Carcass and meat quality of young Angus steers with different growth potential finished exclusively grass-fed or corn supplemented.

Beef cattle production has a large variety of systems with different performance and technology levels. This study compared young Angus steers carcass and meat quality traits of high growth potential early-finished animals fed exclusively on pasture with low growth potential late-finished animals. Besides the grazed forage, the low growth potential group received corn grain at 0.8% of their body weight right after the slaughtering of the first group. Following weaning, the 20 steers grazed pastures composed of oat, ryegrass, and clovers. At winter's onset, animals were moved to native pastures improved with fescue and clovers. The supplemented period of late steers began when the first group of ten early-finished animals reached the requirements for slaughter. Supplementation ended when the animals got the slaughter requirements. The high growth potential steers showed a ribeye area (P < 0.0001) and a Longissimus muscle width highly significantly (P < 0.0001) superior to the low growth group. However, corn-supplemented animals showed better dressing percentage (P < 0.0001), subcutaneous fat thickness (P < 0.0001), marbling (P < 0.0001), and muscle (P = 0.0033) scores, but lower shear force (P = 0.0001). The finishing system did impact fat lightness (L*; P = 0.0234) at the slaughter time. Grass-fed animals showed higher red-green color parameter (a*) values than corn supplemented on fat at 24 h (P = 0.0439) but lower hue angle (P = 0.0418). The consumer panel showed better results for tenderness and general acceptability for supplemented animals beef compared to grass-fed. The supplementation resulted in a higher carcass standardization. It also provided higher PUFA: MUFA and n-6: n-3 ratios, and a higher amount of n-3. Supplemental corn grain did not have adverse effects upon the beneficial effects to human health of the grass-fed beef.

Activation of the Wnt/ß-catenin pathway in pancreatic beta cells during the compensatory islet hyperplasia in prediabetic mice.

The Wnt/ß-catenin signaling pathway, also known as the canonical Wnt pathway, plays a role in cell proliferation and differentiation in several tissues/organs. It has been recently described in humans a relationship between type 2 diabetes (T2DM) and mutation in the gene encoding the transcription factor TCF7L2 associated to the Wnt/ß-catenin pathway. In the present study, we demonstrated that hyperplastic pancreatic islets from prediabetic mice fed a high-fat diet (HFD) for 60 d displayed nuclear translocation of active ß-catenin associated with significant increases in protein content and gene expression of ß-catenin as well as of cyclins D1, D2 and c-Myc (target genes of the Wnt pathway) but not of Tcf7l2 (the transcription factor). Meanwhile, these alterations were not observed in pancreatic islets from 30 d HFD-fed mice, that do not display significant beta cell hyperplasia. These data suggest that the Wnt/ß-catenin pathway is activated in pancreatic islets during prediabetes and may play a role in the induction of the compensatory beta cell hyperplasia observed at early phase of T2DM.

Selection of red deer spermatozoa with different cryoresistance using density gradients.

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa.

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 µM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2 DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO-PRO-1+ and acrosome-reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca(2+) levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.

The Effect of Oxidative Stress on Thawed Bulk-Sorted Red Deer Sperm.

The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0 mm = H000; H2 O2 50 mm = H050; H2 O2 100 mm = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H2 O2 increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.

Experimental and theoretical investigations of electronic structure and photoluminescence properties of ß-Ag2MoO4 microcrystals.

In this paper, we investigate a correlation between theoretical calculations and experimental data to explain the electronic structure and optical properties of silver molybdate (ß-Ag2MoO4) microcrystals synthesized by the microwave-assisted hydrothermal method. X-ray diffraction, Rietveld refinement, and micro-Raman spectroscopy confirmed that these microcrystals crystallize in a spinel-type cubic structure. Field-emission scanning electron microscopy images revealed that the processing temperatures influence in the final shape of microcrystals. Optical properties were analyzed by ultraviolet-visible diffuse reflectance spectroscopy; the increase in the optical band gap energy (Egap) (from 3.24 to 3.31 eV) with processing temperature is associated with the reduction of intermediary energy levels. First-principles quantum mechanical calculations based on the density functional theory at the B3LYP level were conducted. The calculated band structure revealed an indirect Egap of approximately 4.00 and 3.34 eV for the ß-Ag2MoO4 without and with the formation of defects, respectively. Theoretical calculations based on density of states and electron density maps were employed to understand the polarization phenomenon induced by structural defects in the ß-Ag2MoO4 crystals. Finally, photoluminescence properties at room temperature of ß-Ag2MoO4 microcrystals were explained by the charge-transfer mechanism involving tetrahedral [MoO4] clusters.

