Estudo sobre a redução do estresse oxidativo em sêmen equino a partir da adição de quercetina nos diluentes de refrigeração e congelação

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from MO were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5 degrees C under an atmosphere of 5% CO2 with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 +/- 4.8 and 57.3 +/- 4.8, respectively, mean +/- SEM), or among MO (62.3 +/- 5.7%), M3 (56.9 +/- 6.0%), and M6 (66.5 +/- 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture. (C) 2012 Elsevier Inc. All rights reserved.

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.

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.

Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)

(Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)). The objective of this work was to characterize the viability, production and morphology of pollen for different species in the genus Manihot. Floral buds from Manihot accessions were collected from two germplasm banks at Embrapa Cassava & Fruits. The viability of the pollen was assessed via colorimetric, in vitro and in vivo assays. The diameter of the pollen grains was determined by measuring the transversal length of the grain. The experimental design was entirely randomized. Studies on pollen ultrastructure were performed via scanning electron microscopy. Pollen viability was high in the colorimetric tests and intermediate in vivo tests; there was no germination in the in vitro tests. The average production for all accessions was 1,253 pollen grains per floral bud. The size of the pollen grains varied from 132 to 163 pm in the wild accessions, and 129 to 146 pm in the cultivated accessions. The pollen grains for all accessions were very large, apolar, spherical as well as inaperturate, with an exine ornamented with pila organized in a Croton pattern. The wild accessions, in general, produced more and larger pollen grains compared with the cultivated accessions.

Effects of 830 and 670 nm Laser on Viability of Random Skin Flap in Rats

Objective: This study aimed to investigate the effect of 830 and 670 nm diode laser on the viability of random skin flaps in rats. Background data: Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and reducing the inflammatory process after injury. However, the efficiency of such treatment remains uncertain, and there is also some controversy regarding the efficacy of different wavelengths currently on the market. Materials and methods: Thirty Wistar rats were used and divided into three groups, with 10 rats in each. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group, group 2 received 830 nm laser radiations, and group 3 was submitted to 670 nm laser radiation (power density = 0.5 mW/cm(2)). The animals underwent laser therapy with 36 J/cm(2) energy density (total energy = 2.52 J and 72 sec per session) immediately after surgery and on the 4 subsequent days. The application site of laser radiation was one point at 2.5 cm from the flap's cranial base. The percentage of skin flap necrosis area was calculated on the 7th postoperative day using the paper template method. A skin sample was collected immediately after to determine the vascular endothelial growth factor (VEGF) expression and the epidermal cell proliferation index (KiD67). Results: Statistically significant differences were found among the percentages of necrosis, with higher values observed in group 1 compared with groups 2 and 3. No statistically significant differences were found among these groups using the paper template method. Group 3 presented the highest mean number of blood vessels expressing VEGF and of cells in the proliferative phase when compared with groups 1 and 2. Conclusions: LLLT was effective in increasing random skin flap viability in rats. The 670 nm laser presented more satisfactory results than the 830 nm laser.

Fibers from fruit by-products enhance probiotic viability and fatty acid profile and increase CLA content in yoghurts

This study evaluated the effect of the supplementation of total dietary fiber from apple, banana or passion fruit processing by-products on the post-acidification, total titratable acidity, bacteria counts and fatty acid profiles in skim milk yoghurts co-fermented by four different probiotics strains: Lactobacillus acidophilus L10 and Bifidobacterium animalis subsp. lactis BL04, HN019 and B94. Apple and banana fibers increased the probiotic viability during shelf-life. All the fibers were able to increase the short chain and polyunsaturated fatty acid contents of yoghurts compared to their respective controls. A synergistic effect between the type of fiber and the probiotic strain on the conjugated linoleic acid content was observed, and the amount of alpha-linolenic acid was increased by banana fiber. The results of this study demonstrate, for the first time, that fruit fibers can improve the fatty acid profile of probiotic yoghurts and point out the suitability of using fibers from fruit processing the by-products to develop new high value-added fermented dairy products. (C) 2012 Elsevier By. All rights reserved.

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.

Influence of milk type and addition of passion fruit peel powder on fermentation kinetics, texture profile and bacterial viability in probiotic yoghurts

The effect of the addition of passion fruit peel powder (PFPP) on the fermentation kinetics and texture parameters, post-acidification and bacteria counts of probiotic yoghurts made with two milk types were evaluated during 28 days of storage at 4 degrees C. Milks were fermented by Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (CY340), and one strain of probiotic bacteria: Lactobacillus acidophilus (L10 and NCFM), Bifidobacterium animalis subsp. lactis (8104 and HN019). The addition of PFPP reduced significantly fermentation time of skim milk co-fermented by the strains L10, NCFM and HN019. At the end of 28-day shelf-life, counts of B. lactis Bl04 were about 1 Log CFU mL(-1) higher in whole yoghurt fermented with PFPP regarding its control but, in general, the addition of PFPP had less influence on counts than the milk type itself. The titratable acidity in yoghurts with PFPP was significantly higher than in their respective controls, and in skim yoghurts higher than in the whole ones. The PFPP increased firmness, consistency (except for the NCFM strain of L acidophilus) and cohesiveness of all skim yoghurts. The results point out the suitability of using passion fruit by-product in the formulation of both skim and whole probiotic yoghurts. (C) 2012 Elsevier Ltd. 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.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.