Antioxidant Capacity Of Seminal Plasma Research Articles

P-025 Mitochondrial activity in sperm is associated to blastocyst development: a double-donation study

Abstract Study question Do oxidative parameters in sperm affect oocyte fertilization or blastocyst development in double-donation ICSI cycles? Summary answer While intracellular oxidative stress in sperm does not suppose an impact on fertilization or embryo development, mitochondrial activity is related to increased blastocyst development rates What is known already Reactive oxygen species(ROS) are molecules causing a detrimental effect on sperm quality and function, especially when they are imbalanced with respect to the sperm and seminal plasma antioxidant capacity. Previous research have extensively demonstrated that sperm affected by oxidative stress(OS) show reduced fertilizing capacity, and lead to embryos with impaired development after conventional IVF. The role of OS in sperm in the case of ICSI is, however, controversial because many factors may cause a bias. Not only could this explain the inconsistent results reported thus far, but would also support the need of testing healthy donor gametes to reduce variability. Study design, size, duration This research was devised in the context of a collaboration between a sperm bank and a public university, and was set as a blinded, retrospective study that included samples from 45 sperm donors used in a total of 55 oocyte donation cycles. Intracellular levels of total ROS and of superoxides (O2•-), oxidative stress and mitochondrial activity (MA) were evaluated in frozen-thawed sperm donor samples, and related to the embryo outcomes obtained from the same samples. Participants/materials, setting, methods Samples from selected sperm donors were used to fertilize oocytes through ICSI in oocyte donation programs, and fertilization and blastocyst rates were recorded. Sperm viability and intracellular levels of total ROS and superoxides were determined through a combined assay (2’,7’-Dichlorofluorescin diacetate/Hydroethidine/Sytox Red); oxidative stress and sperm viability were assessed through co-staining with CellRox Far Red and propidium iodide (PI); and MA (mitochondrial membrane potential) and sperm viability were evaluated after staining with DiOC6(3) and PI. Main results and the role of chance The average and standard deviation for fertilization and blastocyst rates were 76.8%±17.3% and 67.5%±22.8%, respectively. Taking the viable sperm population into account, percentages of sperm with high ROS and low O2•- were 17.6%±15.9%, those with high ROS and high O2•- were 0.5%±0.4%, percentages of sperm with low ROS and low O2•- were 25.9%±9.2%, and those with low ROS and high O2•- were 54.5%±21.1%. The evaluation of oxidative stress with CellRox revealed that the percentage of viable sperm with high OS was 63.1%±16.5%. Neither intracellular levels of ROS and superoxides, nor oxidative stress in viable sperm were found to be correlated to fertilization or blastocyst rates (P>0.05). In spite of this, the percentage of viable sperm with high MA, which was 20.7%±10.5%, was observed to be positively correlated to blastocyst rates (Rs=0.276; P=0.04). While sperm mitochondria do not participate in further embryo development, a greater mitochondrial activity in post-thawed donor sperm could indicate a healthier sperm function. Mitochondrial activity could, therefore, be used as a predictive biomarker for embryo development. Limitations, reasons for caution The sample size is the main limitation of this study, despite the efforts made to include a high number of sperm donors. Wider implications of the findings Results obtained in the present study show that ICSI evades the detrimental effects of sperm oxidative stress on fertilization and embryo development. Yet, sperm mitochondrial activity could be used as a biomarker to test the ability of a sperm donor to generate healthy embryos that develop up to blastocyst stage. Trial registration number Not applicable

Reduced SIRT1 and SIRT3 and Lower Antioxidant Capacity of Seminal Plasma Is Associated with Shorter Sperm Telomere Length in Oligospermic Men.

