Comparison of chromatin assays for DNA fragmentation evaluation in human sperm

Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT-mediated-dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r > .866; P < .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (< or =15%, >15%-<30%, and > or =30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P < .05) under SCSA (22.0 +/- 1.6 vs 11.8 +/- 1.4), TUNEL (19.5 +/- 1.3 vs 11.1 +/- 0.9) and SCD (20.4 +/- 1.3 vs 10.8 +/- 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P > .05) between infertile men (31.3 +/- 2.4) and donors (32.7 +/- 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.     

The sperm chromatin structure assay. Relationship with alternate tests of semen quality and heterospermic performance of bulls

Data obtained by the sperm chromatin structure assay (SCSA) on spermatozoa from nine bulls were correlated with fertility, measured by heterospermic performance (-0.94, P less than 0.01) and by alternate tests of sperm quality, including motility, acrosome integrity, Sephadex filtration and morphology of spermatozoa (all significant at P less than 0.05 to P less than 0.01). The SCSA uses flow cytometry to determine the susceptibility of nuclear DNA to low pH-induced denaturation in situ as measured by the ratio of acridine orange binding to double- or single-stranded DNA. The error associated with multiple SCSA measurements was relatively low. The primary finding is that the assay of chromatin structure stability performed on killed spermatozoa was as highly correlated with the heterospermic performance of semen as the best of the classical tests for semen quality. The SCSA may therefore be a highly useful technique for evaluation of sperm quality.     

Sperm calcium levels and chlortetracycline fluorescence patterns are related to the in vivo fertility of cryopreserved bovine semen

Cryopreserved bovine semen is less fertile than fresh semen for reasons that have not been fully elucidated. Cryopreservation is known to disrupt the sperm plasma membrane and it induces premature capacitation of a sperm subpopulation, which may be a result of the increased internal calcium levels after thawing. To test the hypothesis that sperm intracellular calcium level is correlated with in vivo fertility, we used the fluorescent calcium indicator, indo-1, and flow cytometry to assess intracellular calcium levels in frozen-thawed sperm from bulls of varying degrees of fertility. We also tested a second hypothesis that the physiological status of sperm, as assessed by the chlortetracycline (CTC) fluorescent assay, is correlated with fertility. As detected by indo-1 fluorescence, the intracellular calcium level is negatively correlated with bull fertility immediately after thawing (P = .0362; n = 3 ejaculates from each of 10 animals). Moreover, there was a significant difference between the 3 most and least fertile bulls over 4 hours of incubation (P < .05; n = 3 ejaculates per bull). Finally, there was a positive correlation between sperm displaying the CTC acrosome reaction pattern and fertility (P = .0014; n = 3 ejaculates from each of 10 bulls).     

精子染色质结构分析与精液参数的相关性研究

目的 :研究精子染色质结构分析 (SCSA)与精液参数的相关性 ,探讨评估精液质量的可靠方法。　方法 :将5 11份精液标本流式细胞术SCSA结果与精液常规分析结果进行多参数的相关性分析。　结果 :与变性精子百分数(COMPαt)呈低度正相关 (r在 0 .10～ 0 .3 0 )的参数是粘度、本次射精间隔时间、精子畸形率、c级精子密度 ;呈低度负相关的参数 (r在 - 0 .3 0～ - 0 .10 )是曲线速度、直线速度、平均路径速度 ;呈中度正相关 (r在 0 .3 0～ 0 .70 )的参数是精子密度、d级精子率和d级精子密度 ;呈中度负相关 (r在 - 0 .70～ - 0 .3 0 )的参数是平均移动角度、a级精子密度和精子存活率。　结论 :流式细胞术能简单、快速、准确地评估损伤和变性精子的百分数 ,其结果 (COMPαt)与精液参数部分相关 ,并不是完全独立于精液常规检查的指标。SCSA对评估生育力具有重要意义     