Understanding sperm heterogeneity: biological and practical implications.

Sperm are the most diverse cell type known. This diversity is thought to reflect adaptation to conditions under which sperm function as a way to ensure the survival of sperm in fertilization environments and to maximize fertilizing capacity thereof. The existence of morphological diversity among species is widely assumed, although this diversity seems less clear as we go deeper (between males, between ejaculates from the same male and even within the same ejaculate), with different theories addressing this heterogeneity. Moreover, the development of assisted reproductive techniques (ART) has led to changes in the physiological conditions in which sperm fertilize, which could lead, ultimately, to a selection towards more favourable sperm design. Regardless of the origin of this diversity, when studying the relationship between shape and function of sperm, it is advisable to assess the degree of heterogeneity of sperm and takes into account to be more likely to identify those morphological characteristics determining the fertile ability of sperm. Otherwise, these relationships could be hidden as a result of considering an average shape not representative of morphological characteristics of sperm. In addition, the knowledge of this morphological diversity in terms of changes arising from modifications in the sperm environment and mechanisms that generate these changes could be useful for understanding the reproductive capacity of males but also in enhancing their fertile ability.

Impaired post exercise heart rate recovery in anabolic steroid users.

Previous study showed that muscle sympathetic nerve activity (MSNA) was augmented in anabolic steroids users (AASU). In the present study, we tested the hypothesis that the heart rate (HR) responses after maximal exercise testing would be reduced in AASU. 10 male AASU and 10 AAS nonusers (AASNU) were studied. Cardiopulmonary exercise was performed to assess the functional capacity and heart rate recovery. MSNA was recorded directly from the peroneal nerve by microneurography technique. Peak oxygen consumption (VO2) was lower in AASU compared to AASNU (43.66±2.24 vs. 52.70±1.68 ml/kg/min, P=0.005). HR recovery (HRR) at first and second minute was lower in AASU than AASNU (21±2 vs. 27±2 bpm, P=0.02 and 37±4 vs. 45±2 bpm, P=0.05, respectively). MSNA was higher in AASU than AASNU (29±3 vs. 20±1 bursts/min, P=0.01). Further analysis showed a correlation between HRR and MSNA (r=- 0.64, P=0.02), HRR at first minute and peak VO2 (r=0.70, P=0.01) and HRR at second minute and peak VO2 (r=0.62, P=0.02). The exacerbated sympathetic outflow associated with a lower parasympathetic activation after maximal exercise, which impairs heart rate recovery, strengthens the idea of autonomic imbalance in AASU.

Characterization and distribution of Teredinidae assemblage in an estuary in Ceará, Brazil's Northeast.

Teredinids are bivalves mollusks considered the most abundant of invertebrates group of marine wood borers performing an important role in the mangrove environment. This study aimed to characterize the Teredinidae species from the Acaraú River estuary in Ceará and analyse the relationship between the mangrove plant structure and the distribution of Teredinidae, according to gradients estuaries: vertical (flooding) and horizontal (salinity). The collection of mangrove logs with Teredinidae happened in three places within the estuary (inner, median, and upper); in each area, three transects were traced in which three plots were lined off, and a total of 40 logs were collected. Teredinidae species were found and identified: Nausitora fusticula; Neoteredo reynei; Teredo turnerae; Teredo cf. bartschi; Bankia bipennata; Bankia gouldi; Lirodus massa and Lyrodus cf. bipartitus. The Lyrodus cf. bipartitus, Bankia gouldi, and Teredo cf. bartschi species were registered for the first time in Ceará. The distribution and species richness of Teredinidae were directly related to the vertical gradient (flooding) and heterogeneity of the mangrove forest habitat. The data presented here are essential for comprehending the mechanisms responsible for the distribution patterns of the Teredinidae species in the mangrove, contributing to biodiversity conservation in Ceará coastal zones.

Molecular analysis of the full-length VP2 gene of Brazilian strains of canine parvovirus 2 shows genetic and structural variability between wild and vaccine strains.