Infertility affects millions of couples worldwide and has a profound impact not only on their families, but also on communities. Telomere attrition has been associated with infertility, DNA damage and fragmentation. Oxidative stress has been shown to affect sperm DNA integrity and telomere length. Sirtuins such as SIRT1 and SIRT3 are involved in aging and oxidative stress response. The aim of the present study is to determine the role of SIRT1 and SIRT3 in regulating oxidative stress, telomere shortening, and their association with oligospermia. Therefore, we assessed the protein levels of SIRT1 and SIRT3, total antioxidant capacity (TAC), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase activity (CAT) in the seminal plasma of 272 patients with oligospermia and 251 fertile men. We also measured sperm telomere length (STL) and leukocyte telomere length (LTL) using a standard real-time quantitative PCR assay. Sperm chromatin and protamine deficiency were also measured as per standard methods. Our results for oligospermic patients demonstrate significant reductions in semen parameters, shorter STL and LTL, lower levels of SOD, TAC, CAT, SIRT1 and SIRT3 levels, and also significant protamine deficiency and higher levels of MDA and DNA fragmentation. We conclude that a shorter TL in sperms and leukocytes is associated with increased oxidative stress that also accounts for high levels of DNA fragmentation in sperms. Our results support the hypothesis that various sperm parameters in the state of oligospermia are associated with or caused by reduced levels of SIRT1 and SIRT3 proteins.

The microbiota of sexual intercourse and its effect on prostatitis.

It is estimated that microorganisms colonize 90% of the body surface. In some tracts, such as the genitourinary tract, the microbiota varies throughout life, influenced by hormonal stimulation and sexual practices. This study evaluated the semen differences and presence of Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae in semen samples from patients with symptoms of chronic prostatitis and men asymptomatic for urogenital infections. Fifty-three semen samples were included: 22 samples from men with symptoms of chronic prostatitis and 31 asymptomatic men (control group). In addition to the presence of L. crispatus, L. iners, G. vaginalis and A. vaginae, semen parameters, total antioxidant capacity of seminal plasma, prostatic antigen and some proinflammatory cytokines were evaluated in each semen sample. Volunteers with symptoms of chronic prostatitis presented a lower percentage of sperm morphology (4.3% vs. control group 6.0%, p = 0.004); in the semen samples of volunteers in the group asymptomatic for urogenital infections, microorganisms associated with the vaginal microbiota were detected more frequently. The presence of bacteria in the vaginal microbiota can also benefit male reproductive health, which undergoes various modifications related to lifestyle habits that are susceptible to modification. Microorganisms associated with the vaginal microbiota, such as L. crispatus, L. iners, G. vaginalis and A. vaginae, may have a protective role against the development of male genitourinary diseases such as prostatitis.

Goji Berry in the Diet of the Rabbit Buck: Effects on Semen Quality, Oxidative Status and Histological Features of the Reproductive Tract.

Goji berry (GB) shows beneficial effects on human health, although its effects on the male rabbit have been little investigated. This study examines the impact of GB dietary supplementation on the semen traits, antioxidant capacity of seminal plasma, and histological features of the reproductive tract of rabbit buck. Eighteen rabbits were distributed into two dietary groups: one receiving a commercial feed (Control), and the other a feed supplemented with 1% of GB (Goji). After a nutritional adaptation period of 60 days, the animals were subjected to semen collection every 15 days. The semen traits, libido, antioxidant, and inflammatory parameters were collected and analyzed. The rabbits were sacrificed after 60 days, and tissues of the genital tract were analyzed. Compared to the Control group, the Goji group showed higher spermatozoa concentration, motility, and vitality (p < 0.05), as well as fewer abnormal spermatozoa and a higher libido (p < 0.1). Histological features such as functional activity and hyperplasia were improved by GB and correlated with some semen traits (p < 0.05). Conversely, antioxidant and anti-inflammatory parameters were unaffected by the diet. These findings suggest that GB acts on the tissues of the reproductive tract positively influencing semen quality, although further studies are needed to understand the effect on oxidative stress.

Effect of a dietary antioxidant supplementation on semen characteristics of the Colombian horse

