Pinar Alkim Ulutas

The influence of cysteine and taurine on microscopic-oxidative stress parameters and fertilizing ability of bull semen following cryopreservation.

Oxidative stress significantly damages sperm functions such as motility, functional integrity, endogenous antioxidant enzyme activities and fertility due to lipid peroxidation induced by reactive oxygen species (ROS). The aim of this study was to determine the effects of antioxidants such as taurine and cysteine in Bioxcell extender on standard semen parameters, fertilizing ability, lipid peroxidation (LPO) and antioxidant activities comprising reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) after the cryopreservation/thawing of bull semen. Nine ejaculates for each bull were included in the study. Three groups, namely taurine (2mM), cysteine (2mM), and control, were designed to analyze the antioxidants in Bioxcell. Insemination doses were processed so that each 0.25-ml straw contained 15 x 10(6) sperm. The addition of cysteine led to higher motility, compared to the other groups (P<0.001). Cysteine showed a greater protective effect on the percentages of acrosome damage and total abnormalities in comparison to the other groups (P<0.001). No significant differences were observed in hypo-osmotic swelling test (HOST), following supplementation with antioxidants during the freeze-thawing process. No significant difference was observed in non-return rates among groups. In biochemical assays, the additives did not show effectiveness on the elimination of malondialdehyde (MDA) formation and maintenance of GSH and GSH-Px activities, when compared to controls. CAT activity (35.1+/-8.1 kU/g) was demonstrated to be significantly higher upon the addition of 2mM taurine (P<0.001), while the level of MDA increased, indicating oxidative stress in this group. SOD activity (21.4+/-2.9 U/g protein) was significantly elevated in the group with cysteine, compared to the other groups (P<0.001).

Effects of different extenders and centrifugation/washing on postthaw microscopic-oxidative stress parameters and fertilizing ability of Angora buck sperm.

The main objective of the current study was to evaluate the effects of extender type and centrifugation/washing prior to cryopreservation on the postthaw sperm parameters, lipid peroxidation, and superoxide dismutase activity of Angora buck (Capra hircus ancryrensis) sperm. Ejaculates collected from three Angora bucks were used in this study. Two consecutive ejaculates from each buck were pooled and split into equal parts in four Falcon tubes. Two tubes were diluted at 37 degrees C and then centrifuged to remove semen plasma. After centrifugation, two sediment parts were diluted with a Tris-based extender and commercial Bioxcell extender, respectively. The remaining two parts, which were not centrifuged/washed, were diluted with the above-mentioned extenders, respectively. Diluted samples were cooled to 5 degrees C and frozen in 0.25-mL French straws to be stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20 sec in a water bath for evaluation. The semen part with centrifugation/washing in the Bioxcell extender (BC) demonstrated a higher rate of subjective motility (58.1+/-3.0%) compared with that of groups with (TC) or without (T) centrifugation/washing in the Tris-based extender (P<0.01). Angora buck sperm frozen with (BC) or without (B) centrifugation/washing in the Bioxcell extender demonstrated higher percentages of motility (60.6+/-2.7% and 54.3+/-4.8%, respectively) compared with that of groups T and TC. The postthaw progressive motility rate (22.3+/-2.7%) was significantly greater for semen parts diluted in B compared with that of other groups. BC gave rise to a lower value of average path velocity (90.0+/-5.2 microm/sec) compared with that of other groups (P<0.01). For straight linear velocity and linearity index, the highest values (103.2+/-4.7 microm/sec, 47.5+/-1.6% and 94.8+/-3.0 microm/sec, 44.8+/-1.1%, respectively) were obtained from B and TC (P<0.001). For sperm acrosome and total abnormalities, TC gave the highest values (11.2+/-0.6% and 26.6+/-1.5%, respectively, P<0.01). In the group frozen in BC, the percentage of membrane integrity assessed by hypo-osmotic swelling test was higher (61.2+/-2.2%) than that of the other groups (P<0.001). With respect to fertility results based on 35-d pregnancy rates, BC gave a higher rate (76.5%) than that of TC (27.8%, P<0.05). Malondialdehyde formation was found to be lower (1.64+/-0.26 nmol/L) in BC than in the other groups after the freeze-thawing process (P<0.001). In the semen part frozen in BC, superoxide dismutase activity was higher (0.18+/-0.02 U/mg protein) compared with that of the other groups (P<0.05). Further studies are required to obtain more precise results for the characterization of oxidative stress parameters and fertilizing ability in cryopreserved buck spermatozoa.

Effects of hypotaurine, cysteamine and aminoacids solution on post-thaw microscopic and oxidative stress parameters of Angora goat semen.

This study was conducted to determine the effects of cysteamine, hypotaurine and aminoacids solution (BME) on standard semen parameters, lipid peroxidation and antioxidant activities of Angora goat semen after the freeze-thawing process. Ejaculates collected from four Angora goats were evaluated and pooled at 37 degrees C. Semen samples, which were diluted with a Tris-based extender containing the antioxidants hypotaurine (5 mM) and cysteamine (5 mM), and an aminoacid solution (13%), and an extender containing no antioxidants (control), were cooled to 5 degrees C and frozen in 0.25-ml French straws in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. Supplementation with cysteamine, hypotaurine and BME caused significant (P<0.05) increases in sperm motility, and significant (P<0.05) decreases in total abnormality rates in comparison to the control group. While all in vitro treatments did not affect the acrosomal abnormality rates, hypotaurine and BME but not cysteamine significantly (P<0.05) increased the HOST results as compared to the control group. Supplementation with antioxidants and BME did not significantly affect MDA levels and CAT activity in comparison to the control group (P>0.05). The antioxidants hypotaurine and cysteamine decreased SOD activity when compared to the BME group and controls (P<0.001).

Effect of glutamine and sugars after bull spermatozoa cryopreservation.

The objective of this study was to evaluate the effects of the addition of different sugars (raffinose, sucrose, and trehalose) on bull spermatozoa cryopreserved in a commercial extender (Optidyl) supplemented with glutamine on semen parameters, fertilizing ability and superoxide dismutase (SOD) activity. Nine ejaculates for each bull were used in the study. Semen was frozen in five different extenders: raffinose 25 mM plus glutamine 3 mM (RGO), sucrose 25 mM plus glutamine 3 mM (SGO), trehalose 25 mM plus glutamine 3 mM (TGO), glutamine 3 mM (GO) and control (O). Insemination doses were processed so that each 0.25 mL straw contained 15 x 10(6)sperm. Groups of GO and RGO resulted in the higher rates of subjective (54.0 ± 1.7% and 64.0 ± 1.1%; P < 0.01) and CASA motilities (53.0 ± 2.7% and 61.0 ± 4.4%; P < 0.001), respectively compared to the other groups. The supplementation of additives did not provide an effect on the level of post-thaw sperm CASA progressive motilities, the sperm motion characteristics and pregnancy rates. GO and RGO provided the better protective effect for sperm acrosome (4.0 ± 0.5% and 12.0 ± 0.6%) and total abnormalities (5.0 ± 0.3% and 13.0 ± 0.7%; P < 0.001), respectively. At the HOST values, the additives did not give to result the protective effect in comparison to Optydil extender without additives (P > 0.05). For pregnancy rates, there were no significant differences among the groups. The supplementation of additives did not provide any significant difference on the level of SOD activity (P > 0.05). It can be also thought that these sugars might have worked with glutamine in a synergy. Thereby, sugars such as raffinose and sucrose with glutamine in freezing extender may be recommended to facilitate bull semen freezability.