Designing the selenium and bladder cancer trial (SELEBLAT), a phase lll randomized chemoprevention study with selenium on recurrence of bladder cancer in Belgium

Background

Growing evidence indicates that oxidative stress can be a primary cause of male infertility. Non-enzymatic antioxidants play an important protective role against oxidative damages and lipid peroxidation. Human seminal plasma is a natural reservoir of antioxidants. The aim of this study was to determine glutathione (GSH) concentrations, trace element levels (zinc and selenium) and the lipid peroxidation end product, malondialdehyde (MDA), in the seminal plasma of men with different fertility potentials.

Methods

Semen samples from 60 fertile men (normozoospermics) and 190 infertile patients (74 asthenozoospermics, 56 oligozoospermics, and 60 teratozoospermics) were analyzed for physical and biochemical parameters. Zinc (Zn) and selenium (Se) levels were estimated by atomic absorption spectrophotometry. Total GSH (GSHt), oxidized GSH (GSSG), reduced GSH (GSHr) and MDA concentrations were measured spectrophotometrically.

Results

Zn and Se concentrations in seminal plasma of normozoospermics were more elevated than the three abnormal groups. Nevertheless, only the Zn showed significant differences. On the other hand, Zn showed positive and significant correlations with sperm motility (P = 0.03, r = 0.29) and count (P < 0.01, r = 0.49); however Se was significantly correlated only with sperm motility (P < 0.01, r = 0.36). GSHt, GSSG and GSHr were significantly higher in normozoospermics than in abnormal groups. We noted a significant association between seminal GSHt and sperm motility (P = 0.03). GSSG was highly correlated to sperm motility (P < 0.001) and negatively associated to abnormal morphology (P < 0.001). GSHr was significantly associated to total sperm motility (P < 0.001) and sperm count (P = 0.01). MDA levels were significantly higher in the three abnormal groups than in normozoospermics. Rates of seminal MDA were negatively associated to sperm motility (P < 0.01; r = -0.24) and sperm concentration (P = 0.003; r = -0.35) Meanwhile, there is a positive correlation between seminal lipid peroxidation and the percentage of abnormal morphology (P = 0.008).

Conclusions

This report revealed that decreased seminal GSH and trace element deficiencies are implicated in low sperm quality and may be an important indirect biomarker of idiopathic male infertility. Our results sustain that the evaluation of seminal antioxidant status in infertile men is necessary and can be helpful in fertility assessment from early stages.     

Motility Related to the Presence of Carnitine/Acetyl-Carnitine in Human Spermatozoa

Total carnitine, acetylcarnitine and carnitine acetyl transferase (E.C. 2.3.1.7) were measured in the plasma and spermatozoa fractions of 41 samples of human semen and the correlation with sperm motility and sperm density examined.
It was confirmed that the concentration of total carnitine as well as of acetylcarnitine was 2–25 times higher and the activity of carnitine acetyl transferase 20–15 fold higher in spermatozoa than in seminal plasma. Sperm motility correlated with the concentration of acetylcarnitine (r = 0.6, P < 0.01) and of total carnitine (r = 0.55, P < 0.01) but not with the concentration of free carnitine nor with the activity of carnitine acetyl transferase in the spermatozoa. No correlation was found between sperm motility and the concentrations of acetylcarnitine, free carnitine or total carnitine in the seminal plasma.
It is concluded that the amount of carnitine present in the spermatozoa probably provides a better index of epididymal function than the carnitine in the seminal plasma as the latter in influenced by af variable contribution from the other accessory sex organs.     