Canine parvovirus 2 (CPV-2), a highly contagious virus, affects dogs worldwide. Infected animals present severe and acute gastroenteritis which may culminate in death. CPV-2 VP2 protein is responsible for important biological functions related to virus-host interactions. Herein we obtained VP2 full-length gene sequences from Brazilian dogs with bloody diarrhea (n=15) and vaccine strains (n=7) produced by seven different laboratories and marketed in Brazil. All wild sequences and one vaccine strain were classified as CPV-2b and six vaccines were the classic CVP-2. Mutations in VP2 protein from vaccine and wild strains obtained in Brazil and worldwide were analyzed (n=906). Amino acid sequences from vaccine strains remarkably diverge from each other, even that classic CPV-2. Phylogenetic analysis based on VP2 gene and conducted with sequences displaying mutations in epitope regions previously described shows that vaccine strains are distantly related from the wide range of wild CPV-2. The impact of amino acid mutations over VP2 protein structure shows that vaccine and wild strains obtained in this study diverge in loop 3, an epitope region that plays a role in the CPV-2 host range. This is the first analysis of CPV-2 VP2 from commercial vaccine strains in Brazil and wild ones from Minas Gerais State, Brazil, and the first detailed attempt to vaccinal VP2 molecular and structural analyses.

Sida cordifolia leaf extract reduces the orofacial nociceptive response in mice.

In this study, we describe the antinociceptive activity of the ethanol extract (EE), chloroform (CF) and methanol (MF) fractions obtained from Sida cordifolia, popularly known in Brazil as "malva branca" or "malva branca sedosa". Leaves of S. cordifolia were used to produce the crude ethanol extract and after CF and MF. Experiments were conducted on Swiss mice using the glutamate and formalin-induced orofacial nociception. In the formalin test, all doses of EE, CF and MF significantly reduced the orofacial nociception in the first (p < 0.001) and second phase (p < 0.001), which was also naloxone-sensitive. In the glutamate-induced nociception test, only CF and MF significantly reduced the orofacial nociceptive behavior with inhibition percentage values of 48.1% (100 mg/kg, CF), 56.1% (200 mg/kg, CF), 66.4% (400 mg/kg, CF), 48.2 (200 mg/kg, MF) and 60.1 (400 mg/kg, MF). Furthermore, treatment of the animals with EE, CF and MF was not able to promote motor activity changes. These data demonstrate that S. cordifolia has a pronounced antinociceptive activity on orofacial nociception. However, pharmacological and chemical studies are necessary in order to characterize the responsible mechanisms for this antinociceptive action and also to identify other bioactive compounds present in S. cordifolia.

Response of thawed epididymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability.

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H(2)O(2)) levels on red deer spermatozoa after cryopreservation, and the role of male-to-male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 µm H(2)O(2) for 2 h at 37 °C. Intracellular reactive oxygen species (H(2)DCFDA-CM) increased with H(2)O(2) concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 µm. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 µm H(2)O(2) and above. In a second experiment, samples from seven males were submitted to 0 and 200 µm H(2)O(2) for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H(2)O(2) presence. We found that the kinematic parameters reflected male-to-male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H(2)O(2) or after incubation with H(2)O(2) . Red deer spermatozoa are relatively resilient to H(2)O(2) after thawing, but it seems to be a great male-to-male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.

Screening of Jatropha curcas genotypes to anthracnose caused by Colletotrichum gloeosporioides.

Anthracnose caused by Colletotrichum gloeosporioides is an important physic nut (Jatropha curcas) disease causing damages on leaves, stems and fruits and consequently a decrease in seed quantity and quality. Physic nut is a native plant of Central America that has gained international attention due to its potential as biodiesel crop. However despite its highest relevance, studies concerning the response of different Jatropha genotypes to infection with this disease are very limited. The main objective of this study is the evaluation of susceptibility of 38 Jatropha curcas genotypes from a worldwide germplasm collection to Colletotrichum gloeosporioides. A simple method using wounded detached leaves inoculated with a 5-mm mycelia plug was used. Observations (lesion diameter and % of infection) were made after 3 and 7 days under controlled conditions (25 degrees C and 85% RH). Results showed significantly differences (P < or = 0.001) in both percentage of infection and lesion diameter among the tested accessions. Two genotypes from Brazil and Cape Verde, presented no lesions after 7 days of treatment, while the others presented between 67 and 100% of infection. Lesion diameter showed an increase with time of incubation and proved to be a useful tool for evaluation of plant susceptibility.

Comparison of the TBARS assay and BODIPY C11 probes for assessing lipid peroxidation in red deer spermatozoa.

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C(11) (B581) and BODIPY 665/676 C(11) (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H(2)O(2) ) 0.1 mM or 1 mM, or tert-butyl hydroperoxide (TBH) 0.1 mM or 1 mM. LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mM of TBH or H(2)O(2). Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.

Nanotechnology in reproduction: Vitamin E nanoemulsions for reducing oxidative stress in sperm cells.

