61 PLANT EXTRACTS REDUCE DNA FRAGMENTATION IN FROZEN-THAWED STALLION SPERM

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
P. D. Burns ◽  
N. Wong ◽  
H. Arnold ◽  
N. Sirs ◽  
R. Romero ◽  
...  
Keyword(s):  
Dna Damage ◽  
Liquid Nitrogen ◽  
Dna Fragmentation ◽  
Plant Extract ◽  
Plant Extracts ◽  
Sperm Quality ◽  
Tail Length ◽  
Practical Method ◽  
Computer Assisted ◽  
Sperm Cells

Mares inseminated with frozen–thawed sperm have reduced pregnancy rates compared with mares inseminated with fresh sperm. Processing mammalian sperm for cryopreservation increases the concentration of free radicals and induces oxidative stress, which can result in DNA damage and may lead to lower fertility. The objective of this experiment was to examine the effects of several plant antioxidant extracts on stallion sperm post-thaw motility and DNA quality. Single ejaculates were collected from 4 stallions and the concentration of sperm cells in each ejaculate was determined spectrophotometrically. Semen was centrifuged at 300g for 10 min at room temperature and seminal plasma was removed. Sperm pellets were resuspended to a final concentration of 200 × 106 cells mL–1 in E-Z Freezin LE (ARS, Chino, CA) extender (control) or extender containing 1 of 3 plant extracts (3% v/v) from 2 different commercial sources. Extended sperm cells were loaded into 0.5-mL straws and frozen over liquid nitrogen vapor for 10 min. Straws were then plunged into liquid nitrogen and stored until further evaluation. Motility and velocity parameters were determined at 0, 30, and 60 min post-thaw using a computer-assisted sperm analyzer. DNA fragmentation was determined immediately after thawing using a single-cell gel electrophoresis (Comet) assay. Motility (total and progressive) and velocity parameters of sperm cells did not differ between controls and plant extract treatments (P > 0.05). However, total Comet length and tail length were reduced in sperm cells stored in extender containing each plant extract (P < 0.05). Tail and olive moment tended to be reduced (P < 0.10) in sperm cells stored in plant extracts. In conclusion, sperm cells stored in plant extracts had reduced post-thaw DNA damage. The addition of plant extracts to commercial freezing extenders may be a practical method for improving sperm quality.

scholarly journals Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells

2007 ◽  
Vol 97 (2) ◽  
pp. 321-328 ◽  
Author(s):  
S. Aisling Aherne ◽  
Joseph P. Kerry ◽  
Nora M. O'Brien
Keyword(s):  
Dna Damage ◽  
Plant Extract ◽  
Plant Extracts ◽  
Antioxidant Status ◽  
Cell Injury ◽  
Membrane Integrity ◽  
Neutral Red ◽  
Dna Integrity ◽  
Cell Membrane Integrity ◽  
Wide Range

Experimental evidence suggests that most herbs and spices possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage. The objectives of the present study were: first, to determine the effects of plant extracts on the viability, membrane integrity, antioxidant status and DNA integrity of Caco-2 cells and second, to investigate the cytoprotective and genoprotective effects of these plant extracts against oxidative stress in Caco-2 cells. The plant extracts examined were rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), sage (Salvia officinalis L.) and echinacea (Echinacea purpurea L.). Cell membrane integrity was assessed by the lactate dehydrogenase release assay. Viability was determined by the neutral red uptake assay (NRUA) and the concentration of compound that resulted in 50 % cell death (IC50) was calculated. Antioxidant status of the cells was assessed by measuring GSH content, catalase activity and superoxide dismutase activity. To examine their cytoprotective and genoprotective effects, Caco-2 cells were pre-treated with each plant extract for 24 h followed by exposure to H2O2. DNA damage was assessed by the comet assay and cell injury was determined by the NRUA. Rosemary was the most toxic (IC50 123 μg/ml) and echinacea the least toxic (IC50 1421 μg/ml). Sage was the only plant extract to affect the antioxidant status of the cells by increasing GSH content. Sage, oregano and rosemary protected against H2O2-induced DNA damage (olive tail moment and percentage tail DNA), whereas protection against H2O2-induced cytotoxicity was afforded by sage only.