流式细胞术在精子功能检测中的应用

目的:研究流式细胞术在精子功能评价中的作用。方法:对104例男性不育患者和10例正常生育男性的精液标本进行精液量、精子密度、活动率、畸形率等精液常规分析(CASA法),对精子存活率、染色质结构和线粒体膜电位等进行流式细胞术分析,并将检测结果通过SAS软件进行统计学分析。结果:成组U检验结果表明:不育患者的精子与正常生育男性精子相比除了具有密度低(U=2.51,P=0.014 3)、活动率差(U=3.44,P=0.001)、畸形率高(U=-5.88,P<0.000 1)等特点外,经流式细胞术测定精子结果表明不育男性的精子质膜完整率(U=4.72,P<0.000 1)、线粒体膜电位正常率(U=-2.53,P=0.030 9)和染色质完整率(αt:U=-3.82,P=0.000 3;SDαt:U=-3.98,P=0.000 1;COMPαt:U=-3.57,P=0.000 5)均显著低于正常生育男性。多元逐步回归分析结果表明:精子质膜完整性与精子活动率(t=1.66,P=0.101 6)、精子的线粒体膜电位与精子活动率(t=3.33,P=0.001 4)、精子染色质的完整性与精子活动率(t=3.24,P=0.001 9)均呈正相关,而精子的线粒体膜电位与精子颈、尾部的畸形率呈负相关(t=-3.44,P=0.001)。结论:流式细胞仪测定对精子质量评定意义显著,对男性不育的诊断和治疗也具有较高的参考价值。     

Quality of sperm obtained by penile vibratory stimulation and percutaneous vasal sperm aspiration in men with spinal cord injury

The aim of this study was to explore sperm chromosomal aneuploidy and DNA integrity in infertile patients with spinal cord injury (SCI). Semensamples were collected from 12 infertile menwith SCI by percutaneous vasal sperm aspiration (PVSA) and from 14 male SCI patients by penile vibratory stimulation (PVS). These semen samples as well as samples from 16 donors were analyzed using the hypo-osmotic swelling (HOS) test, the sperm chromatin dispersion test, terminal deoxynucleotidyl transferase-mediated terminal uridine nick-end labeling assay, and multicolor fluorescence in situ hybridization with probes specific for the chromosomes 13, 18, 21, X, and Y. There were significant differences in the percentages of motile sperm, normal morphologic sperm, normal HOS/eosin staining, and sperm DNA fragmentation between the infertile men with SCI and the control group (P < .05 and P < .01). The sperm forward motility was significantly greater in the PVSA group than in the PVS group (P < .01). The number of round cells per milliliter of semen obtained from the 14 SCI patients by PVS was between 1 million and 12 million. The rate of sperm DNA fragmentation, as identified by the sperm chromatin dispersion test, was higher in the PVS group than in the PVSA group (P < .05). The aneuploidy rates for the SCI patients were 1.5- to 1.6-fold higher for chromosomes 13, 18, and 21, and were 2.3- to 2.4-fold higher for chromosomes X and Y than for patients in the control group (P < .001). These results suggest that for men with SCI, the semen quality is poorer, the prevalence of abnormal HOS/eosin staining is greater, and sperm DNA fragmentation and sperm chromosomal aneuploidies are seen at a higher rate compared with healthy, fertile, and normospermic men.     

Differences in SCSA outcome among boars with different sperm freezability

Spermatozoa from some boars sustain the process of cryopreservation poorly and yield poor fertility after artificial insemination. Poor freezability has not been disclosed using conventional semen analyses. A defective chromatin can, if present in a substantial number of spermatozoa, affect the fertilizing ability of spermatozoa. Here we tested the hypothesis that nuclear DNA instability could explain differences in freezability among boars, and complement or supersede conventional tests for sperm quality such as sperm motility or membrane assessments. Frozen-thawed (FT) spermatozoa from a total of 44 stud boars were assessed by the sperm chromatin structure assay (SCSA), in relation to computer-assisted sperm analysis-derived sperm motility variables and sperm viability (triple fluorescent microscopic staining), including three experiments. The first trial, including 24 boars, evaluated the relationship between the sperm motility and viability with levels of DNA integrity. The SCSA showed that most spermatozoa had intact DNA [levels of DNA fragmentation index (%DFI) ranging from 0.63% to 11.85%] significantly correlated (albeit weakly) with current sperm quality variables. The second trial, on 15 boars, assessed the influence of two different thawing rates (20 s at 37 degrees C vs. 8 s at 70 degrees C) and the post-thaw incubation times (0, 30, 150 and 300 min) at 37 degrees C on FT-boar sperm quality. The highest sperm survival (p < 0.05) and the lowest DNA damage (p < 0.01) were achieved when thawing was carried out at 70 degrees C for 8 s, without any change during the first 150 min of incubation. Finally, the third experiment studied if differences in sperm freezability showed by stud boar semen, as 'good' or 'bad' freezers by conventional analyses, could be attributed to differences in chromatin structure. All SCSA parameters were low, but significantly (p < 0.05-0.001) higher for 'bad' freezers, showing they had less homogeneous sperm chromatin than the 'good' freezers. The results indicate that SCSA outcome complements conventional assessment of FT-boar spermatozoa, disclosing differences in their ability to sustain freezing and thawing. However, the low overall DNA damage observed in FT spermatozoa seems to have poor biological significance.     

