Nuri Baspinar

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage.

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20s in a water bath for the evaluation. The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9±1.3% and 51.3±1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6±2.9% and 54.2±4.9%) and inositol (34.9±2.0% and 47.3±2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06±0.38 mM) than that of control (0.96±0.29 mM) following the freeze-thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.

Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters

The aim of the current study was to evaluate the effects of cysteine and ergothioneine on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities. Semen samples from 5 mature Merino rams were used in the study. Semen samples, which were diluted with a Tris-based extender containing l-Cysteine and l-(+)-Ergothioneine and no antioxidant (control), were cooled to 5°C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37°C for 20s in a water bath for evaluation. Ergothioneine at doses of 2 and 4mM increased percentages of subjective motility, VSL and VCL, compared to controls following the freeze-thawing (P<0.001). Ergothioneine at three different doses led to higher rates of progressive motility and VAP, compared to control groups (P<0.001). Cysteine and ergothioneine at three doses provided the higher rates of ALH, in comparison to no antioxidant group (P<0.001). As regards CASA motility, supplementation with antioxidants did not provide any significant difference on the percentage of post-thaw sperm CASA motilities, in comparison to the control. In regards of sperm membrane integrity, only cysteine 1mM provided a greater protective effect, compared to control (P<0.001). Percentages of sperm with high mitochondrial activity were dramatically increased with cysteine at doses of 1 and 2mM, compared to control (P<0.05). No significant differences were observed in sperm acrosome integrities among groups. CAT activity was increased significantly only in cysteine1mM compared to control group (P<0.001). Cysteine at doses of 2 and 4mM showed a tendency of increased activities of CAT when compared to control. But these increases were not statistically significant. Supplementation with antioxidants did not significantly affect activities of SOD and GPx. Findings of this study showed that ergothioneine supplementation in semen extenders, was of greater benefit to motility and motion characteristics of frozen-thawed ram sperm.

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).

Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen.

The aim of this study was to investigate the effects of methionine and dithioerythritol, added to the Tris extender, on ram sperm motility and LPO (lipid peroxidation) and antioxidant capacities during liquid storage up to 72 h at 5°C. Ejaculates collected from five Merino rams, were evaluated and pooled at 37°C. This study included two experiments. In experiment 1, each pooled ejaculate was divided into four equal aliquots and diluted (37°C) with the base extender, containing 0 (control), 1, 2 and 4 mM methionine, at a final concentration of approximately 4×10(8)sperms/ml (single step dilution), in a 15-ml plastic centrifuge tube. In experiment 2, dithioerythritol, at concentrations of 0 (control), 0.5, 1 and 2 mM, was used as an additive in the extender, and the procedure explained above was applied for the division of aliquots and the dilution of semen. Diluted semen samples were kept in glass tubes and cooled from 37 to 5°C in a cold cabinet, and maintained at 5°C. Sperm motility and LPO and total glutathione (GSH) and glutathione peroxidase (GPx) capacities were determined at 5°C for periods of 0, 24, 48 and 72 h of liquid storage. The extender supplemented with 1 mM methionine led to higher motility percentages (77.0±1.2%), in comparison to the control group (66.0±4.9%), during 72 h of liquid storage (P<0.05). As regards dithioerythritol, it did not statistically improve the motility rates for any of the storage times at 5°C. In biochemical assays, differences in LPO levels between the groups with antioxidants and the control groups were not statistically significant. Compared to the control group, no significant difference was observed in GSH and GPx activities following the addition of methionine, during 72 h of storage. Total GSH and GPx activities did not increase significantly upon supplementation with 0.5 and 1 mM of dithioerythritol, compared to the control group, at any of the time points (P>0.05). Dithioerythritol at 2 mM led (P<0.01) to elevating GSH activity, compared to the control group, during 72 h of liquid storage. GPx activity was approximately 10 times higher for 2 mM of dithioerythritol (P<0.001), compared to that of the control group at all time points. The question regarding the sustainability of sperm survival, LPO and antioxidant capacities following liquid storage of semen remains unanswered. Further studies are required for a better understanding of the biochemical changes and to obtain more information on the determination of lipid peroxidation and antioxidant capacities during cooled storage of ram semen.