Cooling equine semen increases production of reactive oxygen species (ROS). Elevated concentrations of ROS have detrimental consequences by causing changes in membrane permeability. Although there are reports of antioxidant supplementation to improve sperm quality in horses, there are few reports on the Colombian breed. The objective of this study was to evaluate the effect of nutritional supplementation with a variety of antioxidants on semen characteristics of Colombian horses. Six (n=6) Colombian Creole stallions aged between 5 and 15 years were collected 3 times a week using a Missouri artificial vagina for 20 weeks. Stallions were randomly allocated into a control (C) or a supplementation group (SG) with Natural Fertility®. Cobalamin 0.36 mg/day; pyridoxin 16 mg/day; retinoic acid 366799 IU/day; alpha tocopherol 1600 IU/day; ascorbic acid 4000 mg/day; folic acid 1200 mg/day; lysine 640 mg/day; methionine 960 mg/day; threonine 400 mg/day; arginine 400 mg/day; L-Carnitine 10000 mg/day; omega 3 3200 mg/day; zinc 1200 mg/day; copper 44 mg/day, chromium 6 mg/day and selenium 8 mg/day, was added to the basal diet (hay, mineralized salt, 2 kg of grain and water). Repeated measures design with a wash-out period of 2 weeks between treatments was used. During a periodof 8 weeks, semen was analyzed twice a week for motility and kinetics using a Sperm Class Analyzer (SCA®) system; sperm vitality (SV) and acrosomal Integrity (AI) by fluorescent microscopy, abnormal morphology (AM) by supravital stain, and plasmatic membrane Integrity by hypoosmotic test (HOST). Reactive oxygen species and total antioxidant capacity (TAC) by ferric reducing antioxidant power assay (FRAP) were assessed by spectrofluorimetry. Comparison of means was performed using Tukey's test with p≤0.05; using SAS program 3.81. SG increased the beat cross frequency of semen with 36.68 Hz ± 0.89 vs. 32.67 Hz ± 0.73 (C). In addition, a higher AI was found for SG (82.92 ± 0.72 %) compared to C (78.51% ± 0.83). SG also increased the TAC of seminal plasma (15.35± 0.92 μmol Trolox/L) compared to C (18.86± 1.75 μmol Trolox/L). In the individual analysis it was found that SG increased the total and progressive motility of the semen of two horses, however it reduced both parameters for two other individuals, compared to C. In conclusion, supplementation of the diet of horses with a supplement that provides vitamins, minerals, amino acids, and omega 3 can increase the antioxidant capacity of seminal plasma, as well as sperm quality, however this may be subject to a differential effect on each horse.

Mitochondrial metabolism determines the functional status of human sperm and correlates with semen parameters

The diagnosis of male infertility is based essentially on the patient’s medical history and a standard semen analysis. However, the latter rarely provides information on the causes of a possible infertility, emphasizing the need to extend the analysis of the sperm function. Mitochondrial function has been associated with sperm function and dysfunction, the latter primarily through the production of excessive amounts of reactive oxygen species (ROS). We hypothesized that analysis of sperm mitochondrial metabolism together with sperm ROS production could be an additional tool to improve routine semen analysis, after appropriate validations. To test our hypothesis, we performed several experiments using a non-routine method (high-resolution respirometry, HRR) to access mitochondrial function. First, we investigated whether mitochondrial function is related to human sperm motility and morphology. When mitochondrial metabolism was challenged, sperm motility decreased significantly. Additionally, morphological abnormalities in the sperm mid-piece and mitochondria were associated with global sperm defects evaluated by routine methods. Subsequently, sperm mitochondrial function was assessed by HRR. Respiratory control ratio (RCR) was determined and evaluated in the context of classical sperm analysis. In parallel, sperm hydrogen peroxide (H2O2) production and seminal plasma (SP) antioxidant capacity were measured. The percentage of sperm with progressive motility correlated positively with RCR, SP antioxidant capacity, and negatively with the concentration of extracellular H2O2 production ([H2O2]). The percentage of normal sperm morphology correlated positively with RCR and negatively with [H2O2]. Sperm morphology did not correlate with seminal plasma antioxidant capacity. Furthermore, Receiver Operating Characteristic curves were used for the first time to test the diagnostic ability of RCR, [H2O2], and SP antioxidant capacity as binary classifiers. An RCR cut off value of 3.2 was established with a sensitivity of 73% and a specificity of 61%, using reference values considered normal or abnormal in routine semen analysis. The cut off value for [H2O2] was 0.2 μM/106 sperm (sensitivity = 65%, specificity = 60%). There were no reference values for SP antioxidant capacity that distinguished between abnormal and normal sperm samples. We conclude that sperm mitochondrial function indices in combination with [H2O2] may be useful tools to complement the routine semen analysis.

