The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen-thawed, unsorted, and sex-sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P=0.09) or IGF2R genes (P=0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex-sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed. Mol. Reprod. Dev. 79:7784, 2012. (C) 2011 Wiley Periodicals, Inc.

Glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats

Background: Given the established fact that obesity interferes with male reproductive functions, the present study aimed to evaluate sperm production in the testis and storage in the epididymis in a glutamate-induced model of obesity. Methods: Male rats were treated neonatally with monosodium glutamate (MSG) at doses of 4 mg/kg subcutaneously, or with saline solution (control group), on postnatal days 2, 4, 6, 8 and 10. On day 120, obesity was confirmed by the Lee index in all MSG-treated rats. After this, all animals from the two experimental groups were anesthetized and killed to evaluate body and reproductive organ weights, sperm parameters, plasma hormone levels (FSH, LH and testosterone), testicular and epididymal histo-morphometry and histopathology. Results: Significant reductions in absolute and relative weights of testis, epididymis, prostate and seminal vesicle were noted in MSG-treated animals. In these same animals plasma testosterone and follicle-stimulating hormone (FSH) concentrations were decreased, as well as sperm counts in the testis and epididymis and seminiferous epithelium height and tubular diameter. The sperm transit time was accelerated in obese rats. However, the number of Sertoli cells per seminiferous tubule and stereological findings on the epididymis were not markedly changed by obesity. Conclusions: Neonatal MSG-administered model of obesity lowers sperm production and leads to a reduction in sperm storage in the epididymis of adult male rats. The acceleration of sperm transit time can have implications for the sperm quality of these rats.

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Primary antiphospholipid syndrome: morphofunctional penile abnormalities with normal sperm analysis

Objective: To perform a global gonadal and sexual functions assessment in primary antiphospholipid syndrome (PAPS) patients. Methods: A cross-sectional study was conducted in 12 male PAPS patients and 20 healthy controls. They were assessed by demographic data, clinical features, systematic urological examination, sexual function, testicular ultrasound, seminal parameters according to the World Health Organization (WHO), seminal sperm antibodies, and hormone profile, including follicle stimulating hormone (FSH), luteinizing hormone (LH), morning total testosterone, and thyroid hormones. Results: The median of current age and age of spermarche were similar in PAPS patients and controls (37.5 vs. 32.4 years, p = 0.270, and 13.1 vs. 12.85 years, p = 0.224, respectively), with a higher frequency of erectile dysfunction in the former group (25% vs. 0%, p = 0.044). Further analysis of PAPS patients with and without previous arterial thrombosis demonstrated that the median penis circumference was significantly lower in PAPS with arterial thrombosis than in PAPS without this complication (8.1 [6-10] vs. 10.2 [10-11] cm, p = 0.007). In addition, the median penis circumference was significantly lower in PAPS patients with erectile dysfunction than in patients without this complication (7.5 [6-9.5] vs. 9.5 [7.5-11] cm, p = 0.039). Regarding seminal analysis, the median sperm concentration, sperm motility, and normal sperm forms by WHO guidelines were comparable in PAPS patients and controls (141.5 [33-575] vs. 120.06 [34.5-329] x 106/ml, p = 0.65; 61.29 [25-80] vs. 65.42 [43-82]%, p = 0.4; 21.12 [10-42.5] vs. 23.95 [10-45]%, p = 0.45, respectively), and none of them had oligo/azoospermia. No differences were observed between PAPS patients and controls regarding the frequency of antisperm antibodies, testicular volume by ultrasound, or hormone profile (FSH, LH, morning total testosterone, and thyroid hormone) (p > 0.05). Conclusions: Normal testicular function has been identified in PAPS patients, in spite of morphofunctional penile abnormalities. Previous arterial thrombosis may underlie penile anthropometry alteration. Lupus (2012) 21, 251-256.

Validation of a new proposal of quantitative sperm culture for industrialized bull semen

The Brazilian Ministry of Agriculture (MAPA) has discussed the mandatory culture of industrialized semen, both to ensure biosefety, and to prevent in vitro fertilization problems caused by oocyte contamination with ubiquitous and opportunistic bacteria from preputial microbiota. Pour plate, a quantitative technique recommended by the World Organization for Animal Health (OIE), is operationally difficult and costly for routine analysis in Artificial Insemination Centers (AICs). The objectives of this study were to evaluate and validate viable superficial bacteria counts (VSBC), in CFU/mL, compared with pour plate counts, in industrialized bull semen samples from AICs. Semen straws from Projeto Hungria - MAPA bulls were used. VSBC and pour plate were carried out in parallel in serial dilutions of the samples, from 10(-1) to 10(-5). CFU/mL means or medians recorded in each dilution and technique were compared, and no statistical differences were observed between the two techniques regarding the quantification of bacteria in CFU/mL, suggesting that it may be possible to replace pour plate for CBSV, a cheaper and more practical technique.