scholarly journals The in vitro toxicity of atrazine on kinematics and DNA fragmentation in common carp (Cyprinus carpio) sperm cells

2019 ◽  
Vol 70 (3) ◽  
pp. 1639
Author(s):  
M.E. ÖZGÜR
Keyword(s):  
Dna Fragmentation ◽  
Common Carp ◽  
Cyprinus Carpio ◽  
In Vitro Toxicity ◽  
Computer Assisted ◽  
Sperm Cells ◽  
Common Carp Cyprinus Carpio ◽  
Significant Difference ◽  
Carp Cyprinus Carpio

This study investigated the in vitro effects of different concentrations of Atrazine (0.001, 0.01, 0.1, 0.5 mg/L) added to motile and immotile solutions on kinematics quality of sperm cells of common carp, Cyprinus carpio, which is a fish of economic significance. The kinematics of the sperm cells was analyzed by a computer-assisted sperm analysis system (CASA). As a result of the study, while there was a significant difference (P < 0.05) between the groups in terms of the VSL (μm/s) and VCL (μm/s) values after the Atrazine-added immotile solution’s (IMS) and incubation for 3 hours, there was a significant difference (P < 0.05) in only the VSL values directly activated by the Atrazine-added motile solution (MS). DNA fragmentation was evident but not in higher numbers in the 0.1 mg/L atrazine group. Finally, it was determined the effective concentration (EC50) values of the VSL value of the motile and immotile solution as 0.34 mg/L and 0.03 mg/L, respectively.

105 Increasing fertility of interspecific hybrid males using biotechnological approaches

2021 ◽  
Vol 33 (2) ◽  
pp. 159
Author(s):  
A. Vetokh ◽  
A. Tadzhieva ◽  
B. Iolchiev ◽  
N. Volkova ◽  
V. Bagirov
Keyword(s):  
Dna Fragmentation ◽  
Interspecific Hybrid ◽  
Semen Quality ◽  
Sperm Quality ◽  
Semen Analysis ◽  
Differential Staining ◽  
Computer Assisted ◽  
Sperm Dna Fragmentation ◽  
Russian Science

The results of AI depend on many factors, with the quality of semen being one of the most important. Not all male hybrids can meet the requirements for semen quality, because they often have reduced fertility following cryopreservation. Thus, it is necessary to improve semen processing before use in AI. The aim of the study was to evaluate the effectiveness of using the “swim-up” flotation method to improve sperm quality of hybrid males of the Ovis genus. Semen from interspecific hybrid rams (1/4 Argali×3/4 Romanov, n=15; 1/8 Argali×7/8 Romanov, n=15) was freshly obtained, frozen–thawed, and processed by the swim-up method. Evaluation of sperm motility was determined using computer-assisted semen analysis. Statistical analysis was performed using SPSS vs.15.0 (ANOVA and t-test; SPSS Inc.). Semen was collected during the breeding season (October–December) via artificial vagina. Assessment of acrosome integrity was determined using differential staining with a Diachem diff-quick kit (NPF ABRIS+). The degree of sperm DNA fragmentation was determined using the acridine-orange test. The sperm freezing/thawing cycle was accompanied by sperm damage and an increase in the proportion of immobile sperm from 10 to 58%, with non-progressive movement increasing from 9 to 19.3%. The number of spermatozoa with abnormal morphology doubled, and the DNA fragmentation index increased from 16 to 26%. Use of the swim-up procedure allowed us to sort progressively motile spermatozoa. The content of progressively motile spermatozoa in the samples obtained from the supernatant was 86%, which was 2.3 times higher than in frozen–thawed sperm (P≤0,01). The obtained results show the effective use of the swim-up procedure to determine the quality of semen in hybrid rams. These studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.

scholarly journals Effect of Imidocarb Application on Oxidative DNA Damage Caused by Anaplasmosis

2021 ◽  
Vol 9 (12) ◽  
pp. 2308-2311
Author(s):  
Ahmet Cihat Öner ◽  
Adnan Ayan
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Dna Fragmentation ◽  
Oxidative Dna Damage ◽  
Clinical Signs ◽  
Tail Length ◽  
Assay Method ◽  
Control Group ◽  
Tail Moment ◽  
Before And After