DNA integrity is compromised in protamine-deficient human sperm

The objective of this study was to examine the relationship between DNA integrity and protamines in human sperm. One hundred forty-nine male infertility patients were included in an Institutional Review Board-approved study. Sperm were evaluated for DNA fragmentation using the DNA Integrity Assay, a test equivalent to the sperm chromatin structure assay (SCSA). Additionally, nuclear proteins were extracted and the protamine-1/protamine-2 ratio (P1/P2), protamine-1 (P1), protamine-2 (P2), and total protamine concentrations were evaluated. We identified 37 patients with abnormally low P1/P2 ratios, 99 patients with normal P1/P2 ratios, and 13 patients with abnormally high P1/P2 ratios. DNA fragmentation was significantly elevated in patients with low P1/P2 ratios (37.1 +/- 6.02) vs those with normal and high P1/P2 ratios (26.7 +/- 1.9 and 23.8 +/- 3.2, respectively; P < .05) and was inversely correlated with the P1/P2 ratio (R(s) -0.18, P < .05), P1 concentration (R(s) -0.29, P < .001), P2 concentration (R(s) - 0.24, P < .005), and total protamine concentration (R(s) -0.28, P < .001). Furthermore, chi2 analysis revealed a significant increase in the incidence of marked DNA fragmentation in patients with diminished levels of either P1 or P2. The present study is the first to report that human sperm protamine content is significantly related to DNA fragmentation. In particular, sperm P1 and P2 concentrations inversely correlate with DNA fragmentation, indicating a protective role of the protamines against sperm DNA damage. In light of recent studies highlighting the negative effect of sperm DNA damage on ART outcomes, these findings indicate a possible clinical significance for human sperm protamine levels.     

The mast cells in semen: their effects on sperm motility

This study was conducted to evaluate any possible association between mast cells and sperm concentration, morphology, and motility. The study comprised 400 patients who had applied for semen analysis. To evaluate mast cells, 6 smear slides were prepared for each subject and stained with 1% toluidin blue-pyronine (pH 4). The slides revealing any mast cells were labeled as mast+. Concentration and motility was evaluated through a Makler chamber. Kruger's strict criteria were used in morphometric analysis. The mean age of 86 mast+ cases (21% of total patients) was 31+/-6.7; progressive sperm motility rate was 33+/-21.2. The mean concentration was 32+/-30.2 x 10(6)/mL, and normal sperm percentage was 11.8+/-6.5. Progressive sperm motility rate in the mast- cases were 53+/-25. The mean age of mast cell+ patients was higher than that of mast cell- patients (t=3.57, p<.001), while they had lower sperm concentration (p>.05) and lower normal morphologic sperm rate (t=2.26, p<.024), compared to mast cell- patients. The relation between mast cell+ and mast cell- cases and sperm progressive motility was statistically significant (t=6.44, p<.001). It was concluded that sperm parameters were negatively affected by mast cells.     

