Association of vitamin D status with semen quality and reproductive hormones in Iranian subfertile men.

Although vitamin D deficiency is one of the most common health problems throughout the world, including Iran, conflicting information exists on the potential association between serum vitamin D levels and semen quality. This study intended to evaluate the association between serum vitamin D [25(OH) D3] with semen quality and hormones in Iranian subfertile men. We also compared mean vitamin D and hormone levels in normospermic men with oligoasthenoteratozoospermia (OAT) men. This cross-sectional study was conducted on 278 men who were referred to Royan Infertility Clinic (Tehran, Iran) from March to September 2014. The participants were categorized into two groups; of 186 normospermic and 92 OAT patients according to World Health Organization 2010 criteria. Each participant provided informed consent prior to launching research. Participants completed two general questionnaires of nutritional status. Blood and semen samples were obtained for assessment, and all data were adjusted for age, body mass index (BMI), and season. Vitamin D levels were classified according to Institute of Medicine guidelines. Vitamin D deficiency, insufficiency, and normal levels were observed in 8.6%, 43.6%, 47.8% of participants, respectively. No association was found between daily dietary intake of vitamin D and calcium with sperm parameters. Serum vitamin D was inversely correlated with PTH (p < 0.045). In normospermic men, serum vitamin D levels categorized were not correlated with semen parameters and reproductive hormones (FSH, LH, testosterone(T), and FT), whereas sperm motility showed a positive correlation with vitamin D categorized in OAT men (rs = 0.131, p = 0.028). In conclusion, there was a high incidence of deficiency and insufficiency 25(OH) D Levels (<20ng/ml) observed in Iranian men (52.2%). Moreover, our findings showed a correlation between vitamin D levels and sperm motility in OAT men, which requires further studies.

Dietary fatty acids affect semen quality: a review.

Mammalian spermatozoa are characterized by a high proportion of polyunsaturated fatty acids (PUFA) which play a crucial role in fertilization. This review focuses on analysis of sperm fatty acid profiles and the effects of omega-3, saturated and trans dietary and sperm fatty acids on sperm parameters. Two major points have been pivotal points of investigation in the field of sperm fatty acid profiles: first, the comparison between fatty acid profiles of fertile and infertile men and second, the effect of dietary fatty acids on sperm fatty acid profiles as well as sperm quality and quantity. Docosahexaenoic acid (DHA, C22:6n-3), and palmitic acid (C16:0) are the predominant PUFA and saturated fatty acids, respectively, in human sperm cells. Higher levels of DHA are concentrated on the sperm's head or tail varying among different species. However, the human sperm head contains a higher concentration of DHA. Dietary fatty acids influence on sperm fatty acid profiles and it seems that sperm fatty acid profiles are most sensitive to dietary omega-3 PUFA. Although improvements in sperm parameters are a response to omega-3 sources after more than 4 weeks of supplementation in the male diet, time-dependent and dose-dependent responses may explain the failure in some experiments. In human spermatozoa, elevated saturated or trans fatty acid concentration and a low DHA level is a concern. The regulations of the sperm fatty acid mean melting point as well as expression regulation of peroxisome proliferator-activated receptor gamma (PPARG) alongside with spermatozoon assembly, anti-apoptosis effects, eicosanoid formation, and hormone activity are the putative key factors that induce a response by inclusion of omega-3 PUFA.

Chemically assisted somatic cell nuclear transfer without micromanipulator in the goat: effects of demecolcine, cytochalasin-B, and MG-132 on the efficiency of a manual method of oocyte enucleation using a pulled Pasteur pipette.