Relationships between seminal plasma arginase activity and spermatological parameters in rams

This study was conducted to evaluate the correlation between seminal plasma arginase activity and spermatological parameters in rams. In this study, five fertility-proven Awassi rams were used as material. Six ejaculates were collected from each ram by an artificial vagina. Spermatological parameters (semen volume, mass activity, sperm motility and concentration and abnormal sperm rate) were immediately determined in each ejaculate. For enzyme assay, the semen samples were centrifuged and stored at -20 °C for the analysis of arginase activity. The average seminal plasma arginase activity was 0.61 ± 0.20 U (mg protein)(-1) . There was a positive correlation between arginase activity and semen volume (r = 0.412, P < 0.05), semen mass activity (r = 0.610, P < 0.01), sperm motility (r = 0.447, P < 0.05) and sperm concentration (r = 0.808, P < 0.01). However, there was a negative correlation between arginase activity and abnormal sperm rate (r = -0.424, P < 0.05). In conclusion, this study clearly suggests that there is a significant correlation between seminal plasma arginase activity and spermatological parameters. In light of these results, seminal plasma arginase activity may be a biochemical criterion for determining sperm quality besides classical semen analysis parameters in rams.     

Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

Aim： To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality. Methods： Two early apoptotic changes in the semen of 56 men were assessed using Annexin V （AN）/propidium iodide （PI） staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential （MMP）. The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. Results： The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinctive spermatozoa populations. The percentage of AN^-/PI^- spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL （＋） spermatozoa. As for the AN^-/PI^＋ fraction, we found an opposite result in comparison to AN^-/PI^- spermatozoa. The level of early apoptotic AN^＋/PI^- spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN^＋/PI^＋ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL （＋） spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL （＋） spermatozoa. Conclusion： Although early apoptotic AN^＋/PI^- spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa （especially, the percentage of AN^-/PI^- spermatozoa）, and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization. （Asian J Androl 2008 Mar; 10： 227-235）     

Enolase isoforms activities in spermatozoa from men with normospermia and abnormospermia

Total enolase as well as enolase-alphaalpha. (ENO-alphaalpha, ubiquitous) and enolase-S (ENO-S, sperm-specific) activities were measured in total and Percoll-selected sperm from 30 normospermic fertile men and 20 abnormospermic infertile patients. The total enolase activity was significantly higher in total sperm from patients with abnormospermia compared with normospermic patients (11.1 +/- 1.9 vs. 4.8 +/- 0.5 mlU/10(7) sperm P < .05). ENO-alphaalpha activity was significantly higher in total sperm from abnormospermic men than from normospermic men (P < .05). ENO-alphaalpha activity in Percoll-selected sperm was significantly lower compared with total sperm in both group of patients; however, for the same sperm fraction ENO-alphaalpha activity did not differ between normospermic and abnormospermic men. ENO-alphaalpha activity was related to the cell contamination ratio and to the percentage of spermatozoa with abnormal morphology. Furthermore, ENO-alphaalpha was positively correlated with the percentage of immature sperm showing an excess of residual cytoplasm. ENO-S activity was significantly higher in total sperm from normospermic patients than from abnormospermic patients (P < .05). ENO-S activity in Percoll-selected sperm was not significantly different compared with total sperm in both group of patients. However, this activity was significantly lower in Percoll-selected sperm from abnormospermic men compared with normospermic men (P < .05). ENO-S activity was not related to the cell contamination ratio but was significantly correlated with the percentage of spermatozoa with normal morphology. The 2 enolase isoforms seem to reflect 2 opposite aspects of sperm cells quality: ENO-alphaalpha is associated with abnormal spermatozoa, immature spermatozoa, or both; and ENO-S is associated with normal spermatozoa. As an additional index to distinguish normal from abnormal semen, the ENO-S:ENO-alphaalpha ratio was evaluated for total and Percoll-selected sperm in both groups. This ratio seems to be a new, valuable marker of the global sperm quality in a given semen sample, and may represent a predictive index of sperm fertilizing potential.     