The effect of ions and cryoprotectants upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus

The solutions commonly used to dilute or cryopreserve sperm are commonly composed of salts, buffers and cryoprotectants, which may affect gametes and subsequent fertilization success. Here, we have evaluated the effects of several cryoprotectants (methanol; MeOH, dimethyl sulfoxide; DMSO and dimethyl acetamide; DMA at concentrations of 0.25, 0.5 and 1%) and different ions (potassium, calcium and magnesium at concentrations of 1.25, 2.5, 5.0 and 10 mM) as sperm diluents upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus sperm. Our results demonstrated that DMSO (at 1%) decreased sperm motility while calcium and magnesium ions (from 2.5 mM) induced sperm aggregation and reduced sperm motility. Reduced fertilization rates were observed with potassium (from 1.25 mM), calcium (at 10 mM), magnesium (at 10 mM), DMA (at 1%), and DMSO (at 1%). We conclude that specific ions and cryoprotectants, and their relative concentrations caused effect upon loach gametes. These data are important to consider for the preparation of sperm diluents and activating solutions in order to manage gamete quality for artificial propagation. (C) 2012 Elsevier B.V. All rights reserved.

Assessment of field fertility and several in vitro sperm characteristics following the use of different Angus sires in a timed-AI program with suckled Nelore cows

In order to provide information that may help researchers to understand the main cause(s) of differences in bull fertility frequently observed in field trials, this study aimed to investigate conception rates as well as several in vitro sperm characteristics of different sires of unknown fertility utilized in a Timed-AI (TAI) program. Suckled Nelore cows submitted to the same TAI protocol were allocated into eight breeding groups of approximately 120 animals each. Frozen semen doses from three Angus bulls and three different batches from each bull were utilized. Approximately 100 doses from each batch were used in TAI. Sires, batches and AI technicians were equally distributed across breeding groups. Cows were examined for pregnancy diagnosis 40 d after TAI. For in vitro sperm analyses, the same thawing procedure was repeated in the laboratory to mimic field conditions. The following in vitro sperm characteristics were assessed: computerized motility, thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, morphology, morphometry and chromatin structure. No effect of breeding group, body condition score, AI technician and sire was observed. However, some significant differences among bulls were detected in laboratory analyses. Semen from sire presenting numerically lower (P > 0.05) pregnancy/AI also presented lower (P < 0.05) values in all sperm characteristics analyzed in thermal resistance test at 4 h (Total Motility, Progressive Motility, Average Path Velocity, Straight-Line Velocity, Curvilinear Velocity, Amplitude of Lateral Head Displacement, Beat Cross Frequency, Straightness, Linearity, and Percentage of Rapidly Moving Cells), higher (P < 0.05) Major and Total Defects in sperm morphological test, lower (P < 0.05) Length, Ellipticity and Fourier parameter (Fourier 0) in sperm morphometric analysis as well as higher (P < 0.05) chromatin heterogeneity. It was concluded that, although no bull effect was observed in the field experiment, the sire that presented numerically lower pregnancy/AI also presented lower semen quality according to the laboratory analyses performed. (C) 2012 Elsevier B.V. All rights reserved.

Impairment on sperm quality and fertility of adult rats after antiandrogen exposure during prepuberty

This study evaluated the effects of antiandrogen exposure during the prepubertal period on reproductive development and reproductive competence in adults. Male rats were divided into two groups: flutamide, receiving 25 mg/kg/day of flutamide by oral gavage and control, receiving vehicle daily. Dosing continued from PND 21 to 44, and animals were killed on PND 50 or PND 75-80. The epididymis, prostate, vas deferens and seminal vesicle weights were lower in Flutamide group on PND 50, while on PND 80 only seminal vesicle weight was reduced. Fertility assessed by IUI revealed a decrease in the fertility potential in the flutamide-treated adults. Flutamide accelerated sperm transit time through the epididymis, impairing sperm motility and storage. A quantitative analysis of the cauda sperm membrane proteome revealed a few significant changes in protein expression. Thus, exposure to flutamide during the prepubertal period compromises the function of the epididymis along with epididymal sperm quality at adulthood. (C) 2011 Elsevier Inc. All rights reserved.

