DNA integrity

Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n 5 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P 5 0.025), curvilinear velocity (P 5 0.033), straight-line velocity (P 5 0.040), and average path velocity (P 5 0.033), whereas progressive motile sperm (P 5 0.019), curvilinear velocity (P 5 0.006), straight-line velocity (P 5 0.003) and average path velocity (P 5 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P 5 0.002) in VF (36.3% VF-control vs 31%VFvaline). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% 6 12.1) than VF cryopreserved sperm control (16.1% 1 6.2). Supplementing valine to the freezing extender doubled (P 5 0.026) the fertility rate of VF (32.6% 6 12.2) compared with the control (16.1% 1 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat ('good freezer') and Black-Red Andaluza ('bad freezer') showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.

Background and Aim: The reproductive efficiency of livestock, especially indigenous breeds such as Donggala bulls, is pivotal to successful breeding programs. While conventional semen parameters are widely used, molecular markers, such as sperm protein profiles and DNA integrity, are emerging as reliable indicators of fertility. This study aimed to characterize the sperm protein profiles of Donggala bulls and examine their correlation with sperm DNA integrity and protamine deficiency.

Materials and Methods: Frozen semen samples were collected from six Donggala bulls (aged 5–7 years). Pre-freezing evaluations included progressive motility (via computer-assisted sperm analysis), sperm morphology (using Diff-Quik staining), DNA integrity (assessed by the acridine orange assay), and protamine deficiency (assessed by the chromomycin A3 assay). Protein concentrations were determined using the bicinchoninic acid assay, and protein profiling was performed using 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis. Band intensities and distributions were analyzed using ImageJ. Statistical correlations were analyzed using a one-way analysis of variance and Pearson’s correlation coefficients.

Results: Significant individual variation was observed in semen quality among bulls. Progressive motility ranged from 38.3% to 46.1%, DNA integrity from 79.5% to 96.8%, and protamine deficiency from 96.0% to 98.7%. The number of protein bands per sample varied between 8 and 11, with molecular weights ranging from 5 to 175 kilodaltons (kDa). Protein concentration ranged from 8.32 to 20.70 μg/mL. A 35 kDa protein band was notably absent in one bull, which may be linked to lower motility. Strong correlations were observed between sperm motility and DNA fragmentation (r = 0.628), protamine deficiency (r = 0.539), protein concentration (r = 0.658), and protein band expression (r = 0.788).

Conclusion: Sperm protein profiles in Donggala bulls are significantly correlated with DNA integrity and protamine deficiency, indicating their potential as molecular biomarkers for fertility prediction. These findings provide a foundation for integrating protein profiling into breeding soundness evaluations, suggesting that targeted proteomic analysis may enhance reproductive management strategies.


Keywords: DNA integrity, Donggala bulls, fertility biomarkers, protamine deficiency, sperm proteins.

This study aimed to examine how varying concentrations (0.5 %, 1.0 %, 2.0 %, 3.0 %, and 4.0 %) of nano-L-α phosphatidylcholine (nano-L-α-PC) affected the quality of frozen-thawed buffalo bull semen compared to the same concentrations of L-α phosphatidylcholine (L-α-PC), and egg yolk (EY). Ejaculates (n=72) were collected from fertile buffalo bulls (n= 12) for 6 weeks. The ejaculates were pooled and divided into 11 groups (3 aliquots per each group) extended with  20% EY (Group1 without supplement), 0.5%, 1%, 2%, 3%, and 4% nano-L-α-PC (Groups 2-6), and 0.5%, 1%, 2%, 3%, and 4% L-α-PC (Groups 7-11) in Tris buffer. After cryopreservation procedures, semen samples were thawed at 37 °C and evaluated for sperm kinematics, acrosome, plasma membrane, and DNA integrities. The seminal plasma was analyzed for malondialdehyde (MDA) levels, and superoxide dismutase (SOD), glutathione reduced (GSH), and catalase (CAT) enzymes activities. Results demonstrated that semen extended with 1-3% nano-L-α-PC or 1-4% L-α-PC showed high total motility, whereas at 0.5–4% of nano-L-α-PC and L-α-PC showed high progressive motility. Significant (P< 0.05) Wobble were seen at 0.5, 1, and 3% nano-L-α-PC, and distance average path and Velocity average path at 2% L-α-PC. Acrosome and plasma membrane integrities were markedly elevated (P< 0.0001) at 2% nano-L-α-PC and 4% L-α-PC. High DNA integrity metrics were noticed at 0.5–1% nano-L-α-PC, as well as 3% and 4% L-α-PC. Low MDA levels and high SOD, GSH, and CAT activities were recorded at 2% nano-L-α-PC and 4% L-α-PC. In conclusion, the optimum concentration of nano-L-α-PC that improved semen quality was 2%, which is roughly equivalent to the effect of 4% L-α-PC.

