169 CRISP-2 protein and antioxidant enzymes of seminal plasma are positively related to the quality of donkey semen

Seminal plasma (SP) in equids is involved in several events that precede fertilization, such as sperm motility activation, protection, nutrition, transport and its interaction with the oocyte. In addition, SP represents the highest antioxidant defence available to spermatozoa; enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) are the main enzymes with antioxidant properties, which have been related to seminal quality in stallions. Seminal plasma proteins such as cysteine-rich proteins (CRISP) and osteopontin (OST) have been related to sperm maturation and therefore with the acquisition of movement and fertilizing capacity of sperm, as well as with the interaction with the oocyte; however, few studies that evaluate these proteins have been reported in donkeys. The aim of this study was to evaluate the association between SP proteins with the quality of semen in Colombian creole donkeys. Ten Colombian Creole donkeys (Equus asinus) were collected using the artificial vagina method to obtain three ejaculates per animal, for a total of 30 ejaculates. Volume (VOL) and sperm concentration (CON) were evaluated. A fraction of each ejaculate was centrifuged to obtain the SP, in which the concentration of total proteins (TP) was evaluated using the Bradford method; CAT, SOD and GPX were also evaluated by spectrophotometry; and the concentration of the CRISP-2 protein and OST, by the ELISA assay. Sperm motility and kinetics were evaluated using a computerized semen analysis system; sperm vitality (SV) by fluorescence microscopy; abnormal morphology (AM) by eosin-negrosin staining; and the integrity of the plasma membrane (IM) by the HOST test. A descriptive analysis of data and a Pearson correlation analysis were performed. A semen quality index (SQi) was calculated, including the parameters of total motility (TM), progressive motility (PM), SV, AM and IM. Sperm quality results were VOL 51.2 ± 5.6 mL, CON 310.7 ± 21.9 × 10⁶ cells mL⁻¹, TM 86.5 ± 1.8%, PM 57.6 ± 2.9%, SV 81.3 ± 1.5%, AM 25.5 ± 1.6% and IM 74.5 ± 1.6%. Mean values found in SP for TP were 8.5 ± 0.03 mg BSA mL⁻¹, CAT 3902 ± 111.6 U mL⁻¹, SOD 32.9 ± 0.2 U mL⁻¹, GPX 804.0 ± 46.2 U L⁻¹, CRIPS2 0.49 ± 0.02 ng mL⁻¹ and OST 70.4 ± 4.9 ng mL⁻¹. Significant correlations (P < 0.05) were found between OST and VOL (−0.41), and between CAT and CON (0.50), SOD and SV (0.35), and GPX and CON (0.44). CRISP-2 showed significant (P < 0.05) correlations with TM, IM and SQi of 0.41, 0.37 and 0.43, respectively. It is concluded that CRISP-2 protein and antioxidant enzymes of seminal plasma are positively related to the quality of donkey semen.