Lycopene and resveratrol improve post-thaw bull sperm parameters: sperm motility, mitochondrial activity and DNA integrity

We focussed on evaluating the protective effect of lycopene and resveratrol on post‐thaw bull sperm and oxidative stress parameters. Nine ejaculates for each bull were used in the study. Each ejaculate, splitted into three equal aliquots and diluted at 37 °C with base extenders containing lycopene (1 × 10−3 g ml−1) and resveratrol (1 mm), and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The supplementation of the semen extender with lycopene and resveratrol increased the percentages of post‐thawed computer‐assisted sperm analysis (CASA) motility (55.8 ± 3.8 and 61.9 ± 4.0%) and progressive motility (38 ± 2.4 and 37 ± 8.8), compared with the controls (50.7 ± 2.65 and 33.3 ± 3.74%, respectively, P < 0.05). Resveratrol provided a higher ALH (4.3 ± 0.1), in comparison with the control (3.9 ± 0.3, P < 0.05). The supplementation of the semen extender with lycopene and resveratrol produced a higher mitochondrial activity (24.6 ± 2.9 and 30.1 ± 6.5% respectively), compared with that of the control (11.8 ± 9.5%, P < 0.05). It was determined that both antioxidants resulted in a lower percentage of sperm with damaged DNA than that of the control (P < 0.05). Sperm motion characteristics except for ALH, acrosome integrity, sperm viability and oxidative stress parameters were not affected by the adding of lycopene and resveratrol.

Effects of curcumin and dithioerythritol on frozen‐thawed bovine semen

The aim of this study was to determine the effects of curcumin and dithioerythritol added into bull semen extender on sperm parameters, lipid peroxidation, total glutathione and antioxidant potential levels of bull spermatozoa following the freeze/thawing process. Twenty‐seven ejaculates obtained from three bulls were included in the study. Each ejaculate that was splitted into five equal groups and diluted in a Tris‐based extender containing curcumin (0.5 and 2 mm), dithioerythritol (0.5 and 2 mm) and no additive (control) was cooled to 5 °C and frozen in 0.25‐ml French straws. The extender supplemented with 0.5 mm dose of curcumin led to lower percentage of total abnormality (20.40 ± 2.36%) when compared to the control (30.60 ± 1.47%, P < 0.05). Curcumin and dithioerythritol at 0.5 mm provided a greater protective effect in the membrane functional integrity (54.40 ± 2.09% and 50.00 ± 2.68%), in comparison with control (37.20 ± 1.77%, P < 0.001). Supplementation with antioxidants did not significantly affect the lipid peroxidation and antioxidant potential levels, while the maintenance of total glutathione levels in curcumin 0.5 mm was demonstrated to be higher than that of control, following the freeze/thawing (P < 0.05). Supplementation with these antioxidants prior to the cryopreservation process may be recommended to facilitate the enhancement of sperm cryopreservation techniques.

Boron enhances strength and alters mineral composition of bone in rabbits fed a high energy diet

An experiment was performed to determine whether boron had a beneficial effect on bone strength and composition in rabbits with apparent adiposity induced by a high energy diet. Sixty female New Zealand rabbits, aged 8 months, were randomly divided into five groups with the following treatments for seven months: control 1, fed alfalfa hay only (5.91 MJ/kg); control 2, high energy diet (11.76 MJ and 3.88 mg boron/kg); B10, high energy diet+10 mg/kg body weight boron gavage/96 h; B30, high energy diet+30 mg/kg body weight boron gavage/96 h; B50, high energy diet+50mg/kg body weight boron gavage/96 h. Bone boron concentrations were lowest in rabbits fed the high energy diet without boron supplementation, which suggested an inferior boron status. Femur maximum breaking force was highest in the B50 rabbits. Tibia compression strength was highest in B30 and B50 rabbits. All boron treatments significantly increased calcium and magnesium concentrations, and the B30 and B50 treatments increased the phosphorus concentration in tibia of rabbits fed the high energy diet. The B30 treatment significantly increased calcium, phosphorus and magnesium concentrations in femur of rabbits fed the high energy diet. Principal component analysis of the tibia minerals showed that the three boron treatments formed a separate cluster from controls. Discriminant analysis suggested that the concentrations of the minerals in femur could predict boron treatment. The findings indicate boron has beneficial effects on bone strength and mineral composition in rabbits fed a high energy diet.

Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters ☆

The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze-thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10mM raffinose, 5mM hypotaurine, 5mM raffinose +2.5mM hypotaurine (H+R) and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 °C for 25s in a water bath for evaluation. The addition of raffinose led to higher percentages of subjective and CASA motilities (47.5 ± 12.2%, 46.3 ± 13.6%) compared to controls (38.8 ± 13.8%, 30.5 ± 11.7%, P<0.05). For the CASA progressive motility, 5mM raffinose (20.12 ± 8.82%) had increasing effect in comparison to control (10 ± 7.94%, P<0.05) following the freeze-thawing process. Raffinose and hypotaurine led to higher viability (40.8 ± 4.68%, 40.8 ± 4.7%), high sperm mitochondrial activity (29.5 ± 5.4%, 27.3 ± 4.9%) and acrosome integrity (50.8 ± 8.1, 50.7 ± 4.4) percentages, compared to control groups (31.5 ± 3.5%, 9.5 ± 8.2%, 42.8 ± 7.3%, P<0.05). H+R group only led to high sperm mitochondrial activity when compared to control group. In the comet test, raffinose and hypotaurine resulted in lower sperm with damaged DNA (6.2% and 3.9%) than that of control (9.1%), reducing the DNA damage. For TUNEL assay, The TUNEL-positive cell was distinguished by distinct nuclear staining. Raffinose and H+R groups resulted in lower sperm with TUNEL-positive cell (1.5 ± 1.2% and 2.1 ± 0.9%) than that of control (4.9 ± 2.5%) (P<0.05). In conclusion, findings of this study showed that raffinose and hypotaurine supplementation in semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

Effects of dithioerythritol on ram semen after the freeze-thawing process.

The aim of this study was to evaluate the effects of dithioerythritol added to cryopreservation extender on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities of Merino ram sperm. Semen samples from 5 mature Merino rams (1 and 2 years of age) were used in the study. Semen samples, which were diluted with a Tris-based extender containing 0.5, 1, and 2mM dithiothreitol and no antioxidant (control), were cooled to 5°C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37°C for 20s in a water bath for evaluation. The addition of dithioerythritol at 0.5 and 2mM doses led to higher percentages of subjective motility (62.9±4.2% and 63.6±1.8%) compared to control (52.0±4.9%, P<0.05). As regards CASA motility, dithioerythritol 0.25 and 2 mM (60.2±4.5% and 59.6±1.2%) groups were higher from that of control (44.2±8.7%, P<0.05). For the CASA progressive motility, 0.25, 0.5 and 2 mM doses of dithioerythritol (22.0±2.1%, 21.7±2.5% and 24.0±1.2%) had increasing effect in comparison to control (15.0±2.5%). Dithioerythritol at 1 and 2 mM doses for ALH provided higher values compared to the control (P<0.001) following the freeze-thawing process. Supplementation with dithiothreitol did not significantly affect the integrities of sperm membrane and acrosome, and mitochondrial activities. No significant differences were observed in biochemical parameters among the groups (P>0.05). Findings of this study showed that dithioerythritol supplementation in semen extenders, was of greater benefit to sperm motility of frozen-thawed ram sperm.

NMR Based Metabolomics Evaluation in Neonatal Calves with Acute Diarrhea and Suspected Sepsis: A New Approach for Biomarker/S

Metabolic consequences of diarrhea-induced sepsis revealed by plasma 1H-Nuclear Magnetic Resonance (NMR) quantitative metabolomics. Diarrhea and sepsis is generally the most common cause of morbidity and mortality in pre-weaned calves. Traditional biomarkers for sepsis are mainly derived from host immune/inflammatory response. Various high-throughput omics technologies facilitate comprehensive screening of sepsis-specific biomarkers in human medicine. The aim of this first study was to reveal the new potential biomarkers in diarrhea-induced septic neonatal calves evaluating by 1H-NMR based quantitative metabolomics. Clinical and laboratory data revealed all the ill calves had leukocytosis, metabolic acidosis and failure of colostral passive transfer. This study produced quantitative data sets that presented differences between patients with diarrhea–induced sepsis and healthy subjects in the level of the water and lipid soluble metabolites. All the lipid soluble metabolites (e.g., sphingomyeline, various fatty acids, etc.) were significantly decreased in diseased calves. Changes in water soluble metabolites (increases in niacinamide, choline + phosphocholine, 2-methylglutarate and isopropanol, and decreases in formate, lysine-arginine, acetate, creatine) were meaningful for pathogenic mechanisms of sepsis. This pilot study showed the implementation of plasma 1H-NMR quantitative metabolomics because it produced a physiologically relevant metabolite data set that distinguished diarrhea-induced sepsis from healthy ones. The metabolites identified and quantified in the study may be new potential biomarkers for systemic inflammatory response syndrome in calf sepsis.