This study was aimed to evaluate DNA fragmentation by using Comet assay in naturally infected sheep with Anaplasmosis before and after treatment with the Comet method, which shows DNA damage specifically. In the study, blood samples were collected from 10 Anaplosmosis infected and 10 healthy sheep. The anaplosmosis was diagnosed by clinical signs and symptoms. The infection was confirmed by Giemsa staining. The blood was collected from control group and infected group before and after the treatment, from the vena jugularis with the appropriate method. The DNA fragmentation was checked by using the Comet assay of blood cells. The data were analysed throught ANNOVA one-way. The result showed higher DNA fragmentation in sick animals diagnosed with anaplasmosis; tail length and tail moment values were found to be statistically significantly higher than the control group. When the data obtained after imidocarb (IMD) application were compared with obtained during the disease, a decreased DNA damage and tail moment was determined, however, these values higher than control. In this study, DNA damage and the extent of this damage were investigated by the Comet assay method using a healthy control group before and after treatment in animals with Anaplasmosis. When the findings obtained from the study were evaluated, it was seen that Anaplasma agents caused DNA damage and with the imidocarb application given for treatment, DNA damage was reduced and results close to healthy individuals were obtained.

73 SUPPLEMENTATION OF FROZEN BOAR SEMEN WITH β-MERCAPTOETHANOL INCREASES THE INCIDENCE OF INTACT CELLS AFTER THAWING

2008 ◽  
Vol 20 (1) ◽  
pp. 117
Author(s):  
H. Funahashi ◽  
S. Yamaguchi ◽  
W. Fujii ◽  
T. Murakami
Keyword(s):  
Liquid Nitrogen ◽  
Dna Fragmentation ◽  
Egg Yolk ◽  
Molecular Probes ◽  
Sperm Cells ◽  
Freezing And Thawing ◽  
Intact Cells ◽  
Boar Semen ◽  
Post Hoc ◽  
Boar Spermatozoa

During the process of freezing and thawing of boar spermatozoa, a large number of the cells appear to be injured by some stresses such as osmotic forces and oxidation, causing reduced viability and penetrability. β-Mercaptoethanol (bME), a strong reducing agent, may ease oxidative stress and rescue sperm cells from those injuries. The aim of this study was to determine the effect of the presence of bME during freezing and thawing of boar spermatozoa on the viability and acrosome status of the sperm cells. Semen samples were collected from 3 boars; only samples with a high motility (more than 80%) were used for this experiment. Each sample was diluted 1:1 with modified Modena solution and kept overnight at 15�C. After centrifugation at 800g for 10 min, the diluent supernatant was removed; spermatozoa were re-suspended at 2 � 109 cells mL–1 in the first diluent (8.8% trehalose solution containing 20% egg yolk and antibiotic) supplemented with 0, 25, or 50 µm bME, and then cooled to 5�C over 2–3 h. At 5�C, semen samples were further diluted 1:1 with the second diluent (same as the first diluent + 5% glycerin + 1.48% Orvus ES Paste (Equex STM; Minitube, Verona, WI, USA)) supplemented with 0, 25, and 50 µm bME, respectively. After packaging the semen into 0.5-mL straws, it was frozen by keeping the straws 4 cm above the surface of liquid nitrogen for 15 min and then storing them in liquid nitrogen until use. After thawing at 37�C for 30 s, semen samples were re-suspended in 10 mL of BTS solution containing 1.15 mm caffeine and 4 mm Ca chloride, and incubated at 37�C under 5% CO2 in air for 90 min. Viability, DNA fragmentation, and acrosome status of spermatozoa were assessed by flow cytometry after staining with SYBR�14/PI (Molecular Probes, Inc., Eugene, OR, USA), acridine orange, and PNA/PI, respectively. Statistical analyses of data from at least 3 replicated trials were carried out by ANOVA and Fisher's protected least-squares difference (PLSD) post-hoc test. Just after thawing, no differences in viability (45.6–51.1%; P = 0.67), DNA fragmentation (0.7–0.9%; P = 0.76), and acrosome status (intact acrosome: 79.2–83.0%; P = 0.26) of the spermatozoa were observed when sperm cells were frozen and thawed in 0, 25, and 50 µm bME. After culture for 90 min, however, the incidence of spermatozoa with an intact acrosome was significantly higher (P < 0.05) when the semen was frozen and thawed in the presence of 50 µm bME (70.9%), compared with 0 (61.7%) and 25 µm bME (61.0%). Chlortetracycline (CTC) analyses were peformed to confirm that the incidence of intact spermatozoa was higher (P < 0.01) in 50 µm bME (67.6%) than that of non-supplementation controls (51.4%). These results demonstrate that supplementation of semen with 50 µm bME during freezing and thawing processes reduces acrosome damage of boar spermatozoa.