The objectives were to evaluate: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 Ϯ 27.1 g/dL Cu) were similar (P Ͼ 0.05) to concentrations in FF from large (90 Ϯ 20.4 g/dL Cu) and small (82 Ϯ 22.1 g/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 g/mL (P Ͻ 0.01) in the IVM medium (mean Ϯ SEM index of DNA damage was: 200.0 Ϯ 27.6, 127.6 Ϯ 6.0, 46.4 Ϯ 4.8, and 51.1 Ϯ 6.0 for supplementation with 0, 2, 4, and 6 g/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 g/mL Cu (4.7 Ϯ 0.4 pmol in oocytes and 0.4 Ϯ 0.04 nmol/10 6 cumulus cells) and 6 g/mL Cu (5.0 Ϯ 0.5 pmol in oocytes and 0.5 Ϯ 0.05 nmol/10 6 cumulus cells, P Ͻ 0.01) compared with the other classes. Cleavage rates were similar (P Ն 0.05) when Cu was added to the IVM medium at any concentration (65.1 Ϯ 2.0, 66.6 Ϯ 1.6, 72.0 Ϯ 2.1, and 70.7 Ϯ 2.1 for Cu concentrations of 0, 2, 4, and 6 g/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 Ϯ 0.6, 26.4 Ϯ 0.03, and 29.0 Ϯ 1.7% for 0, 2, and 4 g/mL Cu, and was highest (33.2 Ϯ 1.6 %) in oocytes matured with 6 g/mL Cu (P Ͼ 0.01). There was an increase (P Ͼ 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 g/mL Cu relative to 0 Cu (IVM alone) and 2 g/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

Egypt is anticipated to be potentially influenced by the global climate warming. Therefore, the current study aimed to evaluate the influence of age on the fertility potential of Arabian stallions during summer breeding months. Arabian horses grouped according to their age into three groups, each involved six stallions: young (5-6 years), middle (11-12 years) and old (15-20 years) age groups, were weekly sampled during the months of July-August. Ejaculates were collected using artificial vagina, Missouri model, and examined for pH, volume, concentration, motility, livability and morphological abnormalities. Serum samples were harvested and assessed for testosterone, total antioxidant capacity (TAC), lipid profile, and copper and zinc levels. Semen pH (P<0.005), spermatozoa motility (P=0.08), sperm morphology (P<0.001), tail abnormalities (P<0.001), and sperm count per ejaculate differed noticeably between stallions' groups. Testosterone (P=0.07) and TAC (P<0.05) concentrations were markedly affected by stallions' age. Cholesterol correlated negatively with sperm normality, but serum copper and zinc levels correlated positively with semen volume, sperm cell count and spermatozoa livability. These results revealed that the fertility of stallions is age-dependent and is prominently influenced by lipid metabolism and oxidative stress during hot summer breeding season. It is highly advisable to provide animals' house (along with feed and drinking water) with the evaporative cooling system and allow morning or late afternoon outdoor activity to bypass the summer hot climates and sustain stallions' fertility.

Following an application from TA-XAN AG, submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Germany, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to xanthohumol in XERME ® , a xanthohumol-enriched roasted malt extract, and protection of DNA from oxidative damage. The scope of the application was proposed to fall under a health claim based on newly developed scientific evidence. The food proposed by the applicant as the subject of the health claim is xanthohumol in XERME ® , a xanthohumolenriched roasted malt extract. The Panel considers that xanthohumol in XERME ® , a xanthohumolenriched roasted malt extract, is sufficiently characterised. The claimed effect proposed by the applicant is 'helps to maintain the integrity of DNA and protects against oxidative damage in the cells of the body'. The Panel considers that protection of DNA from oxidative damage is a beneficial physiological effect. The only human study from which conclusions can be drawn for the scientific substantiation of the claim did not show an effect of xanthohumol in XERME ® on the protection of DNA from oxidative damage. In the absence of evidence for an effect of xanthohumol in XERME ® on the protection of DNA from oxidative damage, the studies provided by the applicant on the proposed mechanisms by which the food/constituent could exert the claimed effect were not considered by the Panel for the scientific substantiation of the claim. The Panel concludes that a cause and effect relationship has not been established between the consumption of xanthohumol in XERME ® , a xanthohumol-enriched roasted malt extract, and protection of DNA from oxidative damage.