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT cooling rate of -0.55 degrees C min-1 and freezing rate of -19.1 degrees C min-1) and automated (AT cooling rate of -0.23 degrees C min-1 and freezing rate of -15 degrees C min-1), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fishers test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 +/- 1.41% and 30.50 +/- 1.06%, with ethylene glycol was 21.17 +/- 1.66% and 21.67 +/- 1.13% and with dimethyl formamide was 8.33 +/- 0.65% and 9.17 +/- 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 +/- 1.49% (CT) and 15.83 +/- 1.26% (AT) to glycerol, 9.20 +/- 1.31% (CT) and 9.92 +/- 1.29% (AT) to ethylene glycol 4.65 +/- 0.93% (CT) and 5.17 +/- 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Effect of varicocele on sperm function and semen oxidative stress

OBJECTIVE To assess the effect of varicocele on sperm DNA integrity, mitochondrial activity, lipid peroxidation and acrosome integrity. PATIENTS AND METHODS In all, 30 patients with a clinically diagnosed varicocele of grade II or III and 32 men without a varicocele were evaluated for sperm DNA fragmentation (comet assay), mitochondrial activity (3,3'-diaminobenzidine assay), lipid peroxidation (malondialdehyde) and acrosome integrity (fluorescent probe labelled peanut agglutinin). RESULTS The varicocele group showed fewer spermatozoa with intact DNA (grade II, P = 0.040), more cells with inactive mitochondria (class III, P = 0.001), fewer cells with active mitochondria (class I, P = 0.005) and fewer spermatozoa with intact acrosomes (P < 0.001). Finally, no significant differences were observed in lipid peroxidation levels. CONCLUSION Men with varicocele showed an increase in sperm DNA fragmentation and a reduction in mitochondrial activity and acrosome integrity. However, lipid peroxidation levels remained unchanged.

Evaluation of Sperm DNA Peroxidation in Fertile and Subfertile Dogs

Contents Oxidative stress (OS) has been recognized as one of the most important causes of male infertility. The antioxidant activities of seminal plasma and epididymal fluid are not enough to prevent OS, which can damage sperm membranes and DNA, so antioxidant supplementation has been used as a treatment of male infertility. The aim of this experiment was to evaluate the DNA peroxidation before and after antioxidant supplementation with vitamin C and E in dogs with and without fertility problems. A total of eleven dogs were used and were divided in two groups: fertile group (G1), dogs with normal spermiogram (n=5); subfertile group (G2): dogs with low sperm count (<20x106sptz/ml) and/or more than 30% of total sperm pathology (n=6). Both groups received 500mg/day of vitamin C and 500mg/day of vitamin E for 60days. A semen sample was collected before (M1) and after (M2) oral supplementation. Samples were analysed for DNA peroxidation by measuring the 8-hydroxy-2'-deoxyguanosine concentration. No significant difference was observed between groups at either time. Oral supplementation with 500mg/day of vitamin C and 500mg/day of vitamin E did not change the DNA peroxidation in fertile and subfertile dogs.

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): a model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies. (c) 2012 Elsevier Inc. All rights reserved.

Meiotic segregation and interchromosomal effect in the sperm of a double translocation carrier: a case report

Abstract Background Infertility is a natural mechanism of selection intended to prevent the delivery of a child with malformations or mental retardation. Male infertility is closely related to chromosomal abnormalities. This study was focused on the analysis of meiotic segregation involving a Robertsonian translocation, 45,XY,der(13;13) [56]/45,XY,der(13;14) [44] and the evaluation of possible interchromosomal effects. Results Hybridisation with LSI 13q14 and subtelomere 14q probes and WCP13 SpectrumGreen and WCP14 SpectrumOrange probes showed a high proportion of unbalanced gametes, corresponding to 71.2% of the spermatozoa. The disomic frequencies of the sexual chromosomes and chromosome 18 of the patient were higher (5.28% and 2.55%, respectively) than those of the control (0.6% and 0.59%, respectively). Conclusion Meiotic segregation studies in sperm are an important tool for genetic counselling of chromosomal aberrations, allowing for a prediction of the risks and consequent implications for the reproductive life. The patient with this rare translocation exhibited meiotic segregation fidelity, and a high rate of unbalanced gametes with disomic spermatozoa.