scholarly journals The swim-up technique separates bovine sperm by metabolic rates, motility and tail length

2019 ◽  
Author(s):  
Veronika Magdanz ◽  
Sergii Boryshpolets ◽  
Clara Ridzewski ◽  
Barbara Eckel ◽  
Klaus Reinhardt
Keyword(s):  
Sperm Quality ◽  
Tail Length ◽  
Sperm Cells ◽  
Metabolic Rates ◽  
Label Free ◽  
Purification Method ◽  
Bovine Sperm ◽  
Consumption Rates ◽  
Simple Step

AbstractSwim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction but the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms the upper fraction of bull sperm has improved motility compared to the cells in the lower fraction and shows higher metabolic rates. This pattern was consistent across media of two different levels of viscosity. Sperm with longer flagella are selected in the upper fraction. We conclude that the motility-based separation of the swim-up technique is based on metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality, which is likely particularly useful in cases of asthenozoospermia and teratospermia. Furthermore, metabolic measurements of sperm cells can reveal differences in metabolic pathways in different environments.

Probiotic administration improves sperm quality in asthenozoospermic human donors

2017 ◽  
Vol 8 (2) ◽  
pp. 193-206 ◽  
Author(s):  
D.G. Valcarce ◽  
S. Genovés ◽  
M.F. Riesco ◽  
P. Martorell ◽  
M.P. Herráez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Dna Fragmentation ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Bifidobacterium Longum ◽  
Fold Change ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Sperm Analysis

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under similar diet conditions participated in the study. The quality of individual sperm samples was evaluated before (previous to ingestion), during (after 3 and 6 weeks of ingestion) and after probiotic administration (3 and 6 weeks after finishing the treatment). Sperm motility was evaluated by computer-assisted sperm analysis system, DNA fragmentation by sperm chromatin structure assay, cell viability by flow cytometry and measurement of intracellular H2O2 (reactive oxygen species; ROS) by flow cytometry using dichloro-dihydrofluorescein diacetate. Sperm motility was drastically improved after the treatment (approximately 6 fold change), DNA fragmentation was statistically reduced after probiotic administration from (approximately 1.2 fold change) and intracellular H2O2 level was decreased (approximately 3.5 fold change). Cell viability was not affected by the treatment. The significant improvement in sperm motility and the decrease in DNA fragmentation reported in this study provide preliminary evidence that probiotics could be administrated to improve motility and decrease DNA fragmentation and ROS levels in asthenozoospermic human males.

scholarly journals The risk of sperm demage in men with the combined effect of endocrine disruptors

2019 ◽  
Vol 74 (4) ◽  
pp. 229-234
Author(s):  
Stanislav V. Chigrinets ◽  
Gennadiy V. Brukhin
Keyword(s):  
Dna Damage ◽  
Bisphenol A ◽  
Dna Fragmentation ◽  
Endocrine Disruptors ◽  
Seminal Fluid ◽  
Combined Effect ◽  
Sperm Cells ◽  
Median Concentration ◽  
Sperm Dna ◽  
Comparison Groups