The aim of our study was to look for alternative predictive biomarkers for breast cancer management in limited resource setup. A comprehensive analysis of circulating cell-free DNA (CCFD) in serum at baseline was performed to assess its prognostic potential. Quantitative polymerase chain reaction (qPCR) of ALU sequences using ALU115 and ALU247 primers was carried out in patients (N: baseline 148, postoperative 47) and 51 healthy controls. Mean serum DNA integrity, levels of ALU 247 and levels of ALU 115 were significantly higher in patients than in healthy females. No significant differences were observed in the levels ALU 247 and ALU 115 between stage IV and earlier stages of the disease. The DNA integrity was significantly higher in stage IV than earlier stages. A significant decrease in DNA integrity was observed after surgery (pre: 0.55 ± 0.23 vs post: 0.43 ± 0.30; P = 0.002) while no such change could be observed for ALU 247 and ALU 115. Baseline DNA integrity was significantly higher in relapsed patients than in patients who were free of disease (P = 0.005). Higher baseline DNA integrity was also indicated, though statistically not significant, in patients who died (P = 0.14). In contrast, ALU 247 and ALU 115 levels were decreased in died patients as compared to survivors (24.8 ± 34.80 vs 73.5 ± 170.83, P = 0.02 for ALU 247 and 41.0 ± 47.99 vs 159.5 ± 299.54, P = 0.005 for ALU 115). Baseline levels of ALU 115 and ALU 247 were lower in relapsed patients, though statistically not significant. In univariate analysis, the only clinic-pathological parameter associated with disease prognosis was tumor size. The hazards of 5-year overall mortality was 3.60 (95 % CI: 1.03 12.53, P = 0.03) among patients with lower baseline serum levels of CCFD (ALU 247 < 21 and ALU 115 < 41). Similarly the 4 year hazards for recurrence was 2.30 (95 % CI: 0.96 5.52, P = 0.05) among patients with higher DNA integrity. Baseline serum levels of CCFD and its integrity were found to be potential prognostic biomarkers in patients of primary breast cancer at our centre.

Paraquat (PQ) is a commonly used herbicide that induces oxidative stress via reactive oxygen species (ROS) generation. This study aimed to investigate the effects of the antioxidant N-acetylcysteine (NAC) against PQ-induced oxidative stress in mice. Male Balb/C mice (24) were randomly divided into 4 groups and treated for 3 weeks: 1) control (saline), 2) NAC (0.5% in diet), 3) PQ (20 mg/kg, IP) and 4) combination (PQ + NAC). Afterwards mice were sacrificed and oxidative stress markers were analyzed. Our data showed no significant change in serum antioxidant capacity. PQ enhanced lipid peroxidation (MDA) levels in liver tissue compared to control whereas NAC decreased MDA levels (p<0.05). NAC significantly increased MDA in brain tissue (p<0.05). PQ significantly depleted glutathione (GSH) levels in liver (p=0.001) and brain tissue (p<0.05) but non-significant GSH depletion in lung tissue. NAC counteracted PQ, showing a moderate increase GSH levels in liver and brain tissues. PQ significantly increased 8-oxodeoxyguanosine (8-OH-dG) levels (p<0.05) in liver tissue compared to control without a significant change in brain tissue. NAC treatment ameliorated PQ-induced oxidative DNA damage in the liver tissue. PQ significantly decreased the relative mtDNA amplification and increased the frequency of lesions in liver and brain tissue (p<0.0001), while NAC restored the DNA polymerase activity in liver tissue but not in brain tissue. In conclusion, PQ induced lipid peroxidation, oxidative nuclear DNA and mtDNA damage in liver tissues and depleted liver and brain GSH levels. NAC supplementation ameliorated the PQ-induced oxidative stress response in liver tissue of mice.

Paraquat (PQ) is a commonly used herbicide that induces oxidative stress via reactive oxygen species (ROS) generation. This study aimed to investigate the effects of the antioxidant Nacetylcysteine (NAC) against PQ-induced oxidative stress in mice. Male Balb/C mice (24) were randomly divided into 4 groups and treated for 3 weeks: 1) control (saline), 2) NAC (0.5% in diet), 3) PQ (20 mg/kg, IP) and 4) combination (PQ + NAC). Afterwards mice were sacrificed and oxidative stress markers were analyzed. Our data showed no significant change in serum antioxidant capacity. PQ enhanced lipid peroxidation (MDA) levels in liver tissue compared to control whereas NAC decreased MDA levels (p<0.05). NAC significantly increased MDA in brain tissue (p<0.05). PQ significantly depleted glutathione (GSH) levels in liver (p=0.001) and brain tissue (p<0.05) but non-significant GSH depletion in lung tissue. NAC counteracted PQ, showing a moderate increase GSH levels in liver and brain tissues. PQ significantly increased 8-oxodeoxyguanosine (8-OH-dG) levels (p<0.05) in liver tissue compared to control without a significant change in brain tissue. NAC treatment ameliorated PQ-induced oxidative DNA damage in the liver tissue. PQ significantly decreased the relative mtDNA amplification and increased the frequency of lesions in liver and brain tissue (p<0.0001), while NAC restored the DNA polymerase activity in liver tissue but not in brain tissue. In conclusion, PQ induced lipid peroxidation, oxidative nuclear DNA and mtDNA damage in liver tissues and depleted liver and brain GSH levels. NAC supplementation ameliorated the PQ-induced oxidative stress response in liver tissue of mice.

