Neat Semen Samples Research Articles

Viability Tests, Active Caspase‐3 and −7, and Chromatin Structure in Ram Sperm Selected Using the Swim‐Up Procedure

Swim-up (SU) is a technique that permits the obtaining of motile sperm. Based on the sperm motility of neat ejaculates, we analyzed functional parameters, cytoplasmic esterases, and mitochondrial dehydrogenases of ram sperm using calcein acetomethylester and resazurin, respectively. Active caspase-3 and -7 and chromatin structure were evaluated in ram sperm before and after the SU process. There were no changes in any of the viability parameters after SU in neat semen samples with less or more than 25% motility. The percentage of active caspase-3 and -7 decreased after SU (68.8 +/- 4.6 vs 54.2 +/- 6.0), whereas a small but significant increase of chromatin structural abnormalities was observed (DNA fragmentation index [DFI], 287.3 +/- 3.1 vs 297.2 +/- 2.4). For the first time, the location of active caspase-3 and -7 was described for ram sperm. Notably, we found active caspases in the implantation fossa region. The presence of active caspases in neat ejaculates and the diminished presence of active caspases in SU-processed ejaculates suggest a role for caspases in motility and possibly in male fertility. The results of this study indicate that the evaluation of more than one cell-function marker is necessary to appropriately evaluate sperm quality. Furthermore, in semen samples with low motility, a lower percentage of sperm with active caspases is obtained after SU, although these sperm present increased values of DFI.

Effects of cryopreservation on human sperm deoxyribonucleic acid integrity

To evaluate the effect of cryopreservation on sperm motility and viability and to assess sperm DNA fragmentation and oxidation in men undergoing infertility investigation before and after cryopreservation in liquid nitrogen. Analysis of cryopreservation effects on sperm DNA integrity. Laboratory of Histology-Embryology of medicine faculty, Sfax, Tunisia. Fifteen semen samples from men undergoing infertility investigation. Neat semen samples were cryopreserved in liquid nitrogen using a commercial freezing medium (SpermFreeze, Fertipro, Belgium) according to the manufacturer's instructions. Samples were thawed at room temperature. Sperm DNA fragmentation was assessed using terminal deoxynucleotidyl transferase (Tdt) mediated dUTP nick end labeling and sperm DNA oxidation was determined using a fluorescent assay (OxyDNA test) for the detection of 8-oxoguanine. Evaluation of DNA fragmentation and oxidation rates was carried out before and after cryopreservation using flow cytometry. A significant decrease in sperm motility and viability was observed after cryostorage. In addition, sperm DNA fragmentation and DNA oxidative damage increased significantly after cryopreservation/thaw. Cryopreservation has deleterious effects on sperm DNA by inducing DNA fragmentation and oxidation but the mechanisms underlying such damages need to be elucidated by further investigations.

Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men

To define physiologic levels of reactive oxygen species in infertile men and establish a cutoff value of reactive oxygen species level in neat semen with a high sensitivity and specificity to differentiate infertile men from fertile donors (controls). Reactive oxygen species levels were measured in the neat semen samples (n = 51) from fertile donors and infertile patients (n = 54). Reproductive research laboratory at a tertiary care hospital. Infertile patients from male infertility clinic. Reactive oxygen species measurement in neat semen sample using luminol-based chemiluminescence method, receiver operating characteristic curves. Seminal reactive oxygen species levels, cutoff value, sensitivity and specificity, positive and negative predictive values. The best cutoff value to distinguish between healthy fertile donors and infertile men was 0.0185 x 10(6) counted photons per minute/20 x 10(6) sperm. At this threshold, the specificity was 82% and the sensitivity was 78%. This value can be defined as basal reactive oxygen species level in infertile men. Reactive oxygen species levels in neat semen samples as measured by luminol-based chemiluminescence are a highly specific and sensitive test in the diagnosis of infertility. This test also may help clinicians treat patients with seminal oxidative stress.

Effects of radiofrequency electromagnetic waves (RF-EMW) from cellular phones on human ejaculated semen: an in vitro pilot study

To evaluate effects of cellular phone radiofrequency electromagnetic waves (RF-EMW) during talk mode on unprocessed (neat) ejaculated human semen. Prospective pilot study. Center for reproductive medicine laboratory in tertiary hospital setting. Neat semen samples from normal healthy donors (n = 23) and infertile patients (n = 9). After liquefaction, neat semen samples were divided into two aliquots. One aliquot (experimental) from each patient was exposed to cellular phone radiation (in talk mode) for 1 h, and the second aliquot (unexposed) served as the control sample under identical conditions. Evaluation of sperm parameters (motility, viability), reactive oxygen species (ROS), total antioxidant capacity (TAC) of semen, ROS-TAC score, and sperm DNA damage. Samples exposed to RF-EMW showed a significant decrease in sperm motility and viability, increase in ROS level, and decrease in ROS-TAC score. Levels of TAC and DNA damage showed no significant differences from the unexposed group. Radiofrequency electromagnetic waves emitted from cell phones may lead to oxidative stress in human semen. We speculate that keeping the cell phone in a trouser pocket in talk mode may negatively affect spermatozoa and impair male fertility.