不育病人精浆前列腺特异性抗原的检测及意义

目的 :探讨不育病人精浆前列腺特异抗原 (PSA)检测的意义。　方法 :随机选择 85例不育病人 ,采用ELISA法检测不育病人精浆PSA水平 ,并分析其与精浆酸性磷酸酶 (ACP)、精子密度、活率之间的关系。　结果 :35例不液化病人、30例液化不全病人精浆PSA水平、ACP浓度、精子活率 (a +b +c级精子百分率 )均显著低于 2 0例液化正常病人 (P <0 .0 1) ;而精子密度则无差异 (P >0 .0 5 )。精浆PSA水平与精浆ACP浓度、精子活率显著正相关 (P<0 .0 1)。　结论 :精浆PSA水平与精液液化密切相关 ,其质和量的异常会使精子活率降低 ,从而导致男性生育力下降     

Comparison of semen quality in older and younger men attending an andrology clinic

The effects of age on semen quality were analysed in patients referred to an andrology outpatient clinic in a retrospective study covering a period of 3 years. Semen analyses of older men (n = 66; > or = 50 years; median: 53) were compared with those of young patients (n = 134; 21-25 years; median 24). The duration of sexual abstinence was longer among older patients (median: 5.5 vs. 5.0 days; + 10%, P < 0.05). Even after adjustment for duration of sexual abstinence, progressive motility (- 27%, P < 0.01), percentage of morphologically normal spermatozoa (- 44%, P < 0.01) and semen volume (- 29%, P < 0.0005) were significantly lower in older than in younger men. Impaired sperm morphology was mainly attributed to abnormal staining of flagella (+ 50%, P < 0.05) and an increased portion of coiled or bent tails (+ 44%, P = 0.001) indicating epididymal dysfunction. Total sperm count and sperm concentration were unaffected by age. Age-dependent changes in accessory gland function, as indicated by fructose concentration (- 27%, P < 0.05), was correlated with lower semen volume. Moreover, serum testosterone levels were significantly reduced in the group of older men (median: 3.0 vs. 3.6 ng ml-1; -17%, P < 0.005).     

Correlation of biochemical constituents of seminal plasma with semen quality in Teddy goat (Capra hircus) bucks

This study was planned to determine the relationship between semen quality parameters and the levels of biochemical constituents of seminal plasma of Teddy (Capra hircus) buck semen. For this purpose, semen ejaculates were collected from five mature healthy Teddy bucks. All the experimental bucks were kept under natural environmental conditions. Semen was collected twice in a week for the duration of 6 weeks by Artificial Vagina (AV) in the breeding season (February‐April). Two successive ejaculates of single buck were pooled at time of collection, and a total of 60 semen samples were processed for semen analysis. Sperm per cent motility, sperm concentration, dead sperm percentage, morphological abnormal spermatozoa, plasma membrane integrity were correlated with biochemical constituents of seminal plasma. The mean per cent motility (89.18% ± 0.37%), sperm concentration (1.86 ± 0.04 × 10⁹/ml), dead sperm percentage (8.08% ± 0.29%), morphological abnormal spermatozoa (6.05% ± 0.29%) and plasma membrane integrity (88.22% ± 0.34%) were recorded. The seminal plasma contained Na⁺ (144.12 ± 1.59 mEq/L), K⁺ (27.38 ± 0.49 mEq/L), Cl^? (65.73 ± 0.45 mEq/L), Ca⁺⁺ (9.34 ± 0.22 mg/dl), P (19.32 ± 0.97 mg/dl), aspartate aminotransferase (AST; 26.48 ± 1.30 IU/L), alanine aminotransferase (ALT; 168.47 ± 5.18 IU/L), lactate dehydrogenase (LDH; 215.98 ± 6.06 IU/L), albumin (1.90 ± 0.10 g/dl), globulins (2.08 ± 0.11 g/dl) and total protein (3.98 ± 0.20 g/dl). The collected data were analysed by applying Pearson's correlation coefficients. Dead sperm percentage had negative correlation with sodium (r = ?.278, p < .05), albumin (r = ?.294, p < .05), globulin (r = ?.266, p < .05) and total protein (r = ?.295, p < .05). Phosphorus was negatively associated with sperm concentration (r = ?.262, p < .05). AST was negatively correlated with plasma membrane integrity (r = ?.292, p < .05). It was concluded that most of the semen quality parameters of Teddy bucks were positively correlated with biochemical constituents, but opposite trends were found in case of dead sperm percentage. The seminal biochemical constituents dynamically interact with each other.     