Sperm freeze-drying is considered an alternative method to preserve male gametes in refrigeration or at room temperature condition. In order to preserve sperm integrity special protection is required. The aim of our research was to examine the effect of vitamin C (0.5 mM ) and rosmarinic acid (105 μM) on the DNA spermatozoa integrity after freeze-drying and 36 months of preservation at refrigerator temperature. Our results indicates that more than 90% of DNA boar spermatozoa integrity is not affected by long-time preservation with small differences between experimental groups: with +0.59% higher DNA integrity in AR group from Duroc boar, with +2.83% higher DNA integrity in AR group from Landrace boar and with no differences regarding DNA integrity in group supplemented with vitamin C. The main conclusion of these preliminary results is that DNA integrity of boar freeze-dried semen is not affected by longtime preservation and it can be used further for intracytoplasmatic sperm inject...

Ozone is used to treat several medical conditions, while the underlying mechanisms of action are sometimes poorly understood. In the current study, we exposed cultured human epithelial (HeLa) cells acutely and repeatedly to ozone and investigated the effects thereof on cell viability. The involvement of anti-apoptotic pathways in observed adaptive responses to ozone were investigated by employing the Akt inhibitor (-)-deguelin. Cells were exposed to an ozone-saturated physiological solution using various dosing regimens, including acute exposure and various repetitive exposures. Cell viability was determined with Trypan Blue or MTT tests, or by a DNAfragmentation (comet) assay. Acute ozone exposure compromised cell membrane integrity severely, while adaptation to reverse an initial reduction in mitochondrial activity was observed. Repetitive, short-duration exposures followed by a single long-duration exposure to ozone furnished a protective adaptation that was reversed by Akt inhibition. Extracellular and intracellular damage (and adaptation) occurs differentially. While acute ozone may decrease cell viability, multiple preexposures up-regulates cellular plasticity via induction of anti-apoptotic pathways in a treatment regimen-specific manner.

Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n 5 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P 5 0.025), curvilinear velocity (P 5 0.033), straight-line velocity (P 5 0.040), and average path velocity (P 5 0.033), whereas progressive motile sperm (P 5 0.019), curvilinear velocity (P 5 0.006), straight-line velocity (P 5 0.003) and average path velocity (P 5 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P 5 0.002) in VF (36.3% VF-control vs 31%VFvaline). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% 6 12.1) than VF cryopreserved sperm control (16.1% 1 6.2). Supplementing valine to the freezing extender doubled (P 5 0.026) the fertility rate of VF (32.6% 6 12.2) compared with the control (16.1% 1 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.

Reactive oxygen species (ROS) are produced in significant amounts by spermatozoa and leucocytes. They are necessary to carry out various physiological functions such as sperm capacitation, hyperactivation, acrosome reaction, sperm-zona binding and cellular signalling pathways. Oxidative stress (OS) results when the ROS levels overwhelm the available antioxidant reserve. A number of direct and indirect tests have been developed to assess oxidative stress. In this manuscript, we discuss these common direct and indirect tests as well as their advantages and disadvantages. Tests measuring sperm dysfunction secondary to oxidative stress such as lipid peroxidation, DNA fragmentation and protein alterations are also described.