Evaluation of poly (ADP-ribose) polymerase cleavage (cleaved-PARP) in sperm fractions after sperm apoptosis induction

Sperm cell apoptosis is an energy and protein dependent process that results in sperm DNA damage. PARP cleavage (cPARP) is stimulated by DNA breaks for help in repair process, so it is considered as an early marker for apoptosis. Staurosporine (STS) is a PARP inducer while 3-aminobenzamide (3ABA), is a PARP inhibitor. Sperm DNA damage due to oxidative stress is considered as one of the causes of male infertility. Conflicting reports exist regarding the presence of whole or cPARP in human ejaculated spermatozoa. We evaluated: 1) the presence of cPARP in mature ejaculated spermatozoa and 2) if we can control its level in sperm fraction using apoptosis inducers with or without PARP inhibitor. Prospective-controlled study. Semen samples were collected from 10 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation. Mature and immature sperm fractions were resuspended into HTF media and divided in 5 aliquots (control, H2O2 treated, H2O2 + 3ABA, STS treated, and STS + 3ABA) incubated at 37°C with 5 % CO2 for 1 hour. The final concentrations were 2.5 μM for STS, 30 μM for H2O2, and 0.3 mM for 3ABA. After incubation, the mature and immature aliquots were evaluated for the early and late apoptosis by 1) annexin V, activated caspase-3, 2) sperm DNA damage by TUNEL and 3) cPARP by FACS. cPARP was detected in both neat and mature fractions. There were no significant differences between mature versus immature fractions regarding most of molecular apoptosis markers for treated and untreated sperm fractions for cPARP modulation.Table 1Correlation between the measured parameters in different treatment groupsUntreated sperm fractionsTreatmentActivated Caspase-3 (a C-3)TUNEL + sperm%Cleaved PARP + %Early apoptotic sperm %Late apoptotic sperm %rPrPrPrPrPSTS treated sperm0.960.120.30.50.720.90.420.130.60.12STS + 3ABA treated sperm0.940.0040.60.570.80.170.240.040.16<0.001H2O2 treated sperm0.940.110.80.20.80.26-0.190.002-0.13<0.001H2O2 + 3ABA treated sperm0.970.0020.510.560.81-0.7-0.20.003-0.06<0.001Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Open table in a new tab Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Cleaved PARP is present in neat as well as in washed ejaculated human spermatozoa. Its level is related to the extent of sperm DNA damage. PARP inhibitors can not prevent/repair oxidative or chemical-induced sperm DNA damage.

P-986: Evaluation of seminal fractions using the Toluidine blue test and sperm chromatin structure assay

Over the past decade, several techniques have emerged as potential protocols for evaluating the sperm chromatin integrity. Such integrity is of utmost importance for successful fertilization. Most of the techniques used for sperm chromatin evaluation require extensive instrumentation and special expertise. Toluidine blue (TB) test has been reported as an easily accessible alternative test for sperm chromatin assessment. Spermatozoa may either stain as dark violet (DV, abnormal chromatin conformation/DNA integrity) or light blue (LB, normal chromatin conformation). The sperm chromatin structure assay (SCSA) is the one of the most standardized methods for assessment of fragmented sperm DNA and the percentage of sperm with immature nuclear development (high DNA stainability index, %HDS). The objective of our study was to evaluate the Toluidine blue test and SCSA for assessment of sperm DNA damage in different seminal fraction. Prospective-controlled study. Semen samples were collected from 19 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation (90% and 47% layers). Both fractions as well as an aliquot of the neat sample were evaluated using the Toluidine blue test. In addition, the %HDS was measured using the SCSA. The means ± standard errors of the percentages of sperm with normal (light blue) and abnormal (dark violet) chromatin in neat semen, mature and immature fractions are given in the table below. Using the Toluidine blue test, mature sperm fractions showed higher incidence of spermatozoa with normal DNA compared to the immature fractions and neat semen samples (p=0.04 and p=0.006, respectively). Similarly, the mature sperm fractions showed lower incidence of spermatozoa with abnormal DNA compared to the immature fractions and neat semen samples (p=0.01 and p<0.0001, respectively). The %HDS showed a significant positive correlation with the number of DV sperm (r=0.51, p=0.0002) and a significant negative correlation with the number LB sperm (r=-0.59, p<0.0001).Tabled 1 Mature sperm fractions show a lower incidence of abnormal DNA as measured by the Toluidine blue test. The association of %HDS with cells that are stained dark violet suggests that Toluidine blue can identify immature nuclear chromatin with abnormal chromatin conformation.

Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques.

The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa.

Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).

Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index

A new instrument for assessing mammalian semen attributes, the Sperm Quality Analyzer, was evaluated as a potential tool for determining rooster sperm quality. The Sperm Quality Analyzer measures the “activity” of sperm in a semen sample as the sperm motility index (SMI). The SMI is defined as the number and amplitude of deflections in a light path per second as a result of sperm movement within a capillary tube. In the present study, effects of sperm concentration, viability, and motility on the SMI were evaluated. Peterson broiler breeder males (n = 40) were used as semen donors. In the initial experiment, semen was diluted from 2- to 25-fold and SMI readings were obtained. The SMI was very low in neat semen samples but increased when semen was diluted up to threefold. However, at dilutions greater than fivefold, the SMI decreased. Apparently, sperm concentration in undiluted semen is so great that sperm are unable to move freely within the capillary tube. Maximum SMI values were obtained at sperm concentrations of approximately 1 billion sperm per milliliter. When thawed, dead sperm were mixed with incubated, live sperm, the SMI decreased with decreasing sperm viability even though sperm concentration was constant. Obviously, fewer sperm move across the light beam as sperm mortality increases. When motile, aerobically incubated sperm were mixed at different rates with immotile, anaerobically incubated sperm samples, the SMI increased with increasing concentrations of motile sperm, whereas total sperm concentration was static. In addition, the SMI was strongly correlated with motility scores obtained by microscopic analysis. The Sperm Quality Analyzer provides an estimate of the overall quality of sperm from broiler breeder males by reflecting sperm concentration, viability, and motility in a single value, the SMI.

Assessment of the separation of X- and Y-bearing sperm on albumin gradients using double-label fluorescence in situ hybridization

To determine the ratio of X- to Y-bearing human spermatozoa in fractions isolated from discontinuous albumin gradients. The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in albumin-separated fractions from the same samples. Two albumin methods were used: a two-layer method (experiment 1) and a three-layer method (experiment 2). X- and Y-bearing sperm were identified simultaneously using chromosome-specific DNA probes and fluorescence in situ hybridization. Hospital-based university department. Healthy donors with normal semen characteristics. The proportions of haploid cells (X or Y) and cells with two sex chromosomes (XX, YY, or XY) were determined. Labeling efficiencies were > 96% in all samples. Control samples showed a 1:1 ratio of X- to Y-bearing sperm. Fractions isolated on albumin gradients showed a slight, but statistically significant enrichment of X-bearing sperm. This was evident with both albumin methods. Discontinuous albumin gradients do not enrich Y-bearing sperm as previously reported.

Detection of X- and Y-bearing human spermatozoa after motile sperm isolation by swim-up

To assess the ratio of X- to Y-bearing human spermatozoa in motile fractions isolated by the swim-up technique. The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in motile fractions isolated from the same samples by swim-up. X- and Y-bearing sperm were simultaneously identified using chromosome-specific DNA probes and double fluorescence in situ hybridization. Hospital-based university department. Ten healthy donors with normal semen characteristics. The distribution of haploid cells (X or Y), normal size cells with two sex chromosome (XX, YY, or XY), and large cells containing two (XX, YY, or XY) or four (XXYY) sex chromosomes were measured in neat semen samples and in motile fractions prepared by swim-up. Overall, 95% of sperm in the neat semen and swim-up fractions were labeled with the probes. The ratios of X- to Y-bearing sperm were 47.3:46.9 (neat semen) and 48.4:47.1 (swim-up fractions), which were not significantly different from a 1:1 ratio. The frequencies of sperm with normal size nuclei and two sex chromosomes (XX, YY, or XY) in the swim-up fractions were not significantly different from the controls, but there was a significant reduction in the proportion of cells with large nuclei and two (XX, YY, or XY) or four (XXYY) sex chromosomes in the swim-up fractions. The swim-up technique does not selectively enrich either X- or Y-bearing sperm. Because the isolation of motile spermatozoa is an important procedure for routine IUI, IVF-ET, and GIFT, the results of this study are important reassurance that the sex ratio is not altered by this method of sperm preparation.

The concentration of estradiol-17 beta in bovine semen.

This study was conducted to evaluate the influence of age, breed, epididymectomy and semen processing on the concentration of estradiol-17 beta (E2) in bovine semen. Semen was collected either by electroejaculation or with an artificial vagina. Neat semen samples were stored at -20 C until analysis. Processed, frozen semen and an egg yolk-citrate semen extender were obtained from a commercial semen processing firm and stored in liquid nitrogen at -196 C. The concentration of E2 in semen was determined by radioimmunoassay. Semen from mature (greater than 24 mo), fertile Brahman (n = 19), Brangus (n = 16), Charolais (n = 29), Holstein (n = 15) and Santa Gertrudis (n = 25) bulls was analyzed for E2 concentration, and no difference (P greater than .10) between breeds was found. There was no difference (P greater than .10) in seminal E2 concentration between mature, fertile bulls (n = 104) and epididymectomized bulls (n = 22). In semen collected from prepuberal (12 to 16 mo, n = 21), peripuberal (17 to 20 mo, n = 17) and mature (greater than 24 mo, n = 19), Brahman bulls, the mature bulls had a lower (P less than .01) semen E2 concentration than peripuberal and prepuberal bulls. There were no differences (P greater than .10) in seminal E2 concentration among peripuberal Angus (n = 8), Hereford (n = 8) and Brahman (n = 17) bulls.(ABSTRACT TRUNCATED AT 250 WORDS)