Activity of testis angiotensin converting enzyme (ACE) in ejaculated human spermatozoa

Testicular angiotensin converting enzyme (ACE) isozyme is likely to play important functional roles in male reproduction. Several studies have shown that ACE is released from human spermatozoa during capacitation and that ACE is associated with reduced sperm motility. Recently, we established an assay to detect testicular ACE activity in human spermatozoa. The purpose of this study was to determine if testicular ACE activity is related to sperm motility in human ejaculates. Semen samples were collected from 80 infertile patients. According to the semen characteristics, they were divided into four (WHO) categories. Enzyme activities of ACE in spermatozoa (testicular ACE) and seminal plasma (somatic ACE) were spectrophotometrically determined. Total testicular ACE activity in spermatozoa was measured by solubilization of spermatozoa with Triton X-100. Membrane testicular ACE activity was measured in a sperm : PBS suspension. Sperm concentration and sperm motility were 136.6 +/- 154.1 x 10(6)/mL and 58.6 +/- 23.4%, respectively (mean +/- SD). Enzyme activities of membrane testicular ACE, total testicular ACE and somatic ACE were 0.273 +/- 1.219 microU/10(6) spermatozoa, 0.35 +/- 1.34 microU/10(6) spermatozoa and 684.7 +/- 226.6 mU/mL, respectively. A negative correlation was observed between sperm motility and membrane testicular ACE activity (p < 0.05). Membrane testicular ACE activity in 44 normal semen samples was 0.04 +/- 0.02 microU/10(6) spermatozoa, whilst that in 36 abnormal semen samples was 0.24 +/- 0.42 microU/10(6) spermatozoa. There was a significant difference between these two groups (p < 0.01). Membrane testicular ACE in sperm samples from normozoospermic men was significantly lower than that from oligoasthenozoospermic men (p < 0.05). These findings suggest that testicular ACE is released from normal functional spermatozoa for them to have fertilizing ability.     

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.     

人精子体外获能中活率和活力的变化

体外获能中精子的存活与寿命是重要的。本文报告了人精子在普通培基和高离子强度培基中获能1、3、6、12、24、36、48小时中活率和活力变化的发生、发展过程。表明精子活率和活力的变化与获能时间呈显著性负相关(P<0.01),与排射精液的质量有关,并有明显的个体差异。     

Role of granulocyte elastase and interleukin-6 in the diagnosis of male genital tract inflammation

Chronic genital tract inflammations are a frequent cause or at least a concomitant factor of male fertility disturbances. The diagnosis is difficult because of the mostly asymptomatic course of the disease. Therefore, determination of biochemical markers of inflammation in addition to the number of leucocytes in the seminal plasma has been recommended. The aim of the study was to find out whether determination of granulocyte elastase and interleukin-6 provide comparable and reliable results with regard to diagnosis of genital tract inflammation; in addition, the association between genital tract inflammation and semen quality should be evaluated with special focus on potentially disturbed sperm functions like sperm motility and DNA integrity. In a prospective study, the concentrations of interleukin-6 (IL-6) and granulocyte elastase were determined in seminal plasma samples from 340 patients to investigate the relationship with other parameters of genital tract inflammation such as the number of peroxidase-positive cells and conventional semen parameters. Microbiological investigations were included. As post-testicular inflammatory influences may cause sperm DNA damage, the correlation between IL-6 and elastase and DNA integrity was evaluated by the sperm chromatin structure assay. IL-6 and elastase were significantly correlated both with each other (P < 0.01) and the number of peroxidase-positive cells (P < 0.01). IL-6 showed a highly significant negative correlation with sperm vitality (P < 0.01) and a significant negative correlation with sperm motility (P < 0.05). Elastase concentrations were highly significantly associated with the number of peroxidase-positive cells (P < 0.01) and negatively correlated with sperm vitality (P < 0.01). Moreover, there were significantly negative correlations with sperm motility (P < 0.05), progressive motility according to WHO a quality (P < 0.05) as well as sperm morphology (P < 0.05). In addition, a significant negative correlation was observed between elastase concentrations and percentage of spermatozoa with intact DNA, which may suggest the use of anti-inflammatory treatment. It can be concluded that both IL-6 and granulocyte elastase are useful and suitable as markers for silent genital tract inflammation; in contrast to previous contributions there were clear correlations of IL-6 and granulocyte elastase with sperm parameters, the relationship of elastase with semen quality being more marked. Moreover, the results of the study confirm the need for a change of the threshold value of peroxidase-positive cells according to WHO definition to lower levels for definition of silent genital tract inflammation.     