The present study aimed to facilitate widespread application of a previously described manual method of somatic cell nuclear transfer (SCNT) by investigating the effects of demecolcine (a microtubule-depolymerizing chemical), cytochalasin-B (a microfilament-depolymerizing chemical: 2.5µg/ml for 15min) and MG-132 (a proteasome inhibitor chemical) on the (i) incidence of cytoplasmic protrusion of MII chromosomes, (ii) improvement of manual oocyte enucleation, and (iii) in vitro and in vivo developmental competence of SCNT embryos in the goat. Following in vitro maturation, around 65% of goat oocytes contained a characteristic cytoplasmic protrusion of MII-chromosomes. Treatment with demecolcine (0.4µg/ml for 30min) significantly increased this rate to 92.2±4.5%. Treatment with MG-132 (2µM for 30min) could not improve this rate when used alone (61.4±11.5%), but when combined with demecolcine (86.4±8.1%). Treatment with cytochalasin-B completely suppressed this rate whenever used, either alone (7.7±5.1%) or in combination with demecolcine (3.9±1.3%). In a direct comparison, there was no significant difference in quantity and quality of embryos propagated by the manual vs. micromanipulation-based methods of SCNT (cleavage: 85.3±4.5 vs. 89.5±8.9%, blastocyst: 19.5±4.3 vs. 24.3±4.4%, grade 1 and 2 blastocyst: 33.8±7.1 vs. 29.5±6.3%, total cell count: 125±11.1 vs. 122±10.5, respectively). Furthermore, development to live kids at term was not significant between the two SCNT methods. From both technical and economical points of view, the overall in vitro and in vivo efficiency of this manual method of SCNT proved it a simple, fast and efficient alternative for large scale production of cloned goats.

Improvement in in vitro fertilization rate, decrease in reactive oxygen species and spermatozoa death incidence in rams by dietary fish oil.

Our aim was to evaluate the effects of fish oil feeding on sperm classical parameters, level of reactive oxygen spices (ROS), spermatozoa death incidence and in vitro fertilization (IVF) rate in rams. We randomly assigned nine rams, into two experimental groups (isoenergetic and isonitrogenous rations with constant level of vitamin E supplement): control (CTR; n = 5) and fish oil (FO; n = 4, 35 g/day/ram). Diets were fed for 70 days during the physiological breeding season. After a 21-day dietary adaptation period, semen was collected weekly from each ram by an artificial vagina. Sperm classical parameters were determined by the computer-assisted sperm analyzer system (CASA), and it was prepared for IVF process by swim-up technique. These evaluations were performed during the first and last weeks of sampling. Intracellular ROS level and spermatozoa death incidence were detected by flow cytometry on a weekly basis after adaptation. Data were analysed with SPSS 15. The volume, concentration (3.6 and 2.7 × 10(9) /ml) and sperm progressive motility (60 and 48%) were significantly improved in the FO group compared with the CTR (p < 0.05). A comparison of two-cell stage embryos following IVF in the two groups showed a significantly higher fertilization rate in the FO group (56%) compared with the CTR (49%). Superoxide anion (O2 (-) ) rate was significantly lower (p < 0.05) at the third week of sampling in the FO. Although the H2 O2 rate was numerically lower in the FO group compared with the CTR, this difference was not significant. In addition, apoptosis showed a significant difference in the third week of sampling (15 and 30% for FO and CTR, respectively; p < 0.05). Overall, adding fish oil to the ram diet not only improved sperm quality and IVF results, it also could reduce oxygen-free radicals and the incidence of spermatozoa death.

Saturated, omega-6 and omega-3 dietary fatty acid effects on the characteristics of fresh, frozen-thawed semen and blood parameters in rams.

The aim of this study was to investigate the effects of several dietary fatty acids (FAs) on semen quality and blood parameters in rams. We gave diet-supplemented treatments (35 g day(-1) ram(-1)) by C16:0 (palm oil), C18:2 [sunflower oil (SO)] and an n-3 source [fish oil (FO)] to 12 rams, who were fed for 15 weeks during their breeding season. Semen was collected once per week. Semen samples were extended with Tris-based cryoprotective diluents, then cooled to 5 °C and stored in liquid nitrogen. Positive responses were seen with FO after 4 weeks. The mean prefreezing semen characteristics improved with the intake of FO (P < 0.05). Interestingly, maximum sperm output in FO was achieved 7.5 × 10(9) when compared to palm oil 5.3 × 10(9). Rams that received FO had the highest total testosterone concentrations (11.3 ng ml(-1) for FO, 10.8 ng ml(-1) for SO and 10.2 ng ml(-1) for palm oil) during the experiment (P < 0.05). FO also improved the rams' sperm characteristics after thawing (P < 0.05). Although C16:0 is a major saturated FA in ram sperm and all rams have been fed isoenergetic rations, the unique FAs of FO improved fresh semen quality and freezing ability compared to other oils.