The objectives were to evaluate: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 Ϯ 27.1 g/dL Cu) were similar (P Ͼ 0.05) to concentrations in FF from large (90 Ϯ 20.4 g/dL Cu) and small (82 Ϯ 22.1 g/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 g/mL (P Ͻ 0.01) in the IVM medium (mean Ϯ SEM index of DNA damage was: 200.0 Ϯ 27.6, 127.6 Ϯ 6.0, 46.4 Ϯ 4.8, and 51.1 Ϯ 6.0 for supplementation with 0, 2, 4, and 6 g/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 g/mL Cu (4.7 Ϯ 0.4 pmol in oocytes and 0.4 Ϯ 0.04 nmol/10 6 cumulus cells) and 6 g/mL Cu (5.0 Ϯ 0.5 pmol in oocytes and 0.5 Ϯ 0.05 nmol/10 6 cumulus cells, P Ͻ 0.01) compared with the other classes. Cleavage rates were similar (P Ն 0.05) when Cu was added to the IVM medium at any concentration (65.1 Ϯ 2.0, 66.6 Ϯ 1.6, 72.0 Ϯ 2.1, and 70.7 Ϯ 2.1 for Cu concentrations of 0, 2, 4, and 6 g/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 Ϯ 0.6, 26.4 Ϯ 0.03, and 29.0 Ϯ 1.7% for 0, 2, and 4 g/mL Cu, and was highest (33.2 Ϯ 1.6 %) in oocytes matured with 6 g/mL Cu (P Ͼ 0.01). There was an increase (P Ͼ 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 g/mL Cu relative to 0 Cu (IVM alone) and 2 g/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

This study aims to investigate the effect of catechin (CT), green tea extract, as a supplement to Tris extender on semen quality parameters in frozen-thawed of bull sperm. Ejaculates were taken with artificial vagina from Holstein bulls and divided equal five aliquots, diluted to containing different amounts of CT (5, 10, 25 and 50 μg/mL) and no-additive (control). All samples were equilibrated at 4°C for 4 h and were frozen using a digital freezing machine. Post-thawed sperm motility and kinetic parameters were determined using the sperm analyser system. Spermatozoa DNA integrity was evaluated with the single cell gel electrophoresis, abnormal spermatozoa rate was evaluated by fluid fixation test and lipid peroxidation status was evaluated colorimetrically. CT supplementation did not improve motility and kinetic parameters. However, the higher morphological integrity was detected in CT10, 25 and 50 groups compared to control (P<0.05). Regarding chromatin integrity, positive effects of catechin were observed in the treatment groups while in CT 50 group adverse effects were found (P<0.05). Although there was no improvement in malondialdehyde levels, the highest total antioxidant activity was seen in the CT50 group (P<0.05). In conclusion, CT supplementation could be used the protection of morphological and DNA integrity from cryodamage and it has increased the total antioxidant activity depending of the dose in bull semen.

Paraquat (PQ) is a commonly used herbicide that induces oxidative stress via reactive oxygen species (ROS) generation. This study aimed to investigate the effects of the antioxidant N-acetylcysteine (NAC) against PQ-induced oxidative stress in mice. Male Balb/C mice (24) were randomly divided into 4 groups and treated for 3 weeks: 1) control (saline), 2) NAC (0.5% in diet), 3) PQ (20 mg/kg, IP) and 4) combination (PQ + NAC). Afterwards mice were sacrificed and oxidative stress markers were analyzed. Our data showed no significant change in serum antioxidant capacity. PQ enhanced lipid peroxidation (MDA) levels in liver tissue compared to control whereas NAC decreased MDA levels (p&lt;0.05). NAC significantly increased MDA in brain tissue (p&lt;0.05). PQ significantly depleted glutathione (GSH) levels in liver (p=0.001) and brain tissue (p&lt;0.05) but non-significant GSH depletion in lung tissue. NAC counteracted PQ, showing a moderate increase GSH levels in liver and brain tissues....

The study is to evaluate the possible genotoxic and antigenotoxic potential of Lagenandra toxicaria rhizome methanol extract using Allium cepa root tip assay. Methods: The rhizome methanol extract was prepared using Soxhlet apparatus. The A. cepa roots were treated with various concentrations of the extract at different time points and stained with aceto-orcein. The mitotic index (MI) was calculated. Results: A significant decrease in MI and increase in the percentage of clastogenicity was observed in a time-and dose-dependent manner in the roots treated with the extract at 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml, 5 mg/ml, and 10 mg/ml concentration for 1, 2, and 4 h. The field emission scanning electron microscopy and Fourier-transform infrared spectroscopy revealed evident morphological and biochemical changes at 10 mg/ml treatment when compared to control for 4 h. The agarose gel electrophoresis showed loss of DNA integrity at 10 mg/ml extract for 4 h. In situ histochemical staining by Schiff's reagent and nitroblue tetrazolium confirmed the increased lipid peroxidation and free radical generation at 4 h treatment. Subsequently, the possible antigenotoxic potential of the plant extract was explored using H 2 O 2 standard assays. The increased percentage of H 2 O 2 induced nuclear lesions was reduced significantly after the modulatory treatment with extract. Conclusion: The L. toxicaria rhizome methanol extract acts as an antigenotoxic agent at lower doses and at higher doses the extract induces clastogenic effects. Further studies are needed to unravel the active component in the extract that mediates the observed phenomenon in the current study.