In vitro and xenogenous capacitation-like changes of fresh, cooled, and cryopreserved stallion sperm as assessed by a chlortetracycline stain

Like the human female, the mare experiences reproductive tract pathology that may sometimes be circumvented by the use of assisted reproductive technologies (ARTs). One such technology, gamete intrafallopian transfer (GIFT), may be used in mares that exhibit ovulatory, oviductal, or uterine abnormalities that limit the use of common ARTs, such as embryo transfer. Homologous GIFT has been successfully performed in the horse; however, the logistics, costs, and associated risks of surgically transferring gametes to the oviducts of a recipient mare are considerably high. Use of a less costly species in a heterologous or xenogenous procedure would therefore be beneficial. This study represents the preliminary investigation into the use of sheep as recipients for xenogenous GIFT procedures using equine gametes. We investigated the capacitation response of fresh, cooled, or frozen stallion sperm after 1) in vivo incubation in the reproductive tract of estrous and anestrous ewes as well as 2) in vitro incubation in a modified Krebs/ Ringer extender at 37 degreesC with and without the addition of heparin at 10 IU/mL for up to 8 hours. A chlortetracycline (CTC) fluorescent stain was used to assess the capacitation response of sperm. Findings indicated that oviductal fluid samples recovered from estrous ewes had significantly higher numbers of sperm exhibiting capacitation-like staining patterns when compared to samples recovered from anestrous ewes (P < .05). Fresh semen yielded higher capacitation-like staining patterns after in vivo incubation than did frozen-thawed or cooled samples. A transition from majority CTC unreacted sperm to majority CTC non-acrosome intact sperm was demonstrated for both in vivo and in vitro studies. In vitro incubation of stallion sperm with heparin did not result in an increased capacitation-like staining response over time when compared with nonheparinized samples. Results from this study suggest that xenogenous capacitation of stallion sperm may occur in the estrous ewe.     

Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis.

Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were frozen (-20 degrees C) in the absence of cryoprotectants revealed that significantly more cells with fragmented tails of DNA, or comets, occurred among those exposed to 1, 3, and 5 freeze-thaw cycles (65% +/- 6%, 76% +/- 11%, 92% +/- 6%, respectively) compared with fresh, untreated sperm (19% +/- 16%, P < .05). In addition DNA damage was different (P < .05) between the three freeze-thaw treatments. Sensitivity of SCGE on equine sperm was further tested with known ratios of frozen-thawed and fresh cells. The amount of detectable DNA damage was positively correlated with the percentage of cryo-damaged cells in each treatment (r2 = 0.92, P < .05). Potential damage as a result of cooled storage was also investigated and results revealed that sperm stored for 48 hours (at 5 degrees C) had a higher percentage of comets than that of fresh sperm (63% +/- 13.9% and 28% +/- 15.6%, respectively, P < .05). The percentage of viable sperm also decreased linearly over time and was inversely correlated with percent of comets (r2 = 0.805, P < .001). Detection of sublethal and/or uncompensable fertility factors in semen, such as DNA fragmentation, could be useful for detecting male differences in semen for cooling or cryopreservation potential and could provide a tool for monitoring and preserving fertility for individual stallions.     

Fluorescence in situ hybridisation sperm examination is significantly impaired in all categories of male infertility

To study the outcome of FISH sperm examination in cases with sperm pathology and outline the potential correlation with certain chromosomal defects. A retrospective study of prospectively collected data was performed in IAKENTRO, Infertility Treatment Center. Rates of abnormal FISH semen examination were compared between male infertility patients and fertile controls. Detection of abnormal FISH semen examination as well as each chromosomal abnormality detected was correlated with each sperm deficiency (asthenozoospermia, oligozoospermia and teratozoospermia) in a univariate regression model. There were 72 male partners included, of which 52 male infertility patients and 20 controls. The rate of abnormal sperm FISH examination was significantly higher in patients’ group (55.8% vs. 15.0% for controls, p = .002). Asthenozoospermia, oligozoospermia and teratozoospermia were significantly correlated with detection of abnormal FISH examination (p = .004, p = .01 and p < .001 respectively). Teratospermia was significantly correlated with increased aneuploidy rate for chromosome 17 (p = .005), chromosome X (p = .05) and Y (p = .03). FISH examination reveals pathology in a significant proportion of patients with sperm defects and should be recommended to achieve early detection of chromosomal defects that may postpone favourable reproductive outcome.     

The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas

This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (±SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation.     

