Determination of trace elements in bovine semen samples by inductively coupled plasma mass spectrometry and data mining techniques for identification of bovine class

The reproductive performance of cattle may be influenced by several factors, but mineral imbalances are crucial in terms of direct effects on reproduction. Several studies have shown that elements such as calcium, copper, iron, magnesium, selenium, and zinc are essential for reproduction and can prevent oxidative stress. However, toxic elements such as lead, nickel, and arsenic can have adverse effects on reproduction. In this paper, we applied a simple and fast method of multi-element analysis to bovine semen samples from Zebu and European classes used in reproduction programs and artificial insemination. Samples were analyzed by inductively coupled plasma spectrometry (ICP-MS) using aqueous medium calibration and the samples were diluted in a proportion of 1:50 in a solution containing 0.01% (vol/vol) Triton X-100 and 0.5% (vol/vol) nitric acid. Rhodium, iridium, and yttrium were used as the internal standards for ICP-MS analysis. To develop a reliable method of tracing the class of bovine semen, we used data mining techniques that make it possible to classify unknown samples after checking the differentiation of known-class samples. Based on the determination of 15 elements in 41 samples of bovine semen, 3 machine-learning tools for classification were applied to determine cattle class. Our results demonstrate the potential of support vector machine (SVM), multilayer perceptron (MLP), and random forest (RF) chemometric tools to identify cattle class. Moreover, the selection tools made it possible to reduce the number of chemical elements needed from 15 to just 8.

Time sequence of the intensification of the liver glucose production induced by high-fat diet in mice

It is well established that the development of insulin resistance shows a temporal sequence in different organs and tissues. Moreover, considering that the main aspect of insulin resistance in liver is a process of glucose overproduction from gluconeogenesis, we investigated if this metabolic change also shows temporal sequence. For this purpose, a well-established experimental model of insulin resistance induced by high-fat diet (HFD) was used. The mice received HFD (HFD group) or standard diet (COG group) for 1, 7, 14 or 56?days. The HFD group showed increased (P?<?0.05 versus COG) epididymal, retroperitoneal and inguinal fat weight from days 1 to 56. In agreement with these results, the HFD group also showed higher body weight (P?<?0.05 versus COG) from days 7 to 56. Moreover, the changes induced by HFD on liver gluconeogenesis were progressive because the increment (P?<?0.05 versus COG) in glucose production from l-lactate, glycerol, l-alanine and l-glutamine occurred 7, 14, 56 and 56 days after the introduction of the HFD schedule, respectively. Furthermore, glycaemia and cholesterolemia increased (P?<?0.05 versus COG) 14?days after starting the HFD schedule. Taken together, the results suggest that the intensification of liver gluconeogenesis induced by an HFD is not a synchronous all-or-nothing process but is specific for each gluconeogenic substrate and is integrated in a temporal manner with the progressive augmentation of fasting glycaemia. Copyright (c) 2012 John Wiley & Sons, Ltd.

Heterogeneous time-dependent response of adipose tissue during the development of cancer cachexia

Cancer cachexia induces loss of fat mass that accounts for a large part of the dramatic weight loss observed both in humans and in animal models; however, the literature does not provide consistent information regarding the set point of weight loss and how the different visceral adipose tissue depots contribute to this symptom. To evaluate that, 8-week-old male Wistar rats were subcutaneously inoculated with 1 ml (2 x 10(7)) of tumour cells (Walker 256). Samples of different visceral white adipose tissue (WAT) depots were collected at days 0, 4, 7 and 14 and stored at -80 degrees C (seven to ten animals/each day per group). Mesenteric and retroperitoneal depot mass was decreased to the greatest extent on day 14 compared with day 0. Gene and protein expression of PPAR gamma(2) (PPARG) fell significantly following tumour implantation in all three adipose tissue depots while C/EBP alpha (CEBPA) and SREBP-1c (SREBF1) expression decreased over time only in epididymal and retroperitoneal depots. Decreased adipogenic gene expression and morphological disruption of visceral WAT are further supported by the dramatic reduction in mRNA and protein levels of perilipin. Classical markers of inflammation and macrophage infiltration (f4/80, CD68 and MIF-1 alpha) in WAT were significantly increased in the later stage of cachexia (although showing a incremental pattern along the course of cachexia) and presented a depot-specific regulation. These results indicate that impairment in the lipid-storing function of adipose tissue occurs at different times and that the mesenteric adipose tissue is more resistant to the 'fat-reducing effect' than the other visceral depots during cancer cachexia progression. Journal of Endocrinology (2012) 215, 363-373

Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance

Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high-caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high-caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin-induced glucose uptake in 3T3-L1 adipocytes.