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.     

Quantification of seminal plasma motility inhibitor/semenogelin in human seminal plasma

Semenogelin I and II (Sg I and II) are the major components of human semen coagulum. The protein is rapidly cleaved after ejaculation by the chymotrypsin-like protease prostate-specific antigen (PSA), which results in the liquefaction of the semen coagulum and the progressive release of motile spermatozoa. One of the cleavage products of the protein, a 14-kDa protein, is a sperm motility inhibitor (seminal plasma motility inhibitor [SPMI]). We developed a monoclonal antibody (mAb) that is specific to the fragment of Sgs, SPMI, and a sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of Sgs using this mAb. Then, we measured SPMI/Sg levels in human seminal plasma from healthy male volunteers (n = 100, aged 18-24 years). The mean level of SPMI/Sg in seminal plasma was 19 +/- 13 mg/mL (range, 4-68 mg/mL). Log-transformed SPMI/Sg levels were negatively correlated with the sperm motility (r = -0.229, P =.0220) and positively correlated with the total protein concentration (r = 0.793, P <.0001). This result supports that SPMI, one of the fragments of Sg, has its inhibitory effect on ejaculated spermatozoa in liquefied semen under physiological conditions.     

捐精者精子冷冻复苏率影响因素分析

目的:探讨季节、血型及精液参数等对捐精者精子冷冻复苏率的影响。方法:回顾性分析陕西省人类精子库捐精者4 088份精液标本,研究季节、血型、禁欲时间、精液量、精子形态、冷冻前精子活力及浓度对精子冷冻复苏率的影响。结果:捐精者精子冷冻复苏率随着精子浓度增高而增加,相关性分析提示精子浓度与冷冻复苏率呈正相关(r=0.247,P0.01)。而精子冷冻前活力和精子冷冻复苏率呈负相关(r=-0.262,P0.01)。禁欲第6天组的精子冷冻复苏率[(70.2±5.4)%]明显高于其他禁欲时间组(P0.01)。精子正常形态率20%组的精子冷冻复苏率[(71.4±5.1)%]要高于其他各组(P0.01)。A型血精子冷冻复苏率明显高于B型血[(69.1±4.8)%vs(69.8±4.7)%,P0.01];季节、精液量与精子冷冻复苏率之间无明显相关性(P0.05)。结论:捐精者的精子浓度、活力、形态及禁欲时间对于预测精子冷冻复苏率有一定的价值,而季节、血型、精液量与捐精者精子冷冻复苏率无明显相关性。     

Controlled cooling during semen cryopreservation does not induce capacitation of spermatozoa from two portions of the boar ejaculate

Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear which part of the process affects the spermatozoa the most. The present study monitored, along the entire process of cryopreservation, the stability (PMS) of the architecture of the lipid plasma membrane and its integrity (PMI), as well as the kinetics of the processed spermatozoa using two portions from the boar ejaculate (P1 = the first 10 mL of the sperm-rich fraction, SRF; P2 = the rest of the ejaculate), frozen in a recently developed package, the MiniFlatPack (MFPs, 0.5 x 10(9) sperm/dose). Evaluation was made at four specific stages, viz. S1 = after collection (suspended in Beltsville thawing solution, BTS); S2 = at 15 degrees C (suspended in lactose-egg yolk, LEY); S3 = at 5 degrees C (suspended in LEY plus glycerol); and S4 = post-thaw. Both sperm kinetics (using computer-assisted sperm analysis, CASA) and PMS [i.e. the degree of lipid disorder and of the exteriorization of phosphatidylserine (PS) in the plasma membrane, measured by flow cytometry using Merocyanine-540 (M-540), and Annexin-V (AV) respectively], as well as plasma membrane integrity [PMI, i.e. the degree of membrane damage, measured using Yo-Pro-1 or propidium iodide (PI)] were assessed after incubation in BTS at 38 degrees C. Moreover, spermatozoa were challenged by incubation in modified Brackett-Oliphant medium (mBO+) with 37 mm of bicarbonate at 38 degrees C for 30 min, and their PMS and PMI further explored. Total sperm motility was significantly higher in P1 than in P2 along the entire process (S1-S4; p < 0.01), decreasing significantly at S4 for both fractions (p < 0.0001). The proportion of spermatozoa showing linear motility (LinM) was similar between ejaculate portions (P1 and P2), with a significant increase post-thaw (S4; p < 0.0001). During cooling (S1-S3) but not post-thaw (S4), lateral head displacement (LHD) differed between portions and changed along the stages (p < 0.01). Sperm velocity differed between portions in S1 (p < 0.01), but remained similar, independently of the portion, thereafter (S2-S4). Both PMS and the total number of live spermatozoa remained similar between S1 and S3 while incubated in BTS for both ejaculate portions. Sperm mortality increased post-thaw (S4) in both portions but the degree of lipid disorder remained low in the live cells (1.28% for P1; 1.55% for P2). Exposure to mBO+, on the other hand, significantly increased membrane lipid disorder along cooling (S1-S3; p < 0.0001), increasing the percentages of dead spermatozoa, especially post-thaw (around 70%, both portions). PS-exteriorization (AV) was not evident along the cryopreservation process in control (BTS) samples and exposure to mBO+ only induced minor variations. The data showed that kinetics, PMS and PMI of boar spermatozoa suspended in BTS (S1), LEY (S2) or LEY plus glycerol (S3) were maintained during controlled cooling but were altered by thawing, showing more characteristics of cell injury than of sperm capacitation. The spermatozoa were able to capacitate but the bicarbonate challenge destabilized the plasma membrane during initial cooling and accelerated membrane changes post-thaw. We conclude that capacitation of boar spermatozoa does not occur during controlled cooling.     

Fertility and its relationship to motility characteristics of spermatozoa in ewes after cervical, transcervical, and intrauterine insemination with frozen-thawed ram semen

The fertility of ewes after artificial insemination and the relationship between fertility and motility characteristics assessed by a computerized motility analysis system were examined with ram semen frozen in diluents reported to improve postthaw motility. The percentages of motile and progressive spermatozoa were better when frozen in proline- or glycine betaine-containing or HEPES-based, rather than Tris-based, diluents (P < 0.01). The fertility of spermatozoa frozen in diluents containing proline or glycine betaine was slightly reduced, whereas when both compatible solutes were present, the reduction was more pronounced, in comparison with semen frozen in Tris- or HEPES-based diluents (9.5 versus 71.1 and 66.6%; P < 0.01). Fertility of frozen-thawed spermatozoa was higher after laparoscopic insemination than after cervical or transcervical insemination (P < 0.01). Similarly, higher fertility was obtained after cervical insemination with fresh than with frozen-thawed semen (32.4 versus 11.3%; P < 0.01). Furthermore, loss of embryos was lower after laparoscopic insemination of ewes with semen frozen in a Tris diluent than with semen frozen in proline diluents, in glycine betaine diluents, or in proline-plus-glycine betaine diluents (0.0 versus 26.0, 38.5, and 60.0%; P < 0.001). A wide variation in the postthaw percentage of motile (31.6-59.7%) and progressive (22.6-43.1%) spermatozoa and in the fertility of spermatozoa from individual rams was also observed after laparoscopic (29.2-59.7%) or cervical insemination (8.7-30.5%). Postthaw motility results from immediately after thawing and fertility results from experiments where intrauterine insemination was performed with semen frozen in proline- or glycine betaine-containing or HEPES- or Tris-based diluents were pooled and subjected to a pairwise correlation procedure. The correlation analysis showed relationships between some of the motility characteristics (P < 0.01), but there were no relationships between the motility characteristics and fertility.     

