Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa

P. 1073-1084The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 × g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1 mM of the antioxidant Trolox, 100 μM of the oxidant Fe (with ascorbate), or both. The 3 × 4 treatments were incubated at 37°C and assessed each hour up to 3 h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4 h was assessed using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30 ± 3.52% to 67.94 ± 5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.S

Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa

P. 225–235Fe2+/ascorbate, hydrogen peroxide (H2O2), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 μM Fe2+ (hydroxyl radical generator); 1 mM, 100, and 10 μM H2O2; and 100, 10, and 1 mU/ml XOD (superoxide and H2O2 generator), incubated at 37 °C for 180 min. Intracellular reactive oxygen species (ROS; H2DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Δψm) were considerably decreased by H2O2 (1 mM and 100 μM) and XOD (100 and 10 mU/ml). Only 1 mM H2O2 reduced viability. The antioxidant Trolox (10 μM) reduced intracellular ROS, but could not prevent the H2O2 or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H2O2 increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Δψm loss, confirming that H2O2 was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H2O2 increased their proportion after 60 min. There were important differences between ROS generators, H2O2 being the most cytotoxic. Although H2O2 and XOD caused Δψm dissipation, this was not reflected in increasing apoptotic markers.S

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

P. 160-168The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.S

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

P. 1661-1672Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen–thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.S

Sperm Cell Population Dynamics in Ram Semen during the Cryopreservation Process

[EN] Background: Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. Methodology/Principal Findings: We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. Conclusions/Significance: Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.SIThis work was supported by the Education and Science Council of Castilla-La Mancha (PCC08-0105-8042). Manuel Ramón was supported by the DOC- INIA program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A colorectal cancer genome-wide association study in a Spanish cohort identifies two variants associated with colorectal cancer risk at 1p33 and 8p12

Background: Colorectal cancer (CRC) is a disease of complex aetiology, with much of the expected inherited risk being due to several common low risk variants. Genome-Wide Association Studies (GWAS) have identified 20 CRC risk variants. Nevertheless, these have only been able to explain part of the missing heritability. Moreover, these signals have only been inspected in populations of Northern European origin. Results: Thus, we followed the same approach in a Spanish cohort of 881 cases and 667 controls. Sixty-four variants at 24 loci were found to be associated with CRC at p-values <10-5. We therefore evaluated the 24 loci in another Spanish replication cohort (1481 cases and 1850 controls). Two of these SNPs, rs12080929 at 1p33 (Preplication=0.042; Ppooled=5.523x10-03; OR (CI95%)=0.866(0.782-0.959)) and rs11987193 at 8p12 (Preplication=0.039; Ppooled=6.985x10-5; OR (CI95%)=0.786(0.705-0.878)) were replicated in the second Phase, although they did not reach genome-wide statistical significance. Conclusions: We have performed the first CRC GWAS in a Southern European population and by these means we were able to identify two new susceptibility variants at 1p33 and 8p12 loci. These two SNPs are located near the SLC5A9 and DUSP4 loci, respectively, which could be good functional candidates for the association signals. We therefore believe that these two markers constitute good candidates for CRC susceptibility loci and should be further evaluated in other larger datasets. Moreover, we highlight that were these two SNPs true susceptibility variants, they would constitute a decrease in the CRC missing heritability fraction

BMP2/BMP4 colorectal cancer susceptibility loci in northern and southern european populations

Genome-wide association studies have successfully identified 20 colorectal cancer susceptibility loci. Amongst these, four of the signals are defined by tagging single nucleotide polymorphisms (SNPs) on regions 14q22.2 (rs4444235 and rs1957636) and 20p12.3 (rs961253 and rs4813802). These markers are located close to two of the genes involved in bone morphogenetic protein (BMP) signaling (BMP4 and BMP2, respectively). By investigating these four SNPs in an initial cohort of Spanish origin, we found substantial evidence that minor allele frequencies (MAFs) may be different in northern and southern European populations. Therefore, we genotyped three additional southern European cohorts comprising a total of 2028 cases and 4273 controls. The meta-analysis results show that only one of the association signals (rs961253) is effectively replicated in the southern European populations, despite adequate power to detect all four. The other three SNPs (rs4444235, rs1957636 and rs4813802) presented discordant results in MAFs and linkage disequilibrium patterns between northern and southern European cohorts. We hypothesize that this lack of replication could be the result of differential tagging of the functional variant in both sets of populations. Were this true, it would have complex consequences in both our ability to understand the nature of the real causative variants, as well as for further study designs

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the iberian red deer

Using Iberian red deer as model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to 6 stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, this data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a ierarchical and a multi-step clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing por freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.This work was supported by the Education and Science Council (PBI-05-011), by the Spanish Ministry of Education and Science (RZ2006-00006-C3), and by the Agriculture Council (PREG-05-004) of Junta de Comunidades de Castilla-La Mancha (JCCM). Olga García-Álvarez and Alejandro Maroto Morales were recipients of scholarships from INIA and JCCM, respectively. Felipe Martínez-Pastor, María Rocío Fernández-Santos, and Milagros C. Esteso were supported by the Juan de la Cierva program from the Spanish Ministry of Education and Science.Peer reviewe