Diet is a key factor in determining genomic stability is more important than previously imagined because it impacts on all relevant pathways like exposure to dietary carcinogens, DNA repair, DNA synthesis, epigenetic damage and apoptosis. Recent research focuses into how a single micronutrient deficiency is leading to genomic instability and development of degenerative diseases in various stages of life.The study aimed at finding the nutrigenomic mechanism of how a marginal deficiency of any single micronutrient is interrupting in DNA repairing, methylation and synthesis by taking nutrient-nutrient and nutrient-gene interaction into consideration. It also focuses on how recommended dietary allowance is important in achieving DNA integrity and genome stability to prevent degenerative diseases.Exhaustive review of research papers in genome health nutrigenomicsis involved in this study to explore, assimilate and analyze data to understand the importance of micronutrient in maintaining ...

The aim of this study was to examine effects of sodium pyruvate on viability as well as on synthesis of reactive oxygen species (ROS), lipid peroxidation and DNA integrity of cryopreserved bovine sperm. In each of 23 Simmental AI bulls three ejaculates were collected. In a split sample design ejaculates were diluted by using Triladyl ® extender without and with the addition of 5 mM sodium pyruvate. Both aliquots were equilibrated for 24 h before freezing. Frozen sperm samples were thawed, and examined immediately after thawing (0 h) as well as after 3, 6, 12, and 24 h incubation at 37°C. The percentages of rapidly motile sperm (RMS), plasma membrane and acrosome intact sperm (PMAI), sperm with a high mitochondrial membrane potential (HMMP), amounts of ROS synthesis (dichlorofluorescein-diacetate (DCFH), CellROX Deep Red Reagent ® probe (CellROX)) and lipid peroxidation of sperm (LPO) and percentage of sperm with a high degree of DNA fragmentation (%DFI) were determined. Overall, sperm diluted with the extender containing sodium pyruvate showed higher levels of RMS, PMAI and HMMP, CellROX and lower %DFI values (P < 0.001) compared to sperm frozen in the extender without sodium pyruvate. However, there was no effect (P > 0.05) of sodium pyruvate on LPO and DCFH. The results of this study show that the addition of sodium pyruvate to the semen extender improved the viability as well as DNA integrity of cryopreserved sperm and did not affect their lipid peroxidation, although it increased the synthesis of some ROS.

Telomeres shorten with physiological aging but undergo substantial restoration during cancer immortalization. Increasingly, cancer studies utilize the archive of formalin-fixed, paraffin-embedded (FFPE) tissues in diagnostic pathology departments. Conceptually, such studies would be confounded by physiological telomere attrition and loss of DNA integrity from prolonged tissue storage. Our study aimed to investigate these two confounding factors. 145 FFPE tissues of surgically-resected, non-diseased appendixes were retrieved from our pathology archive, from years 2008 to 2014. Cases from 2013 to 2014 were categorized by patient chronological age (0-20 years, 21-40 years, 41-60 years, > 60 years). Telomere lengths of age categories were depicted by telomere/chromosome 2 centromere intensity ratio (TCR) revealed by quantitative fluorescence in situ hybridization. Material from individuals aged 0

Marine phytoplankton are highly diverse with different species possessing different cell coverings, posing challenges for thoroughly breaking the cells in DNA extraction yet preserving DNA integrity. While quantitative molecular techniques have been increasingly used in phytoplankton research, an effective and simple method broadly applicable to different lineages and natural assemblages is still lacking. In this study, we developed a bead-beating protocol based on our previous experience and tested it against 9 species of phytoplankton representing different lineages and different cell covering rigidities. We found the bead-beating method enhanced the final yield of DNA (highest as 2 folds) in comparison with the non-bead-beating method, while also preserving the DNA integrity. When our method was applied to a field sample collected at a subtropical bay located in Xiamen, China, the resultant ITS clone library revealed a highly diverse assemblage of phytoplankton and other micro-eu...

The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome. To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status. This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA. Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P &lt; 0.05...

Sperm freeze-drying is considered an alternative method to preserve male gametes in refrigeration or at room temperature condition. In order to preserve sperm integrity special protection is required. The aim of our research was to examine the effect of vitamin C (0.5 mM ) and rosmarinic acid (105 μM) on the DNA spermatozoa integrity after freeze-drying and 36 months of preservation at refrigerator temperature. Our results indicates that more than 90% of DNA boar spermatozoa integrity is not affected by long-time preservation with small differences between experimental groups: with +0.59% higher DNA integrity in AR group from Duroc boar, with +2.83% higher DNA integrity in AR group from Landrace boar and with no differences regarding DNA integrity in group supplemented with vitamin C. The main conclusion of these preliminary results is that DNA integrity of boar freeze-dried semen is not affected by longtime preservation and it can be used further for intracytoplasmatic sperm inject...

