fistula in a patient with cryptosporidiosis and AIDS. CT demonstration.

A case of an enterovesical fistula demonstrated by computed tomography (CT) scan in an AIDS patient with cryptosporidiosis is reported. Other studies including barium enema and endoscopy were unsuccessful in detection of the fistula. The value of CT in both the AIDS patient and the patient with suspected enterovesical fistula is reviewed.

Synchronization of estrus in dairy goats given norgestomet and estradiol valerate at various stages of the estrous cycle.

Dairy goats were given subcutaneous implants with 3 mg of norgestomet (NOR) and IM injections of 0.625 mg of estradiol valerate and 0.375 mg of norgestomet on day 0 of the estrous cycle (estrus; NOR 0, n = 18), on postestrus day 4 (NOR 4, n = 18), or on postestrus day 11 (NOR 11, n = 15). Ear implants were removed after 9 days. Mean (+/- SE) hours from removal of ear implants to onset of estrus and proportion of goats responding were 36 +/- 3.8 and 83%, 33 +/- 4.0 and 61%, and 36 +/- 2.7 and 93% for groups NOR 0, NOR 4, and NOR 11, respectively. There were no significant differences between treatment groups in time to onset of estrus. The percentage of goats in group NOR 11 that had signs of estrus was significantly greater than the percentage of goats in group NOR 4. Of the goats in groups NOR 0, NOR 4, and NOR 11 that had signs of estrus, 53, 55, and 86%, respectively, had onset of behavioral estrus between 24 and 48 hours after implant removal. All goats that had signs of estrus had onset of behavioral estrus between 12 and 72 hours after implant removal. Mean (+/- SE) hours from removal of ear implants to time of peak concentrations of luteinizing hormone (LH) were 49 +/- 4.1, 49 +/- 3.8, and 49 +/- 4.0 for groups NOR 0, NOR 4, NOR 11, respectively (not different). The percentage of goats in group NOR 11 that had LH peaks was significantly greater than the percentage of goats in group NOR 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Synchronization of estrus in dairy goats treated with prostaglandin F at various stages of the estrous cycle.

Dairy goats were given IM injections of 125 micrograms of cloprostenol sodium on day 6 of the estrous cycle (prostaglandin F [PGF] 6, n = 22) or day 12 of the estrous cycle (PGF 12, n = 26). Mean +/- SE hours from injection to onset of behavioral estrus and proportion of goats responding were 46 +/- 4.2 (range, 12 to 88 hours) and 95% and 48 +/- 2.9 (range, 34 to 68 hours) and 100% for groups PGF 6 and PGF 12, respectively. There was no significant difference between the groups in mean time to onset of estrus, but variances about the means were different. Of the does in groups PGF 6 and PGF 12, 67 and 85%, respectively, had signs of onset of estrus between 36 and 60 hours after administration of PGF. Mean (+/- SE) hours from injection to time of peak concentrations of luteinizing hormone (LH) were 62 +/- 3.1 and 64 +/- 2.1 for groups PGF 6 and PGF 12, respectively. Of the does in groups PGF 6 and PGF 12, 86 and 100%, respectively, had LH peaks. Of the does in groups PGF 6 and PGF 12, 68 and 77%, respectively, had peak concentrations of LH between 48 and 72 hours after administration of PGF. All does in groups PGF 6 and PGF 12 had concentrations of progesterone greater than or equal to 1.0 ng/ml on the day of administration of PGF. Concentrations decreased to less than 1.0 ng/ml by 48 hours after injection in all does except 1 in group PGF 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Squamous cell carcinoma of the vulva in a cow.

An 8-year-old Holstein cow was evaluated because of a slowly enlarging vulvar mass that had impaired artificial insemination attempts. Vulvar squamous cell carcinoma was diagnosed. Clinical cases of vulvar squamous cell carcinoma have not been as widely reported as those of ocular squamous cell carcinoma in cattle. Treatment modalities for vulvar squamous cell carcinoma have not been as thoroughly investigated as for those of the ocular condition. Cryosurgery and radio-frequency hyperthermia appear to be the optimal therapeutic modalities, each providing up to 90% tumor remission.

Medical management of urinary calculi in a stallion with breeding dysfunction.

A 9-year-old Thoroughbred stallion was examined because of breeding dysfunction and possible urethritis. The stallion had good libido and readily obtained an erection, mounted, and intromitted but did not thrust and ejaculate. After mounting the mare, the stallion would squeal and dismount. Endoscopic examination of the urethra and bladder revealed irregular, spiculate yellow crystals (< 1 cm in size) and sabulous deposits; numerous calculi were embedded in the mucosa of the bladder. Because the horse was at the start of a breeding season, the owner would not give permission for general anesthesia. Medical management was attempted, because postoperative convalescence after surgical removal of calculi might have curtailed breeding activities, and the calculi were small. Every 1 to 3 days, the bladder was lavaged with saline solution containing acetic acid, and anti-inflammatory and antimicrobial drugs were administered. The stallion was able to return to breeding mares, and sperm numbers and semen quality were good. However, urine contamination of the ejaculate was detected, suggesting that the stallion may have had a primary neurologic deficit affecting bladder control and function that was causing calculi to form secondarily because of delay in movement of urine through the urinary tract.

Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses.

The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited (P<0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited (P<0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.