Various physical stress factors on rat sperm motility, integrity of acrosome, and plasma membrane

The objective of this study was to determine the effects of various physical interventions such as centrifugation regimes, Percoll gradient separation, and repeated pipetting on various viability parameters of epididymal sperm of Fischer 344 (F-344) and Sprague-Dawley (SD) rat strains. Three experiments were conducted. In experiment 1, sperm motility and acrosomal and membrane integrity were compared after exposing sperm samples to 200, 400, 600, and 800 x g centrifugal forces for 5, 10, or 15 minutes. In experiment 2, sperm motility and acrosomal and membrane integrity were compared after passing them through a Percoll separation using centrifugal forces of 600, 800, 1000, and 1200 x g for either 15 or 30 minutes. In experiment 3, the effect of repeated pipetting (2, 4, 6, 8, and 10 times) on motility and membrane integrity of rat sperm was compared with that on mouse, ram, bull, and boar sperm. The results revealed that both F-344 and SD rat sperm motility and membrane integrity were significantly affected by centrifugation (P < .05). The acrosomal integrity of SD rat sperm was affected after using 800 x g centrifugation force for 10 or 15 minutes (P < .05), whereas F-344 rat sperm acrosomal integrity was not affected by any centrifugation regimes (P > .05). Sperm from SD rats also had higher motility and membrane integrity loss than did sperm from F-344 rats after centrifugation and pipetting (P < .05). Percoll gradient separation did not cause significant motility loss or acrosomal damage to either F-344 or SD sperm (P > .05). Repeated pipetting had a dramatic adverse effect on both rat and mouse sperm motility (P < .05) as compared with sperm from bull, boar, and ram, which were not affected at all (P > .05). These data suggest that rat sperm have unique properties that need to be considered during centrifugation, Percoll gradient separation, and pipetting procedures.     

吸烟对精子染色质结构完整性影响的探讨

目的:探讨吸烟对精子染色质结构完整性的影响。方法:784例男性不育患者从病例库中选取,根据吸烟与否及每日吸烟支数(≤10、11~19、≥20)和吸烟年限(≤5、6~9、≥10)进行分组,比较各组患者精液常规检测参数及精子染色质结构受损和精子核成熟情况。精子染色质结构完整性采用流式细胞仪检测,DNA损伤程度和不成熟精子指数分别以DNA损伤指数(DFI)和高DNA着色性(HDS)来表示。结果:吸烟≥20支/日和吸烟年限≥10年的患者组精液量、前向运动精子比例低于其他组而精子畸形率高于其他组(P<0.05);吸烟组男性的DFI和HDS值均升高(P<0.05);HDS与前向运动精子比例呈负相关(r=-0.18,P<0.05),DFI与HDS均与精子畸形率呈正相关(r=0.31与r=0.39,P均<0.05)。结论:日吸烟量≥20支或吸烟年限≥10年对男性的精液量、前向运动精子比例和精子畸形率都有影响,吸烟影响男性精子DNA完整性和精子核成熟。     

Implication of calmodulin-dependent phosphodiesterase type 1 during bovine sperm capacitation

Phosphodiesterases (PDEs) are enzymes that degrade cyclic nucleotides. The calcium-calmodulin dependent PDE type 1 (PDE 1) and the cyclic adenosine monophosphate (cAMP)-specific PDE type 4 (PDE 4) have been implicated in sperm function. We tested the hypothesis that specific PDEs regulate capacitation of bovine sperm in a manner independent of those that mediate motility. Our objectives were to determine the effects of inhibiting PDE 1 and PDE 4 on capacitation and motility, and to compare these effects to those of heparin, which is necessary for capacitation of bull sperm in vitro. Fresh sperm were supplemented either with 15 microg/mL heparin (positive control) or the PDE inhibitors vinpocetine (specific for PDE 1) and rolipram (specific for PDE 4), and then incubated for 5 hours. At 0, 3, and 5 hours, samples were assayed for capacitation and motility parameters according to the chlortetracycline (CTC) fluorescent pattern B and computer-assisted sperm analysis, respectively. A higher percentage of CTC pattern B sperm relative to heparin controls was observed at 0 and 3 hours when sperm were incubated with vinpocetine. After 5 hours, the percentage of heparin- and vinpocetine-treated sperm showing pattern B did not differ (P >.05). Rolipram did not affect CTC patterns (P >.05; n = 4). Vinpocetine and heparin both reduced the percentage of progressively motile sperm after 3 and 5 hours, but vinpocetine reduced it more than heparin (P <.05; n = 4). Rolipram transiently increased linearity versus sperm with heparin (P <.05; n = 4). To further test the hypothesis that PDE 1 inhibition permits capacitation, we conducted in vitro fertilization. Vinpocetine did not support the ability of sperm to penetrate homologous oocytes (n = 5). Although cAMP regulation by PDE 1 may occur early during capacitation, downstream events appear to prevent full capacitation from occurring prematurely.     