The objectives were to evaluate: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 Ϯ 27.1 g/dL Cu) were similar (P Ͼ 0.05) to concentrations in FF from large (90 Ϯ 20.4 g/dL Cu) and small (82 Ϯ 22.1 g/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 g/mL (P Ͻ 0.01) in the IVM medium (mean Ϯ SEM index of DNA damage was: 200.0 Ϯ 27.6, 127.6 Ϯ 6.0, 46.4 Ϯ 4.8, and 51.1 Ϯ 6.0 for supplementation with 0, 2, 4, and 6 g/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 g/mL Cu (4.7 Ϯ 0.4 pmol in oocytes and 0.4 Ϯ 0.04 nmol/10 6 cumulus cells) and 6 g/mL Cu (5.0 Ϯ 0.5 pmol in oocytes and 0.5 Ϯ 0.05 nmol/10 6 cumulus cells, P Ͻ 0.01) compared with the other classes. Cleavage rates were similar (P Ն 0.05) when Cu was added to the IVM medium at any concentration (65.1 Ϯ 2.0, 66.6 Ϯ 1.6, 72.0 Ϯ 2.1, and 70.7 Ϯ 2.1 for Cu concentrations of 0, 2, 4, and 6 g/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 Ϯ 0.6, 26.4 Ϯ 0.03, and 29.0 Ϯ 1.7% for 0, 2, and 4 g/mL Cu, and was highest (33.2 Ϯ 1.6 %) in oocytes matured with 6 g/mL Cu (P Ͼ 0.01). There was an increase (P Ͼ 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 g/mL Cu relative to 0 Cu (IVM alone) and 2 g/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

Preservation of sperm by freezing allows breeding dogs that are separated over long distances. To increase the fertility of frozen and then thawed spermatozoa, they must be able to survive the process. The current study aimed to evaluate the sperm motility and DNA integrity of Kintamani dogs extended in extenders with one-step and two-step dilution techniques. Ejaculates collected from four dogs were used in the current study. The semen was divided into two equal parts and diluted with extenders using two different dilution techniques, namely One-step dilution in Tris egg yolk containing 7% glycerol, and a two-step dilution technique diluted in an initial 2:1 with an extender, containing 20% egg yolk without glycerol. The same volume of the second extender was added, including 14% glycerol. The sample was loaded into 0.25 ml straws, cooled to 4°C for 4 hours, equilibrated, and then plunged into the liquid nitrogen. The sperm motility was evaluated using Computer-Assisted Sperm Analysis (CASA), and DNA integrity was assessed using Acridine Orange (AO) stained. Results showed that the sperm motility of Kintamani dogs in extenders using two-step dilution was significantly higher compared to the one-step dilution technique. In addition, the obtained results indicated that two types of dilution steps in Kintamani dog semen were not detrimental to the sperm DNA integrity during the freezing process. In conclusion, extenders with two types of dilution techniques could maintain sperm motility above 30%, and no difference between one and two steps dilution was detected.

Telomeres shorten with physiological aging but undergo substantial restoration during cancer immortalization. Increasingly, cancer studies utilize the archive of formalin-fixed, paraffin-embedded (FFPE) tissues in diagnostic pathology departments. Conceptually, such studies would be confounded by physiological telomere attrition and loss of DNA integrity from prolonged tissue storage. Our study aimed to investigate these two confounding factors. 145 FFPE tissues of surgically-resected, non-diseased appendixes were retrieved from our pathology archive, from years 2008 to 2014. Cases from 2013 to 2014 were categorized by patient chronological age (0-20 years, 21-40 years, 41-60 years, > 60 years). Telomere lengths of age categories were depicted by telomere/chromosome 2 centromere intensity ratio (TCR) revealed by quantitative fluorescence in situ hybridization. Material from individuals aged 0

We evaluated the effects of opiate consumption on semen quality, sperm function, seminal plasma antioxidant capacity, and sperm DNA integrity. A total of 142 opiate addict men (group 1) were enrolled in the study and 146 healthy age matched male volunteers (group 2) served as controls. Two semen analyses were performed in all participants. Sperm chromatin structure assay (SCSA) was used to identify sperm DNA integrity. The mean ± SD sperm concentration in opiate users and in control subjects was 22.2 ± 4.4 and 66.3 ± 8.3 million per ml, respectively (P = 0.002). A significant increase in the amount of fragmented DNA was found in opiate consumers compared with that in controls (36.4 ± 3.8% vs. 27.1 ± 2.4%, P = 0.004). Significantly decreased levels of catalase-like and superoxide dismutase-like (SOD) activity were observed in group 1 compared with group 2. Opiate consumption has significant adverse effects on semen quality. In cases of unexplained infertility in men, opium consumption should be considered as a possible factor.