Background: The most frequent ultrastructural change in sperm cells is nuclear DNA fragmentation. Many authors believe that DNA damage to sperm cells can serve as a diagnostic marker of a negative paternal effect in the pre-implantation period of human development. A number of studies have shown a direct correlation between the high percentage of sperm DNA damage and the frequency of spontaneous abortions. On the other hand, it is known that the damaging effect of endocrine disruptors, for example, triclosan and bisphenol A, is based on their ability to induce oxidative stress, which is considered as one of the factors in cell DNA damage. Aims: to assess the risk of sperm DNA fragmentation in men with the combined effect of bisphenol A, triclosan and 4-nonylphenol in seminal fluid. Materials and methods: 84 samples of seminal fluid of men with normo-and patozoospermia were studied. The concentration of bisphenol A, triclosan and 4-nonylphenol in gas was determined by gas chromatography with mass spectrometry (GC-MS). The comparison groups were divided according to the degree of DNA fragmentation of spermatozoa into two groups: the 1st group of patients with DNA fragmentation of spermatozoa 15% (n=18) and the 2nd group 15% (n=29). The spermiological study was carried out according to the WHO recommendations (2010) taking into account the assessment of the number of spermatozoa, their motility and morphology, as well as the degree of fragmentation of the sperm DNA. Results: Bisphenol A was found in 100% of the ejaculate samples with a median concentration of 0.150 ng/ml. Triclosan and 4-nonylphenol were detected in 84.3 and 98.1% of ejaculate samples with a median concentration of 0.11 and 0.16 ng/ml respectively. Comparison groups were statistically significantly distinguished by the concentration of bisphenol A and triclosan p=0.014; p0.001 respectively. Triclosan with an increase in concentration in seminal fluid by 0.1 ng/ml increased the chance of development of the degree of DNA fragmentation 15% by 2.9 times. The odds ratio of bisphenol A and 4-nonylphenol to DNA damage was not statistically significant. The prognostic model of the joint effect of endocrine disruptors on DNA damage, constructed using multiple logistic regression analysis, was also found to be statistically significant only with respect to the effect of triclosan. Conclusions: The risk of damage to sperm DNA in men is primarily associated with the effect of triclosan in seminal fluid. At the same time, it is necessary to assume the absence of a synergistic effect of bisphenol A, triclosan and 4-nonylphenol on the DNA damage of spermatozoa in men.

scholarly journals The Cryoprotective Potential of Propolis Supplemented in Frozen-Thawed Bull Semen: Biochemical and Physiological Findings

2021 ◽  
Author(s):  
Deniz Yeni ◽  
Mehmet Fuat Gülhan ◽  
Muhammed Enes İnanç ◽  
Fatih Avdatek ◽  
Şükrü Güngör ◽  
...  
Keyword(s):  
Dna Damage ◽  
Kinetic Parameters ◽  
Colorimetric Method ◽  
Tail Length ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Bull Semen ◽  
Sperm Abnormalities ◽  
Phenolic Components ◽  
Negative Effect

Abstract In this study, the cryoprotective effect of different doses of propolis (P) on bull semen, which has strong pharmacological properties thanks to its rich phenolic components, was investigated biochemically and physiologically. Semen samples were collected from Simmental breed bulls via the artificial vagina and pooled. After dividing into five groups, control (C: no additive), and four different dose P (200, 100, 50, and 25 μg/mL) groups, the final concentration was diluted to 16×106 per straw. Semen samples were equilibrated at 4°C for approximately 4 hours, then placed in French straws and frozen. After thawing, sperm motility and kinetic parameters, DNA integrity by single-cell gel electrophoresis, sperm abnormalities by liquid fixation, and lipid peroxidation levels by the colorimetric method was analyzed by Computer-Assisted Semen Analyzer. P added to the diluent showed no effect on motility and kinetic parameters at P25 and P50 (p &gt;0.05), while P100 and P200 had a negative effect (p &lt;0.001). The addition of P (25 and 50) had a treatment effect on tail abnormality compared to C (p &lt;0.05). Especially P50 had a positive effect on tail length, tail DNA, and tail moment, while P100 and P200 caused DNA damage (p &lt;0.001). MDA levels increased in all P dose groups compared to C (p &lt;0.001). This study has clearly demonstrated that P25 and P50 supplements could be used therapeutically in the treatment of sperm abnormalities and prevention of DNA damage in post-thawed bull sperm.

Factors affecting storage of Slovak native rabbit semen in the gene bank

Zygote ◽  
2017 ◽  
Vol 25 (5) ◽  
pp. 592-600
Author(s):  
Barbora Kulíková ◽  
Marta Oravcová ◽  
Andrej Baláži ◽  
Peter Supuka ◽  
Peter Chrenek
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Semen Quality ◽  
Sperm Quality ◽  
Objective Assessment ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Quality Traits ◽  
Plasma Membrane Integrity

SummaryIn this study, fresh and frozen–thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen–thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen–thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen–thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen–thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

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