Grade 3 Varicocele in Fertile Men: A Different Entity

Purpose: Although varicocele size has an inverse relationship with baseline semen parameters and a direct relationship with seminal reactive oxygen species in infertile patients, to our knowledge the effect of varicocele grade in fertile men is unknown. We evaluated the impact of varicocele grade on seminal parameters, testicular size and seminal reactive oxygen species in fertile men. Materials and Methods: We prospectively evaluated 194 men from July 2004 to April 2010. Of the men 156 were fertile and classified by presence of varicocele. A total of 38 infertile patients with varicocele as the only identifiable cause of infertility comprised the control group. Physical examination, semen parameters and seminal reactive oxygen species were compared between the groups. Results: Of 156 fertile men 43 (24.3%) had clinical varicocele, which was grade 1 to 3 in 22, 11 and 10, respectively. The remaining 113 men (72.7%) had no varicocele. Infertile men had smaller testes, decreased semen parameters and higher seminal reactive oxygen species than the fertile groups. Testicular size, reactive oxygen species and semen parameters did not differ between fertile men with vs without varicocele. Fertile men with varicocele grade 3 had higher seminal reactive oxygen species than those with lower grade varicocele. As varicocele grade increased, seminal reactive oxygen species increased and sperm concentration decreased. Conclusions: Although fertile men have more efficient defense mechanisms to protect against the consequences of varicocele on testicular function, these mechanisms may not be sufficient in those with varicocele grade 3. Further research is needed to clarify whether they are at increased risk for future infertility.

A novel spermatozoan ultrastructure in the shrimp Hippolyte obliquimanus Dana, 1852 (Decapoda: Caridea: Hippolytidae)

The aim of this study was to describe and illustrate the morphology of the spermatozoon of the Western Atlantic shrimp, Hippolyte obliquimanus. Individuals were sampled from Itagua Beach (Ubatuba, southern Brazil). The male reproductive system was dissected and morphological analysis was undertaken using a stereomicroscope, a light microscope, and transmission electron and scanning electron microscopes. When viewed from the nuclear or acrosomal poles, each spermatozoon has many translucent radiating arms (about 20) from a denser cell body, while laterally the cell body and arms resemble a "cnidarian medusa", with all the arms projecting away from the bell-like cell body. This sperm morphology is distinct from the "thumbtack"-shaped spermatozoa observed in the majority of carideans but has similarities to the spermatozoa of Rhynchocinetes spp. The morphology of sperm of several species of the genus Hippolyte resembles the spermatozoon of H. obliquimanus with the presence of posterior nuclear arms, but it is necessary to study other Hippolyte species to place these arms in the context of the genus.

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.

Development of spermatogenesis in captive-bred Spix's yellow-toothed cavy (Galea spixii)

The aim of this study was to evaluate the phases of sexual development and spermatogenesis of Spix's yellow-toothed cavy (Galea spixii) based on analyses of the structural components of the testes. The testes of animals from 0 to 150 days of age were collected by orchiectomy, weighed, and processed for analysis by light microscopy. At 45 days of age, spermatozoa were seen in the tubular lumen. Spermatogenesis was not established in animals from 45 to 150 days of age. The stages of sexual development may be classified into the following phases: from birth to the age of 15 days (immature); 30 days of age (prepubertal); 45-105 days of age (pubertal); and 120 and 150 days of age (postpubertal). This is the first study to address the male reproductive biology of Spix's yellow-toothed cavy.

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.

Effect of sequence of insemination after simultaneous thawing of multiple semen straws on conception rate to timed AI in suckled multiparous Nelore cows

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 degrees C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated. (C) 2012 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.

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.

Quantitative chemical profile and multivariate statistical analysis of alembic distilled sugarcane spirit fractions

As concentrações de 39 compostos orgânicos foram determinadas em três frações (cabeça, coração e cauda) obtidas da destilação em alambique do caldo de cana fermentado. Os resultados foram avaliados utilizando-se análise de variância (ANOVA), teste de Tukey, análise de componentes principais (PCA), agrupamento hierárquico (HCA) e análise discriminante linear (LDA). De acordo com PCA e HCA, os dados experimentais conduzem à formação de três agrupamentos. As frações de cabeça deram origem a um grupo mais definido. As frações coração e cauda apresentaram alguma sobreposição coerente com sua composição em ácidos. As habilidades preditivas de calibração e validação dos modelos gerados pela LDA para a classificação das três frações foram de 90,5 e 100%, respectivamente. Este modelo reconheceu como coração doze de treze cachaças comerciais (92,3%) com boas características sensoriais, apresentando potencial para a orientação do processo de cortes.