We evaluated the effects of opiate consumption on semen quality, sperm function, seminal plasma antioxidant capacity, and sperm DNA integrity. A total of 142 opiate addict men (group 1) were enrolled in the study and 146 healthy age matched male volunteers (group 2) served as controls. Two semen analyses were performed in all participants. Sperm chromatin structure assay (SCSA) was used to identify sperm DNA integrity. The mean ± SD sperm concentration in opiate users and in control subjects was 22.2 ± 4.4 and 66.3 ± 8.3 million per ml, respectively (P = 0.002). A significant increase in the amount of fragmented DNA was found in opiate consumers compared with that in controls (36.4 ± 3.8% vs. 27.1 ± 2.4%, P = 0.004). Significantly decreased levels of catalase-like and superoxide dismutase-like (SOD) activity were observed in group 1 compared with group 2. Opiate consumption has significant adverse effects on semen quality. In cases of unexplained infertility in men, opium consumption should be considered as a possible factor.

BRAF, EGFR or CDK4, change the interactions and links to other networks. Lastly, in terms of protein structure and conformation, we need to establish common principles that govern alternative signaling within and between networks by way of alternative hub physical connectivity. A prominent characteristic of cellular signaling complexes is that they are chiefly enriched in

We evaluated the effects of opiate consumption on semen quality, sperm function, seminal plasma antioxidant capacity, and sperm DNA integrity. A total of 142 opiate addict men (group 1) were enrolled in the study and 146 healthy age matched male volunteers (group 2) served as controls. Two semen analyses were performed in all participants. Sperm chromatin structure assay (SCSA) was used to identify sperm DNA integrity. The mean ± SD sperm concentration in opiate users and in control subjects was 22.2 ± 4.4 and 66.3 ± 8.3 million per ml, respectively (P = 0.002). A significant increase in the amount of fragmented DNA was found in opiate consumers compared with that in controls (36.4 ± 3.8% vs. 27.1 ± 2.4%, P = 0.004). Significantly decreased levels of catalase-like and superoxide dismutase-like (SOD) activity were observed in group 1 compared with group 2. Opiate consumption has significant adverse effects on semen quality. In cases of unexplained infertility in men, opium consumption should be considered as a possible factor.

We evaluated the effects of opiate consumption on semen quality, sperm function, seminal plasma antioxidant capacity, and sperm DNA integrity. A total of 142 opiate addict men (group 1) were enrolled in the study and 146 healthy age matched male volunteers (group 2) served as controls. Two semen analyses were performed in all participants. Sperm chromatin structure assay (SCSA) was used to identify sperm DNA integrity. The mean ± SD sperm concentration in opiate users and in control subjects was 22.2 ± 4.4 and 66.3 ± 8.3 million per ml, respectively (P = 0.002). A significant increase in the amount of fragmented DNA was found in opiate consumers compared with that in controls (36.4 ± 3.8% vs. 27.1 ± 2.4%, P = 0.004). Significantly decreased levels of catalase-like and superoxide dismutase-like (SOD) activity were observed in group 1 compared with group 2. Opiate consumption has significant adverse effects on semen quality. In cases of unexplained infertility in men, opium consumption should be considered as a possible factor.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: a b s t r a c t We evaluated the effects of opiate consumption on semen quality, sperm function, seminal plasma antiox-idant capacity, and sperm DNA integrity. A total of 142 opiate addict men (group 1) were enrolled in the study and 146 healthy age matched male volunteers (group 2) served as controls. Two semen analyses were performed in all participants. Sperm chromatin structure assay (SCSA) was used to identify sperm DNA integrity. The mean ± SD sperm concentration in opiate users and in control subjects was 22.2 ± 4.4 and 66.3 ± 8.3 million per ml, respectively (P = 0.002). A significant increase in the amount of fragmented DNA was found in opiate consumers compared with that in controls (36.4 ± 3.8% vs. 27.1 ± 2.4%, P = 0.004). Significantly decreased levels of catalase-like and superoxide dismutase-like (SOD) activity were observed in group 1 compared with group 2. Opiate consumption has significant adverse effects on semen quality. In cases of unexplained infertility in men, opium consumption should be considered as a possible factor.