Vitamin E is considered a powerful biological antioxidant; however, its characteristics such as high hydrophobicity and low stability limit its application. We propose to use nanotechnology as an innovative tool in spermatology, formulating nanoemulsions (NE) that accommodate vitamin E, protecting it from oxidation and promoting its release into the medium. The protective effect of the NE against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from eleven stags were thawed and extended to 400 × 106 sperm/ml in Bovine Gamete Medium (BGM). Once aliquoted, the samples were supplemented with the NE at different concentrations (0, 6 and 12 mM), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. Motility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) were assessed. After 2 and 4 h of incubation, the NE were able to prevent the deleterious effects of oxidative stress, thus improving total and progression motility (P ˂0.05). Moreover, the highest concentration tested (12 mM) improved almost every sperm kinematic variable (P ˂0.05) and preserved sperm viability in samples subjected to oxidative stress. In addition, 12 mM of NE protected the acrosomes integrity, maintained and protected mitochondrial activity, prevented sperm lipoperoxidation and reduced ROS production (P ˂0.05) in samples subjected to oxidative stress. This work indicates for the first time that vitamin E formulated in NE could be a new approach against sperm oxidative damage. This could be highly relevant for sperm physiology preservation in the context of assisted reproduction techniques.

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity.

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 degrees C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 microm Fe(2+)/1 mm ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 +/- 2.9%) and OXI (11.6 +/- 7.6%) (p < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI (p < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 +/- 0.8% OXI vs. 17.4 +/- 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.

Heterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa.

A heterologous in vitro fertilization (IVF) test using calf oocytes with zona pellucida was employed to assess the fertility of thawed ram sperm samples. Six males with significant differences in fertility (P=0.003) were used. The males were classified as having high fertility (>or=42%) and low fertility (

Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation.

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 x 10(6) sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 mm vitamin C. The remaining epididymides and the diluted samples were stored at 5 degrees C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.

Identification of sperm-head morphometric subpopulations in iberian red deer epididymal sperm samples.

Computer-assisted sperm morphometry analysis (CASMA) was used in this study to identify sperm morphometric subpopulations in Iberian red deer epididymal sperm samples. Epididymal sperm samples were collected from 37 mature stags and were divided. One portion was diluted in a Tris-citrate-egg yolk medium. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours using a conventional protocol. After thawing, sperm smears were prepared as described for extended samples. All slides were air-dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 145 sperm-heads were analyzed from each sample by means of the Sperm-Class Analyser, and the mean measurements recorded. Each sperm-head was measured for four primary sperm-head parameters, and five parameters of head shape. All sperm morphometric parameters evaluated were placed in a statistical database and a multivariate cluster analysis was performed. The clustering analyses, based on 10 867 individual spermatozoa, revealed the existence of three subpopulations (SP(1), SP(2), SP(3)) of spermatozoa with different morphometric characteristics (p < 0.001). The proportion of spermatozoa present in any of the three subpopulations remained constant (p > 0.05) through the cryopreservation process. Pre-freeze and post-thaw sperm quality was in vitro evaluated by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. In conclusion, our results show that applying the CASMA techniques and multivariate cluster analyses, it was possible to determine that three subtle subpopulations of spermatozoa with different morphometric characteristics coexist in red deer semen.

DNA status on thawed semen from fighting bull: a comparison between the SCD and the SCSA tests.

The assessment of sperm chromatin status is compulsory in a complete spermiogram. Here we applied the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test to assess the chromatin status of three fighting bulls. Cryopreserved semen (two straws/bull) were analysed by duplicate after thawing and after 6 h at 37 degrees C with and without oxidative stress (1 mm FE(2+)). Results (SCD: percentage of spermatozoa with halo; SCSA: SD-DFI, %DFI and HDS) were analysed for differences between bulls and treatments, sensitivity and specificity (receiver operating characteristic curves) and repeatability (repeatability coefficients as 2SD of duplicate differences).%DFI for the three bulls was below 2% at 0 h, indicating no risk for fertility according to previous reports. It increased slightly for two of the bulls after FE(2+) treatment (%DFI < 5%) and more pronouncedly for the other bull (C, %DFI approximately 10%), which merits further investigation. SCD rendered higher percentage of halos for bull C, but could not discriminate between samples with and without oxidizing treatment (AUC: 0.52). SCSA (%DFI) showed a high discriminating ability between treatments (AUC: 0.96). The repeatability coefficient was also higher for SCD (5.9) than for %DFI (1.8), indicating lower repeatability for SCD. Overall, %DFI might be the most useful parameter for assessing sperm chromatin on fighting bull. SCD might yield different information than SCSA, hence further research is warranted.