Fresh and cryopreserved semen, minerals, hormones and health characteristics in response to reciprocal combinations of vitamin D3 and 25-hydroxyvitamin D3 in the mature and prepubertal Holstein bulls' diet

The objective of this study was to evaluate the effect of supplementation of 25-hydroxyvitamin D3 (25(OH)VD3) instead of vitamin D3 on the quality and seminal plasma antioxidant indicators of fresh and cryopreserved semen, serum minerals, vitamin D3 status, hormone levels, blood biochemistry, antioxidant and immune function in different age of bulls. Thirty-two Holstein bulls (16 bulls aged 24–36 months and weighted 727 ± 99.0 kg; 16 bulls aged 12 months and weighted 332 ± 24.9 kg) randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement with 2 sources of vitamin D3 (vitamin D3 or 25(OH)VD3) fed at 300 IU/kg dry matter intake and two different age of bull (maturity or prepuberty) through a 60 day period. Bulls were tied in individual pens without access to sunlight. Bulls fed 25(OH)VD3 had greater semen concentration (P = 0.03), sperms motility (P = 0.02), viability (P < 0.001), membrane integrity (P < 0.001) and seminal plasma antioxidant capacity (P < 0.05) at fresh stage compared with bulls fed vitamin D3. In addition, 25(OH)VD3 increased sperms motility (P = 0.02) and membrane integrity (P = 0.04) in cryopreserved semen. Feeding 25(OH)VD3 increased serum total calcium (P = 0.03), total phosphorus (P < 0.001), 25(OH)VD3 (P < 0.001), 1,25-dihydroxyvitamin D3 (P < 0.001) compared with dietary vitamin D3. Feeding 25(OH)VD3 improved concentrations of serum estradiol (P = 0.02), testosterone (P = 0.003) and growth hormone (P < 0.001) and decreased serum cortisol (P = 0.01). Lower blood urea nitrogen (P = 0.005), creatinine (P = 0.02), aspartate aminotransferase (P = 0.005) and higher serum glucose (P = 0.03) were observed in the 25(OH)VD3. Dietary 25(OH)VD3 increased serum glutathione (P = 0.001) and immunoglobulin M (P = 0.03) and decreased malondialdehyde (P = 0.04) compared to those fed vitamin D3. The total antioxidant capacity was affected by interaction of age and vitamin D source (P < 0.001). In conclusion, replacing vitamin D3 with 25(OH)VD3 improved semen quality and benefited health of the mature and prepubertal Holstein bulls.

Evaluation of Current Antioxidant Profile in Semen

The freezing and storage of the sperm are used cryopreservation of germplasm in livestock breeding, genetic improvement of indigenous species, preservation of rare races, successful tolerance to environmental changes and international germplasm exchanges. Both the freezing and thawing process causes large changes in the volume of the cell fluid. Spermatozoon removes most of its cytoplasm at differentiation stages and lacks the cytoplasmic component that contains antioxidants that counteract the harmful effect of reactive oxygen species and lipid peroxidation. Therefore, the sensitivity of spermatozoa to lipid peroxidation increases during the freezing and thawing of the sperm, which creates a significant mechanical stress on the cell membrane. Oxidative stress is caused by oxygen and oxygen-derived oxidants, commonly known as ROS, and is known as an imbalance between the ability of biological systems to easily detoxify or repair damaged reagents. Uncontrolled ROS production, which exceeds the antioxidant capacity of seminal plasma, causes oxidative stress that is harmful to spermatozoa. All cellular components, including lipids, proteins, nucleic acids, and sugars, are potential targets of oxidative stress. Antioxidants control the chemical degradation of the substrate caused by oxidation, neutralizing free radicals, thereby it is used to minimize the risk of damage to spermatozoa during cryopreservation.

26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P&amp;lt;0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.

