ABSTRACT
Mammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE-sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility-inhibitory factor of sheep I and II (MIFS-I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS-I and II, respectively. Specific activities of the partially purified MIFS-I and II were 563 and 261 U/mg of protein, while the fold-purification of the activity were 5119 and 2373, respectively. Both the proteins were heat-labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS-I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS-I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross-reactivity of MIFS-I with polyclonal anti-human SEMA3D antibody. It was concluded that the MIFS-I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation.
Subject(s)
Epididymis , Semaphorins , Humans , Male , Animals , Sheep , Cattle , Swine , Sperm Motility , Semen , Antibodies , Tyrosine , MammalsABSTRACT
BACKGROUND AND AIM: Lecithin based diluent such as AndroMed, is a semen diluent made without animal components to prevent the risk of disease transmission, while glutathione (GSH) is an intracellular non-enzymatic antioxidant that prevents cell damage due to reactive oxygen species. Meanwhile, the specific impact of AndroMed and GSH combinations on spermatozoa abnormalities has not been fully studied. Therefore, this study aims to determine the effect of using cauda epididymal plasma-2 (CEP-2) and AndroMed diluents with or without the addition of GSH on the abnormalities of sexing semen of Ongole crossbred bulls in cold storage. MATERIALS AND METHODS: This study used a factorial completely randomized design 2×2, the first factor was types of diluent and the second was with or without the addition of GSH. Observation of spermatozoa abnormalities was carried out at a storage time of 0-5 days using 297 ejaculations of liquid semen, with 100 spermatozoa observed per smear of each ejaculate. Furthermore, the data were analyzed using a two-way analysis of variance and the significant threshold (p-value) for statistical analysis was set at <0.05. RESULTS: The results showed that there was a significant difference (p<0.05) between AndroMed and CEP-2 in minimizing the abnormalities of upper layer spermatozoa (X), with parameters DH and AD on day 0, damage of spermatozoa (DMR) on days 1-5, and dag-like defect (DLD) on day 5. Furthermore, spermatozoa abnormalities in the lower layer (Y) showed a significant difference between diluents in the parameters of AD on day 1, DMR on days 0-5, and DLD on days 1-5. The significant difference between with or without the addition of GSH in the X sperm was observed in the DH parameters on day 0 and DMR on 5, while there was no significant difference in the Y sperm. CONCLUSION: Based on the results, AndroMed has the potential to minimize spermatozoa abnormalities compared to CEP-2 diluent in sexed liquid semen. Therefore, AndroMed diluents with or without the addition of 1 mM GSH have no significant effect on spermatozoa abnormalities.
ABSTRACT
Cauda epididymis in mammals is known to store mature sperm largely in quiescent state for several weeks without significantly affecting fertility. Hence, the aim of this study was to evaluate the effects of mimicking cauda epididymal plasma (CEP)-like conditions in extender on liquid preservation of ram semen at 3-5°C. Four experiments were conducted in this study: (1) evaluation of physicochemical properties of ram CEP, (2) effect of hyperosmotic solution on sperm motility and functional membrane integrity (FMI), and the effects of (3) CEP-like hyperosmolality (390 vs. 360 mOsmol/kg) and (4) pH in extender (pH 6.5 vs. 6.8) on liquid preservation of ram semen. Sperm treatment with hyperosmotic solution (450 mOsmol/kg) resulted in a decline (P < 0.05) in mass motility (3.5 ± 0.2 vs. 4.3 ± 0.2) and FMI (30.4 ± 3.2 vs. 52.1 ± 2.1%) compared to that with isoosmotic solution (360 mOsmol/kg). Overall, sperm viability, acrosomal integrity, and progressive motility were similar (P > 0.05) while straight-line velocity (77.8 ± 3.1 vs. 71.3 ± 2.7µm/s), linearity (47.4 ± 0.4 vs. 39.5 ± 0.9%), straightness (79.7 ± 0.5 vs. 74.0 ± 0.5%) and beat cross frequency (28.6 ± 0.8 vs. 26.0 ± 0.5 Hz) were higher (P < 0.05) and FMI (65.7 ± 1.5 vs. 75.4 ± 1.1%) was lower (P < 0.05) following liquid-preservation in hyperosmotic extender compared to that in isoosmotic extender. Both total motility (83.3 ± 1.8 vs. 75.4 ± 1.5%) and progressive motility (51.7 ± 2.3 vs. 39.5 ± 1.9%) were higher (P < 0.05) at 48 h of storage in hyperosmotic extender compared to the control. Overall, the seminal attributes were similar (P > 0.05) between the two pH's of the extender. In conclusion, semen extender having CEP-like osmolality but not the pH was superior to extenders having conventional osmolality and pH for liquid preservation of ram semen.Abbreviations: AI: artificial insemination; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; CASA: computer-assisted semen analyzer; CEP: cauda epididymal plasma; ELON: elongation; EYTF: egg yolk-Tris-citrate-fructose; FMI: functional membrane integrity; GLM: general linear model; GPC: glycerophosphatidylcholine; HOS: hypoosmotic swelling; LIN: linearity; pHe: external pH; PROG: progressive motility; S.E.M.: standard error of the mean; SLTF: soya lecithin-Tris-fructose extender; SP: seminal plasma; STR: straightness; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight-line velocity; TM: total motility.