Kit ligand and glial-derived neurotrophic factor as alternative supplements for activation and development of ovine preantral follicles in vitro.

In vitro growth of preantral follicles has the potential to produce considerable numbers of competent oocytes for use in medicine, agriculture, and even wildlife conservation. The critical regulatory role of growth factors and hormones in the development of preantral follicles has been established. This study investigated the effect of glial-derived neurotropic factor (GDNF) and kit ligand (KL) on the in vitro development of ovine preantral follicles. Results indicated that both GDNF and KL significantly improved activation of primordial follicles, similar to co-addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), which are commonly used for in vitro follicular development. Importantly, GDNF had a more profound effect on follicle health, development, and differentiation compared with KL alone. Furthermore, the combination of GDNF and KL in the presence of EGF and bFGF had a positive, synergic effect on health, development, and differentiation of preantral follicles, as determined by histological and hormonal assessments. The results of this study may provide a foundation for further studies that will unravel the molecular mechanisms of follicular development to further improve the current status of in vitro preantral follicle culture.

Development of an optimized zona-free method of somatic cell nuclear transfer in the goat.

The purpose of this study was to develop an improved zona-free method of goat somatic cell nuclear transfer (SCNT) that has both ease of operation and efficiency. The main steps involved were: (1) optimization of in vitro oocyte maturation, (2) parthenogenetic activation of zona-free oocytes, (3) SCNT of zona-free anaphase II-telophase II (AII-TII) oocytes that subverted the need for long term UV-exposure of the oocytes, and (4) in vitro culture of groups of cloned embryos in wells in a highly efficient continuous serum-free embryo medium to the blastocyst stage before transfer to the recipients. Percentages of transgenic blastocyst production were 22.3 and 33.1% for adult and fetal cell lines, respectively. After transfer of cloned and transgenic blastocysts, 28.6 and 36.4% of the recipients were confirmed pregnant and 75 and 33.3% of the pregnancies resulted in the delivery of viable offspring, respectively. To our knowledge, this is the first report of successful live and survived birth of cloned and transgenic offspring through a whole procedure of in vitro oocyte maturation and embryo development to the blastocyst stage, and in this study the in vitro efficiencies of cloned and transgenic embryo production were higher than the available reports.

Optimized combined electrical-chemical parthenogenetic activation for in vitro matured bovine oocytes.

Sperm-mediated oocyte activation is a complex procedure, both in steps and duration, not yet been completely mimicked during in vitro studies, e.g., parthenogenesis or somatic cell nuclear transfer. Furthermore, parthenogenetic studies have been recognized as a suitable model for studying activation efficiency for nuclear transfer cloning. This study, therefore, was conducted to develop an optimized artificial activation method, based on bovine cloning. In vitro matured bovine oocytes were initially exposed to electrical pulse, used for cell fusion during cloning, and then treated with 15 temporal sequential combinations of 3 chemical activators [calcium ionophore (CI), strontium (SR) and ethanol (ET)], followed by exposure to a protein kinase inhibitor or used for in vitro fertilization as control group. Treated and naturally fertilized oocytes were further cultured for up to 8 days. Embryo development was scored daily and blastocyst cell counting was carried out using differential staining at day 8 of culture. Among 15 temporal sequential combinations of three chemical activators, the best cleavage rates were associated with double (SR-CI, 84.4%), triple (CI-SR-ET, 79.4%) and single (CI, 73.7%) compounds, respectively, which were not significantly different with each other and with in vitro fertilized (IVF) (85.5%). The highest blastocyst rates were gained with ET-SR (24.5%), SR-CI-ET (20.4%) and CI (24.5%) accordingly which were not significantly different with each other but significantly lower than IVF (47%). Embryo cell counting further confirmed reasonably better quality of blastocysts produced using double, triple and single compounds. Although most of the sequential artificial activation compounds induced high cleavage rate, close to IVF, but this did not assure comparable further embryo development to the blastocyst stage. Nevertheless, the results suggest exposure of in vitro matured bovine oocytes to electrical pulse, followed by exposure to CI-6-dimethylaminopurine (6-DMAP) or ET-SR-6-DMAP could be regarded as the optimal artificial activation protocol for in vitro development of parthenogenic bovine oocytes or as a step for activation protocol in cloning procedure.