Activities of antioxidant seminal plasma enzymes (SOD, CAT, GPX and GSR) are higher in jackasses than in stallions and are correlated with sperm motility in jackasses

This study compared the activities of four antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; and glutathione reductase, GSR) in the seminal plasma of stallions and jackasses. Eighteen stallion ejaculates and 24 jack ejaculates were collected through an artificial vagina. Seminal plasma was obtained by several centrifugations at 3000×g and 4 °C for 10 min, and activities of SOD, CAT, GPX and GSR were subsequently determined. We also evaluated whether the collecting season had any influence on the activities of these four enzymes in both stallions and jackasses. Antioxidant capacity of seminal plasma was significantly higher in jackasses than in stallions (mean ± SEM, SOD: 1707.7 ± 195.9 U/mL vs. 231.9 ± 29.6 U/mL; CAT: 9094.7 ± 1292.9 U/L vs.1682.7 ± 525.9 U/L; GPX 845.4 ± 106.0 U/L vs. 469.7 ± 60.3 U/L; GSR: 50.3 ± 5.1 U/L vs. 20.7 ± 4.6 U/L). Furthermore, whereas season had no effect on the activity of these four enzymes in stallions, the activities of CAT and GPX in jack seminal plasma were significantly higher in the summer than in the other seasons. In addition, the activities of SOD and CAT were found to be significantly correlated with the percentages of progressively motile spermatozoa, and with the percentages of linearity and straightness, respectively, in jackasses. In contrast, the activities of these four enzymes were not correlated with sperm quality parameters in stallions. Finally, while SOD, CAT, and GPX activities but not those of GSR were correlated in jackasses, the activities of all four enzymes were correlated each other in stallions. We can thus conclude that the activities of SOD, CAT, GPX and GSR differ between the seminal plasma of stallions and donkeys, and vary between seasons in jackasses.

Total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs.

The aim of this study was to evaluate the total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs. The study was conducted on 10 infertile and 10 fertile dogs of various breeds. Infertility was defined as conception failure at least three matings with different bitches. Semen was collected by manual manipulation. The sperm concentration and motility parameters were evaluated using CASA Hamilton Thorne, Vers. IVOS 12.3. The morphology of spermatozoa and the percentage of live and dead sperm cells were assessed microscopically, total antioxidant capacity and the content of SH-groups in seminal plasma were determined spectrophotometrically, the contents of protein peroxidation markers in seminal plasma, bityrosine and formylokinurenine, were determined using spectrofluorimetric methods. Sperm concentration and total sperm count were significantly (p<0.05) lower in infertile dogs than in fertile dogs (99.92±3 0.05×106 /ml vs. 282.07±48.27×106 /ml; 214.19±114.74×106 vs. 747.57±210.94×106 , respectively). The percentage of spermatozoa with normal morphology and the most determined motility parameters differed significantly (p<0.05) between both groups. The mean values of total antioxidant capacity in the seminal plasma were significantly (p<0.05) lower (19.95±20.94 vs. 25.66±23.18µmol/g protein), whereas the mean contents of bityrosine and formylokinurenine in seminal plasma were significantly (p<0.05) higher in infertile dogs than in fertile dogs (3.71±4.83µg/mg protein vs. 1.55±2.00µg/mg protein and 0.37±0.45µg/mg protein vs. 0.14±0.08µg/mg protein, respectively). In conclusion, the obtained results suggest that the poor semen quality and infertility in dogs could be associated with lowered total antioxidant capacity and increased protein peroxidation in seminal plasma as a consequence of oxidative stress.

Association between seminal plasma neopterin and oxidative stress in male infertility: A case-control study

Neopterin is a significant and sensitive marker in estimating the activity of cellular immune system. Oxidative stress plays a role in the etiology of male infertility. Increased reactive oxygen species is accompanied with increase in neopterin level. Hence neopterin may be involved in male infertility. The objective of this case-control study was to determine neopterin level in idiopathic infertile and normospermic men; furthermore, to identify its relationship with oxidative stress markers including total oxidant, malondialdehyde, sperm DNA fragmentation, and total antioxidant capacity of seminal plasma. Forty seven infertile and forty three normospermic males were selected according to WHO criteria. Their semen and blood samples were taken; subsequently, the levels of neopterin, total oxidant, total antioxidant, malondialdehyde, and sperm DNA fragmentation were measured. The levels of neopterin, total oxidant, and malondialdehyde in seminal plasma of infertile males were significantly higher than those of normospermic group (p=0.038, 0.018, and 0.028, respectively). Furthermore, sperm DNA fragmentation in infertile men was higher than that of control group (p<0.001). Moreover, total antioxidant capacity of seminal plasma in infertile males was significantly lower than that of normospermic subjects (p=0.002). No significant difference was observed in serum neopterin, total oxidant, and malondialdehyde between the infertile and normospermic groups. The significant inverse correlation between seminal plasma neopterin and total antioxidant in the infertile males supports a possible role of neopterin in male infertility. Neopterin can be suggested as a marker in monitoring and diagnosis of idiopathic male infertility.