Effect of l‐arginine addition on long‐term storability of ram semen

This study was conducted to investigate the effect of l ‐arginine addition on long‐term storability of ram semen. Six Akkaraman rams were used as material. Semen samples were collected. Pooled samples were diluted and were divided into six equal aliquots. While aliquot 1 was kept as control, the stock solutions including 0.1, 0.5, 1, 5 and 10 mm l ‐arginine were added to other aliquots. All aliquots were routinely frozen in 0.25‐ml straws at ?130°C liquid nitrogen vapour and stored in liquid nitrogen ?196°C until being analysed. The equilibrated and thawed sperm motility, membrane integrity and arginase activity were evaluated. While the 10 mm l ‐arginine supplementation significantly (p < .001) decreased equilibrated sperm motility, the 5 mm significantly (p < .05) increased the membrane integrity and arginase activity in comparison with the control group. The motility (p < .001) and membrane integrity (p < .01) were determined to be highest in 0.5 mm group, while significant reductions were observed in motility (p < .001) of 10 mm group and arginase activity (p < .05) of 1, 10 mm groups as compared to the control group. It was concluded that in vitro addition of 0.5 mm l ‐arginine to ram semen may be useful, but 10 mm may be harmful to spermatozoa quality during long‐term storage.     

Study on correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation

This investigation was carried out to study the correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation. Semen samples were evaluated for sperm parameters (mass motility [MM], concentration [CON], progressive motility [PM], viability [VIB], acrosomal integrity [AI] and hypo‐osmotic swelling [HOS] response), antioxidants (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx] and total antioxidant capacity [TAC]) and oxidants (Lipid peroxidation [LPO] and reactive oxygen species [ROS]) at fresh, pre‐freeze and post‐thaw stages. Sperm parameters (PM, VIB, AI and HOS response) and antioxidants (SOD, CAT and TAC) were significantly (p < .05) reduced at fresh stage, and oxidants (LPO and ROS) were significantly (p < .05) increased at pre‐freeze and post‐thaw stages. At fresh stage, MM was negatively correlated with LPO (p < .05), and CON was positively correlated with SOD, TAC and CAT, negatively correlated with LPO and CAT was positively (p < .01) correlated with VIB and HOS response. At pre‐freeze stage, CAT was positively correlated with PM and AI (p < .05), and AI was negatively (p < .05) correlated with ROS. At post‐thaw stage, CAT was positively correlated with PM, VIB, HOS response and AI,, and LPO was negatively correlated with HOS, AI and VIB. The study of correlations of these parameters at different preservation stages with bull fertility may play an important role in developing models for predicting future fertility of bulls in the absence of conception rate data.     

正常生育力和精子活力低下不育者精子运动计算机辅助分析

用精子活力分析仪对７３名正常生育力男性射出精子进行了精子动力学指标分析；用上述指标分析了２００例精子活力低下不育男性射出精子动力学指标及各项运动参数间相互关系；用己酮可可碱作用精子活力低下不育男性射出精子，观察精子各项运动参数改变。结果：（１）正常生育力组，前向运动精子在总运动精子中比例与精子活动率间不呈相关性；随精子活动率提高，快速相和慢速相精子分布分别逐渐增加和减少，精子ＶＡＰ、ＶＣＬ、ＶＳＬ、ＢＣＦ、ＡＬＨ增加，ＬＩＮ和ＳＴＲ降低。（２）精子活力低下不育组，随精子活动率下降，主要表现在运动速度下降，与正常组比较，精子前向运动能力和运动速度均有明显下降（Ｐ＜０．０５），但ＬＩＮ和ＳＴＲ不管在组内还是与正常组相比均无明显差别（Ｐ＞０．０５），反映出这种前向运动能力的减低是由速度下降引起，速度分布变化以中速相明显。（３）己酮可可碱能明显提高精子活力低下不育者精子的活动率，前向运动率和精子运动快速相的分布；对精子ＬＩＮ，ＳＴＲ影响不明显。