Comparison of two processing techniques on the sperm quality of semen contaminated with urine.

Urospermia in stallions can occur intermittently, consistently, or as an isolated event, and may result in reduced sperm quality which is often assumed to reduce fertility. Although sperm quality declines in urospermic ejaculates, fertility has not been assessed in mares bred with urine contaminated semen. The aims of this study were to compare sperm quality after simple dilution (SD), cushioned centrifugation (CC) alone, or cushioned centrifugation combined with a 40 % silane-coated silica solution (SC) in semen contaminated with 0, 20, or 40 % (v/v) urine. Sperm quality values tended to decrease as the percent urine increased within all treatments (SD, CC, SC) after 24 h of cooled storage. However, SC treated groups had higher sperm quality compared to SD and CC when exposed to 20 or 40 % (v/v) urine. Differences in pregnancy rates among treatment groups (SD with 0 or 40 % (v/v) urine, or 40 % (v/v) urine followed by SC) were unable to be detected.

Single-Layer Colloid Centrifugation as a Method to Process Urine-Contaminated Stallion Semen After Freezing-Thawing.

Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey's test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.

Acute exposure to hyperosmotic conditions reduces sperm activation by urine in the yellowtail tetra Astyanax altiparanae, a freshwater teleost fish / Exposição aguda a condições hipereosmoticas reduz ativação do sêmen por urina em lambari do-rabo-amarelo (Astyanax altiparanae), um teleósteo de água doce

In freshwater fish with external fertilization, sperm sampling can be contaminated with urine, which triggers motility and gives rise to decreased fertilization success. The maintenance of freshwater fish in hyperosmotic conditions may reduce urine production and improve sperm quality. Thus, the aim of this work was to verify if acute exposure to various NaCl concentrations improves sperm quality in the yellowtail tetra Astyanax altiparanae. Spermiation was induced using a single dose of carp pituitary gland (5 mg kg-1) and the males were maintained at various NaCl concentrations: NaCl 0.00% (control), NaCl 0.45% (hypoosmotic), NaCl 0.9% (isosmotic) and NaCl 1.0% (hyperosmotic) for 6 h at 26 °C. Sperm was collected and verified for activation by urine and motility traits. At 0.00%, 0.45%, and 0.90%, the sperm was motile just after sampling, indicating activation by urine. Surprisingly, at hyperosmotic conditions, no activation was observed. Other sperm and motility parameters did not show any statistical differences, including sperm viability (P = 0.7083), concentration (P = 0.9030), total motility (P = 0.6149), VCL (curvilinear velocity; P = 0.1216), VAP (average path velocity; P = 0.1231) and VSL (straight-line velocity; P = 0.1340). Our results indicate that acute maintenance at hyperosmotic conditions eliminates sperm activation by urine and maintains sperm quality. Such a new procedure is interesting for both basic and applied sciences, including reproductive practice in fish.(AU)

Em peixes de água doce com fertilização externa, a amostragem de espermatozoides pode ser contaminada pela urina, o que desencadeia motilidade e gera menor sucesso na fertilização. A manutenção de peixes de água doce em condições hiperosmóticas pode reduzir a produção de urina e melhorar a qualidade do esperma. Assim, o presente trabalho foi delineado para verificar se a exposição aguda a várias concentrações de NaCl melhora a qualidade do esperma no tetra-amarelo Astyanax altiparanae. A espermiação foi induzida usando uma dose única de hipófise da carpa (5 mg kg-1) e os machos foram mantidos em várias concentrações de NaCl: NaCl 0,00% (controle), NaCl 0,45% (hipoosmótico), NaCl 0,9% (isosmótico) e NaCl 1,0% (hiperosmótico) por seis horas a 26 °C. O esperma foi colhido e verificado quanto à ativação por urina e traços de motilidade. Em 0,00%, 0,45%, 0,90% os espermatozóides eram móveis logo após a amostragem, indicando ativação pela urina. Surpreendentemente, em condições hiperosmóticas, nenhuma ativação foi observada. Outros parâmetros espermáticos e de motilidade não mostraram diferenças estatísticas, incluindo viabilidade espermática (P = 0,7083), concentração (P = 0,9030), motilidade total (P = 0,6149), VCL (Velocidade Curvilinear; P = 0,1216), VMD (Velocidade Média de Deslocamento; P = 0,1230) e VLR (Velocidade em linha Reta; P = 0,1340). Nossos resultados indicam que a manutenção aguda em condições hiperosmóticas elimina a ativação do esperma pela urina e mantém a qualidade do esperma. Esse novo procedimento é interessante para as ciências básicas e aplicadas, incluindo a prática reprodutiva em peixes.(AU)

Effect of urine contamination on stallion semen freezing ability.