Seminal immunoreactive relaxin in domestic animals and its relationship to sperm motility as a possible index for predicting the fertilizing ability of sires

Although immunoassayable relaxin has been detected in human and boar seminal plasma, there is no evidence suggesting the existence of immunoreactive relaxin in the seminal plasma of other domestic animals. The first objective of this study was to determine whether immunoreactive relaxin was present in the seminal plasma of bulls, rams and he-goats. In addition, the correlation of immunoreactive relaxin with sperm motility as an index for predicting the fertilizing ability of bull sires was investigated. Semen with normal sperm motility was collected from bulls, rams and he-goats, and the relaxin immunoreactivity of the semen samples was measured using a time-resolved fluoroimmunoassay (TR-FIA) for porcine relaxin that we developed. The presence of relaxin immunoreactivity was demonstrated in seminal plasma from bulls, rams and he-goats. The level of immunoreactive relaxin in seminal plasma was the highest in bulls followed by humans, rams, boars and he-goats in that order, when relaxin levels in boar and human semen having normal sperm motility were also assayed under the same conditions. When the correlation between the seminal plasma level of immunoreactive relaxin and sperm motility was examined in bull semen samples as an index for predicting fertilizing ability, it was found that the relaxin level was significantly correlated with the percentage of spermatozoa showing the most intensive motility (r = 0.64, p < 0.05). These results indicate that immunoreactive relaxin is widely found in the seminal plasma of domestic animals and that measuring the relaxin concentration of seminal plasma may be useful to identify subfertile sires or predict the fertility potential of individual sires.     

An in vitro study on reproductive toxicology of Deltamethrin on rat spermatozoa

Recent findings indicate that synthetic pyrethroid insecticide may induce toxic manifestations by enhancing the production of reactive oxygen species and disrupting the balance between pro‐oxidants and antioxidants as a result of lipid peroxidation (LP) of cell membranes. The aim of the study was to examine the potency of Deltamethrin (Del) to induce oxidative stress response in rat spermatozoa in vitro. Spermatozoa were incubated with different concentrations (0, 10, 50, 100 and 200 μm ) of Del for 3 h at 37 °C. After that, sperm parameters (motility, viability and abnormal morphology), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined. We found that in vitro exposure to Del caused a significant decline of sperm motility and viability and increase of abnormal sperm morphology, MDA, SOD and CAT levels at different concentrations of Del. This study demonstrated that Del caused deterioration in sperm motility and viability, and induction in LP, abnormal morphology of spermatozoa and antioxidants enzyme activities.     

Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME)

With the motile sperm organelle morphology examination (MSOME), spermatozoa morphology may be assessed directly on motile spermatozoa at high magnification (up to 6600×). This procedure describes more precisely spermatozoa abnormalities, especially head vacuoles. However, no consensus has been established concerning normal or abnormal MSOME criteria. The aim of our study was to define MSOME vacuole criteria assessed objectively with a digital imaging system software to establish a potential relationship between conventional semen parameters. A total of 440 semen samples were obtained from males consulting in Rouen University Hospital Reproductive Biology Laboratory. Conventional semen analysis (volume, sperm concentration, progressive motility, vitality and morphology) and MSOME assessment {sperm head length, width and area as well as vacuole number, vacuole area and relative vacuole area to sperm head [RVA (%) = [vacuole area (μm(2))/head area (μm(2))] × 100)]} were performed for each semen sample. Among our 440 males, 109 presented normal conventional semen parameters and 331 abnormal ones. Sperm head vacuoles were significantly larger in abnormal semen samples (p < 0.0001). RVA was the most discriminative MSOME criterion between normal and abnormal semen samples according to ROC curves analysis, and was negatively correlated with poor sperm morphology (r = -0.53, p < 0.0001). We concluded to (i) the normal occurrence of vacuoles in sperm head whatever the normality or abnormality of semen parameters, (ii) the discriminative function of the RVA to distinguish semen samples with normal and abnormal parameters, and (iii) the strong correlation between high RVA and poor sperm morphology.