Revisiting The Relationship between The Ejaculatory Abstinence Period and Semen Characteristics.

Variation in the ejaculatory abstinence period suggested by different guidance bodies have resulted in a growing concern among researchers and clinicians over what the precise period of ejaculatory abstinence ought to be for an optimal semen sample. Several studies have thus been undertaken to examine the association between the length of sexual abstinence and semen characteristics. Not all studies, however, have arrived at the same conclusions. This study aims to review all existing literature published during the past few decades pertaining to the influence of ejaculatory abstinence on semen quality. For the purpose of this systematic review, all data related to sexual abstinence duration and seminal parameters were re-analysed to homogenize the current data. Thorough PubMed, MEDLINE and Google Scholar, a literature search was conducted using the keywords “sexual abstinence”, “ejaculatory abstinence”, “semen”, “spermatozoa”, “semen analysis”, “sperm parameters”, “motility”, “reactive oxygen species (ROS)” and “DNA fragmentation”. After carefully reviewing all the literature, 30 relevant papers, both written in English and published between January 1979 and December 2016, were included in this review. The weight of the evidence suggests that the decline in semen volume and sperm concentration with shorter abstinence periods is accompanied by a substantial improvement in sperm motility characteristics, especially progressive motility and velocity. Nevertheless, available data are insufficient to support definitive conclusions regarding the influence of the ejaculatory abstinence period on advanced semen parameters (ROS, DNA fragmentation and seminal plasma antioxidant capacity) and pregnancy rates. In conclusion, taking all data into account, shortening of the abstinence period may be beneficial to sperm quality. Furthermore, we recommend that the current guidelines regarding the prescribed abstinence period should be revisited.

The AhRR-c.565C>G transversion may increase total antioxidant capacity levels of the seminal plasma in infertile men.

The Aryl hydrocarbon receptor (AhR)-repressor (AhRR) is a regulator of the AhR pathway, which plays an important role in xenobiotic and reactive oxygen species (ROS) metabolism. Total antioxidant capacity (TAC) is a major factor in semen quality that protects sperm against ROS stress. Malondialdehyde (MDA) is the indicator of lipid peroxidation damage that is occurred due to ROSs. In this study, we determined and compared the MDA and TAC levels of infertile men's semen and blood plasma regarding genotype groups of AhRR-c.565C>G transversion. Semen and blood samples of 123 infertile males were collected from the Fatemeh Zahra IVF Centre, Babol, Iran. TAC and MDA levels of seminal and blood plasma were measured by TBARS and FRAP methods, respectively. Cases were genotyped by the PCR-RFLP method. The frequency of c.565C>G genotypes was determined as CC (34.14%), CG (55.28%) and GG (10.58%). Mean levels of TAC μm/L and MDA nmol/mL in semen plasma of CC, CG and GG groups were (1365.7, 1.28), (1542.8, 1.51) and (1860.2, 0.82), respectively. Also, mean levels of TAC μm/L and MDA nmol/mL in blood plasma samples in CC, CG and GG genotypes were (806.14, 1.168), (727.1, 1.006) and (635.7, 0.83), respectively. Comparison of marker levels between genotypes revealed that the TAC level of semen plasma in the GG genotype was significantly higher than its level in the CC group (p<0.05). Our findings showed that in seminal plasma of infertile men with the GG genotype of AhRR-c.565C>G transversion, the level of total antioxidant capacity is significantly higher in comparison with the CC genotype. Then, the G allele of AhRR-c.565C>G transversion may have a role in the increase in antioxidant capacity of seminal plasma.