Urospermia is a common ejaculatory dysfunction of stallions. Current practice suggests that urine contaminated semen should not be used for cryopreservation. The aim of this study was to determine effects of urine contamination on semen freezing. Sixty-five ejaculates from eight stallions were divided into no urine (CONT), low (20% urine, LOW), and high (50% urine, HIGH) samples. Semen was extended with a commercial cooling extender, cushion-centrifuged, resuspended to 200 million/mL in a commercial egg-yolk based extender, and cryopreserved in liquid nitrogen. A subset of ejaculates (n = 20) were split in two after cushion-centrifugation, and one half of the ejaculate was submitted to a single-layer gradient centrifugation before cryopreservation. Sperm motility parameters were assessed pre- and post-freezing with an automated sperm analyzer. Semen pH, creatinine, and urea concentrations were assessed in raw samples, after urine contamination and after centrifugation and extension. Statistical analyses were performed with ANOVA and Tukey's posthoc. There were significant reductions in total and progressive sperm motilities (i.e., %TM and %PM, respectively) with increasing urine contamination pre-freezing (%TM 67 ±â€¯1.7, %PM 50 ±â€¯2.2, CONT), (%TM 60.3 ±â€¯1.7, % PM 42.5 ±â€¯2.1, LOW), and (%TM 41.3 ±â€¯2, %PM 21.3 ±â€¯1.5, HIGH). Post-thaw motilities for CONT (%TM 54 ±â€¯2.3, %PM 40.8 ±â€¯3.3) and LOW (%TM 51.7 ±â€¯1.8, %PM 36.2 ±â€¯2.1) were not different, but were higher than the HIGH (%TM 31.5 ±â€¯1.2, %PM 17.1 ±â€¯1.0) (p < 0.05). Post-thaw sperm viability was significantly lower in the HIGH (54.7 ±â€¯2.4) than in the CONT (63.8 ±â€¯2.3) or LOW (64.6 ±â€¯3.4) groups. Semen creatinine and urine levels were significantly higher with increasing urine contamination and were significantly decreased after centrifugation and resuspension in freezing extender. Pre-treatment semen pH was significantly lower than semen contaminated with low or high amounts of urine, and pH decreased significantly after centrifugation and resuspension. Gradient centrifugation did not improve %TM in the control group, but it did improve pre-freeze %TM and %PM in the low and high groups and improved significantly post freezing %TM and %PM in the high urine contaminated group. Semen contaminated with a small amount of urine may be suitable for freezing, whereas highly contaminated semen might not be usable. Although urine was mostly removed in this fashion, the initial exposure to high quantities was sufficient to decrease sperm motility pre- and post-freezing, whereas low urine contamination was not as detrimental.

The effects of urine concentration, and cushion centrifugation to remove urine, on the quality of cool-stored stallion sperm.

Urine-contaminated stallion semen is a clinical problem due to a variety of causes. The effect of the level of urine contamination on the longevity of sperm quality has not been evaluated. The aim of this study was to determine the effects of urine concentration level (0%, 10%, 20%, 30%, and 40%) and cushioned centrifugation and resuspension of the sperm pellet in fresh extender, on measures of sperm quality, immediately after semen collection (T0), after 1 hour of storage at room temperature (T1), and after 24 hours of cooled storage (T24). In general, most sperm quality measures declined with increasing urine concentration starting at T0. Cushioned centrifugation (CC), but not simple dilution, generally maintained sperm quality at T24 as compared with T1. At T24, total sperm motility was higher in all urine-contaminated CC samples compared with uncentrifuged samples (P < 0.05); sperm viability was lower in CC than uncentrifuged at a urine concentration of 20%, but higher at 30% and 40% (P < 0.05); and DNA quality was decreased (higher % cells outside the main population) in all urine concentrations (P < 0.05). Immediate extension in semen extender, followed by cushioned centrifugation and resuspension of the sperm pellet in fresh extender, provided the best option for preserving sperm quality of urospermic semen.

Diagnosis and effects of urine contamination in cooled-extended stallion semen.

Urospermia is known to affect semen quality in many mammals, including stallions. Determinations of semen pH and creatinine and urea concentrations have been used to diagnose urine contamination in raw stallion semen. Unfortunately, practitioners suspecting urine contamination in cooled-shipped samples have no proven means to confirm the presence of urine. Therefore, the objectives of this study were (1) to assess the effects of urine contamination on sperm motility of extended fresh and cooled-stored stallion semen, (2) to evaluate the usefulness of semen color, odor, pH, and creatinine and urea concentrations for urospermia diagnosis, and (3) to evaluate the accuracy of a commercial blood urea nitrogen test strip in diagnosing urine contamination in extended-cooled stallion semen. Thirty-seven ejaculates were obtained from 11 stallions with no history of urospermia before division into 5 mL aliquots, and contamination with stallion urine. Each resulting sample was assessed for sperm motility, color, odor, pH, creatinine, and urea nitrogen concentration using both a semiquantitative test strip (Azostix), and a quantitative automated analyzer before and after cooling for 24 hour. Sperm motility parameters, pH, and creatinine and urea concentrations were analyzed using mixed models. Urine contamination decreased total and progressive motility in all samples before and after cooling (P < 0.05). Mean control total motility was 80% at 0 hour and 67% at 24 hours, whereas urine-contaminated samples ranged from 30% to 71% at 0 hour and 27% to 61% at 24 hours. Control mean urea (29 mg/dL) and creatinine (0.6 mg/dL) concentrations were significantly different (P < 0.05) from all urine-contaminated samples (158 mg/dL and 11.6 mg/dL, respectively) at 0 hour. Similarly, control mean urea (8 mg/dL) and creatinine (0.9 mg/dL) concentrations were significantly different than all urine-contaminated samples at 24 hours. Odor assessment presented moderate sensitivity (65%) and high specificity (100%), while color assessment presented low sensitivity (47%) and moderate specificity (79%) for urine in extended semen. Azostix strips were highly sensitive (95%) and specific (97%). Assessment of color, odor, and pH are not reliable methods to diagnose urine in experimentally contaminated cooled-stored stallion semen. Sperm motility parameters (in raw and cooled semen) are significantly reduced by the presence of urine in a concentration dependent. The results of the present study indicated that determination of urea and creatinine concentrations can be used to diagnose urospermia and that Azostix can be used as a point care method for diagnosing urine contamination in extended cooled stallion semen.