Diagnóstico e efeitos da contaminação por urina em sêmen canino refrigerado

Abstract

Currently, the method used to diagnose contamination by urine in semen is the observation of color, odor and pH of the sample. However, this presents low sensitivity when the contamination is small. The objective of the present work was to evaluate the sensitivity of urea and creatinine for the diagnosis of urine contamination in semen of dogs collected by digital manipulation and to evaluate the effects of intentional contamination by urine in refrigerated semen using urea and creatinine dosage. In experiment 1, semen (second fraction – F2 and third fraction – F3) and urine (SU) were collected from 6 clinically healthy adult dogs (2-7 years old) and the levels of creatinine, urea and pH of the samples were measured. The mean and standard deviation of urea of F2, F3 and SU were, respectively, 75.4±93.8 mg/dL; 35.4±8.35 mg/dL; 2175±497 U/L, creatinine was 3.48±4.49 mg/dL; 0.6±0.34 mg/dL; 269±99.9 mg/dL and pH were 5.57±0.53; 5.8±0.84 and 6.92±1.28. There was a difference between the pH of the F2 samples with SU (p<0.05) and the F3 samples with SU (p<0.05), being higher in urine than in semen. The statistical difference in urea occurred between F2 and F3 in relation to SU (p<0.001) and there was a difference between all samples in relation to creatinine (p<0.05). In experiment 2, the semen of nine adult male dogs was collected by digital manipulation and diluted in CaniPlus Chill ST as recommended by the manufacturer, and each sample was divided into 4 eppendorfs: without contamination (S0), contaminated with 5% (S1), 10% (S2) and 20% (S3) of urine and evaluated at the initial time T0, after 6 hours (T1), 12 hours (T2) and 24 hours (T3) during refrigeration. Values of creatinine, urea and pH of fresh semen (SF), other treatments and urine were evaluated. The values obtained were 1.53±0.95mg/dL (SF), 0.41±0.16 mg/dL (S0); 16.5±7.3 mg/dL (S1); 30.8±14.2 mg/dL (S2); 52.3±22.2 mg/dL (S3) for creatinine and 37.2±23.1mg/dL (SF); 6.98±7.31mg/dL (S0); 110±76.3mg/dL (S1); 208±167mg/dL (S2); 317±194mg/dL (S3), respectively. The pH showed no difference between fresh semen, treatments and urine. Motility showed an immediate decline in S3 compared to S0 (86.7±3.5% and 94±3.2%, respectively), in T1 there was an accentuation of the loss of motility in S3 (25.7±22.7% ), in T2 there was a difference between treatments S2 and S3 compared to S0 (36.1±20.4%; 13±13.2% versus 82.78±6.7%). In T3 the treatments with contamination by urine showed difference with S0 (61.7±12% in S1; 21.1±16.4% and 2.72±4.6% versus 78.9±4.8%). There was a difference in vigor from T1 in S2 and S3 (1.89±0.49 and 1.39±0.33) compared to S0 (2.67±0.43), remaining in T2 and T3 ( 1.56±0.39 and 1.11±0.22 at T2; 1.28±0.27 and 1±0 at T3, respectively) compared to S0 (2.5±0.5 at T2 and 2 .28±0.51 at T3). Acrosomal integrity differed only at T2 between S3 (92.7±2.3%) and S0 (95.7±1.1). Plasma membrane integrity and percentage of minor, major and total defects did not differ (p>0.05) between treatments over time. The effects of urine on semen were aggravated in samples with higher levels of creatinine and urea resulting from higher levels of urine contamination. Urinary compounds may be responsible for deleterious effects on semen and contact time is a relevant factor in these effects. The use of fructose and glucose-based extenders can attenuate the negative effects on the integrity of the plasmatic and acrosomal membrane, as well as on sperm defects. It is possible to affirm that the measurement of urea and creatinine for the diagnosis of contamination of canine semen by urine proved to be a sensitive method and that the deleterious effects of contamination by urine in samples of canine semen are dosedependent and aggravated according to the time in contact with urine.