Are oxidative stress markers associated with unexplained male infertility?

Male infertility can be responsible for up to 20% of the cases attending fertility consultation facilities; nonetheless, the underlying molecular mechanisms that could explain it are still elusive. Therefore, we aimed to evaluate conventional and functional parameters of semen samples from patients who presented with male infertility of unknown origin. Conventional semen parameters and functional parameters (i.e. intracellular reactive oxygen species production, mitochondrial membrane potential, sperm chromatin structure assay, sperm membrane lipid peroxidation and antioxidant capacity of seminal plasma) were evaluated on semen samples from 54 healthy donors, 23 patients with idiopathic infertility and 34 fertile controls. No significant differences were observed in the conventional seminal parameters between the fertile and infertile men. However, increased intracellular reactive oxygen species (ROS) production and DNA fragmentation were observed in the infertile patients compared to the fertile group. Alterations in intracellular ROS production and DNA fragmentation could be associated with male idiopathic infertility. These parameters could eventually distinguish both groups more accurately than the conventional parameters. Our current results are encouraging, and the efficacy of these parameters in the clinical settings needs to be further assessed to establish their predictive potential as a marker of unexplained male infertility.

Diagnostic application of total antioxidant capacity in seminal plasma to assess oxidative stress in male factor infertility.

This study was undertaken in order to establish a new reference value for the total antioxidant capacity (TAC) in seminal plasma as a predictor of fertility. This study also aims to propose a detailed protocol for the TAC assay including calculation of assay results and assessment of sensitivity and specificity over possible cutoff values in infertile men and controls with proven and unproven fertility. Seminal plasma from 279 infertile patients and 46 normal healthy men referred to a male infertility testing laboratory were tested to measure TAC by a colorimetric assay kit. Receiver operating characteristics (ROC) curves were generated to establish cutoff values, sensitivity, and specificity, and the distribution of cutoff values in controls and infertile patients was calculated. Infertile patients showed significantly lower levels (mean ± SEM) of total antioxidants (micromolar Trolox equivalents) in their seminal plasma (1863.84 ± 27.16μM) compared to those from fertile men (2013 ± 56.04μM, P = 0.019). A preferred cutoff TAC value of 1947μM could facilitate better diagnosis of oxidative stress (OS) in men with male factor infertility. At this threshold, the specificity of TAC assay was 63.0% and the sensitivity 59.5% with a positive predictive value of 90.7% and a negative predictive value of 20.4%. Our results establish a new diagnostic cutoff TAC value of 1947μM in seminal plasma to distinguish prevalence of OS in infertile patients compared to healthy men. This study provides a robust reference value of seminal plasma TAC that may provide an important diagnostic tool to the physicians for managing OS and male factor infertility in such patients.

Activity of Enzymatic Antioxidants and Total Antioxidant Capacity in Seminal Plasma of Murrah Bulls during Cryopreservation

Semen samples with mass motility of 4+ or more and an initial progressive motility ≥ 80% collected from murrah buffalo bulls were utilized for study. Semen samples were evaluated for various seminal attributes, enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and total antioxidant capacity at fresh, pre-freeze and post-thaw stage. Seminal attributes (progressive motility, viability, acrosomal integrity and hypo-osmotic swelling response) were significantly (p<0.05) higher in bull II as compared to bulls (I & III) at pre-freeze and post-thaw stage. Superoxide dismutase was significantly higher in bulls (II & III) than bull I at fresh stage (p<0.05) and significantly (p<0.01) higher in bull II as compared to bulls (I & III) at pre-freeze and post-thaw stage. Catalase was significantly higher in bull II than bulls (I & III) at fresh (p<0.05) and pre-freeze (p<0.01) stage. Significantly, higher total antioxidant capacity was noticed at pre-freeze (p<0.05) and post-thaw (p<0.01) stage in bull II and bulls (II & III), than bulls (I & III) and bull I, respectively. On the basis of our findings, it could be concluded that seminal enzymatic antioxidants (superoxide dismutase & catalase) and total antioxidant capacity varied among bulls at various stages of cryopreservation. There was progressive reduction in activity of enzymatic antioxidants and total antioxidant capacity from fresh to post-thaw stage.

High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility.

The study attempted to clarify the role of total antioxidant capacity of seminal plasma (SP-TAC) on boar sperm survival and fertility after artificial insemination (AI). SP-TAC differed (P < 0.001) among boars (n° = 15) and, to a lesser degree, among ejaculates within male (4 ejaculates/boar). SP-TAC also differed (P < 0.001) among ejaculate fractions (43 ejaculates and 3 fractions per ejaculate), of which the sperm-peak portion of the sperm rich ejaculate fraction (SRF) had the highest SP-TAC. SP-TAC was not correlated with sperm quality (motility and viability) or functionality (intracellular ROS generation and lipid peroxidation) of liquid AI-semen samples stored at 17 °C for 72 h (90 AI-samples), but the decline in sperm quality was larger (P < 0.05) in ejaculates with low, compared with high SP-TAC (hierarchically grouped). The SP-TAC differences among ejaculate portions agree with sperm cryosurvival rates (14 ejaculates from 7 boars), showing sperm from sperm-peak portion better (P < 0.01) post-thaw quality and functionality than those from the entire ejaculate (mainly post-SRF). Boars (n° = 18) with high SP-TAC (hierarchically grouped) had higher (P < 0.05) fertility outcomes (5,546 AI-sows) than those with low SP-TAC. Measurement of SP-TAC ought to be a discriminative tool to prognosis fertility in breeding boars.

Reference values of reactive oxygen species in seminal ejaculates using chemiluminescence assay.

A physiological balance exists between seminal reactive oxygen species (ROS) and antioxidant capacity. An overproduction of ROS which exceeds the seminal plasma antioxidant capacity results in oxidative stress (OS). The aim of the present study was to describe a detailed protocol to measure ROS in a diagnostic laboratory and revise our previous cutoff value of ROS in seminal ejaculates in a larger cohort of infertile men and controls with proven and unproven fertility. A total of 258 infertile men and 92 controls were enrolled in the study. Following initial semen analysis, ROS measurement in whole ejaculates was carried out using luminol-based chemiluminescence assay. Chemiluminescence was measured for 15 min with a Berthold luminometer. Results were expressed as relative light units (RLU/s/10(6) sperm). The test's specificity, sensitivity, and cutoff values were calculated using the receiver operating characteristic (ROC) curve. Significantly higher ROS levels were seen in infertile men compared to controls (p < 0.001). The optimal cutoff value to differentiate between controls and infertile men was 102.2 RLU/s/10(6) sperm. At this cutoff value, the test was 76.4% sensitive and 53.3% specific. The positive and negative predictive values of the test were 82.1% and 44.5%, respectively. A total of 76.4% infertile population was above this cutoff value compared to 46.7% of controls. The luminol-based chemiluminescence assay can be used in routine diagnostic screening to test for male infertility diagnosis in a clinical setting. The current ROS cutoff value substantially distinguishes infertile from normal controls. Patients with elevated ROS must be evaluated for the underlying cause of ROS production.

Total antioxidant capacity of honeybee haemolymph in relation to age and exposure to pesticide, and comparison to antioxidant capacity of seminal plasma

Oxidative stress is defined as a disturbance in the balance between the production of reactive oxygen species and antioxidant defences. We measured total antioxidant capacity (TAC) in honeybee haemolymph and seminal plasma and analysed TAC of haemolymph in relation to age and exposure to pesticide. TAC of haemolymph increased with age of bees (1.18 vs 1.97 mM of (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) for 1- and 30-day-old bees, respectively, P ≤ 0.05). Exposure to imidacloprid (IMD) affected TAC of haemolymph of 1-day-old but not 30-day-old honeybees. TAC in haemolymph of 1-day-old bees was lower in treatments with the addition of 5 and 200 ppb IMD (1.57–1.46 mM of Trolox in treated bees compared with 2.37 mM of Trolox in controls; P ≤ 0.05). In conclusion, antioxidant protection of honeybees seems to be related to age and may be disturbed by exposure to IMD. Older bees with higher antioxidant protection seem to be less susceptible to IMD toxicity. The toxic effect of pesticide seems to be particularly dangerous in early life stages of honeybees.