Sperm creatine phosphokinase activity as a measure of sperm quality in normospermic, variablespermic, and oligospermic men

We have found a significant inverse correlation between sperm concentrations and sperm creatine N-phosphotransferase (CPK) activities in oligospermic and normospermic human specimens. In the present work, we carried out serial CPK determinations to assess whether there is a relationship between fluctuating sperm concentrations and sperm quality in consistently oligospermic and variablespermic (sperm concentrations are occasionally in the greater than 20 million/ml range) husbands of 65 couples (23 normospermic men/51 samples, 25 consistently oligospermic men/80 samples, and 17 variablespermic men/68 samples). The sperm CPK activities were significantly lower in the normospermic vs. the oligospermic or variablespermic groups (p less than 0.001), but there were no differences between the latter two (p greater than 0.25). The mean CPK values of migrated sperm fractions in both the oligospermic and variablespermic populations were improved (at least 20% decline in CPK values) compared to those of the initial specimens (1.27 +/- 0.38 vs. 0.68 +/- 0.37 and 0.77 +/- 0.32 vs. 0.46 +/- 0.24 SEM U/100 million sperm, respectively, p less than 0.001 in both pairs) and the incidence of the "failed-to-improve" samples was also similar in the two groups (44/36 vs. 45/23, p greater than 0.2). The lack of differences in the mean CPK activities, in the distribution of CPK values under and over 0.250 U/100 million sperm level, and in the ratio of migrated samples with improved or with failed-to-improve CPK activities suggests that sperm quality is not different between men who are consistently oligospermic and those who occasionally produce normospermic specimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa

We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39°C) in a modified Tyrode's solution (TALP) containing (1) Chol-free liposomes (—Chol, 50 × 10⁶ sperm and 600 nmol phospholipid/ml); (2) liposomes containing 30 mol% Chol (+ Chol, 2 × 10⁸ sperm and 300 nmol total lipid/ml); or (3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with —Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 × 10⁶) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-μl droplets of TALP under medical fluid in an atmosphere of 5% CO₂ in air (3 h, 39°C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined. Percent penetration (%P) of HO (X ± SEM) for 30 and 40 μg of LPC/mg BSA was 59.4 ± 5.3 and 82.9 ± 5.4; 38.5 ± 5.6 and 52.3 ± 4.7; and 16.0 ± 4.6; and 23.2 ± 5.6 for —Chol, Control, and + Chol treatments, respectively (n = 3). %P of BO (X ± SEM) for 30, 35, and 40 μg of LPC/mg BSA was 43.3 ± 5.4, 70.7 ± 7.5, and 81.5 ± 5.1 for —Chol and 16.4 ± 6.9, 36.2 ± 6.9, and 44.2 ± 8.6 for Control treatments, respectively (n = 3). In a second set of experiments %P of BO (X ± SEM) was 63.6 ± 6.8, 31.8 ± 4.9, and 10.5 ± 3.4 for —Chol, Control, and +Chol treatments, respectively, when 40 μg LPC/mg BSA was added (n = 2). %P and the number of sperm per fertilized ovum were consistently higher for the —Chol treatment for both HO and BO (P < .01). These results demonstrate that Chol removal from bovine sperm facilitates penetration of ova in vitro suggesting a potential role in bovine sperm capacitation.     

Intérêt du dosage de l’inhibine B et de l’AMH dans le plasma séminal: étude préliminaire

Objectives

The aim of this study was to establish reference values for seminal inhibin B and AMH concentrations in patients with normal and abnormal sperm parameters. Preliminary analysis was performed to evaluate the predictive value of these markers for retrieving testicular sperm in non-obstructive azoospermic men.

Methods

Seminal inhibin B and AMH concentrations were assayed by an enzyme-linked immunoassay in three groups of men: 47 patients with normal sperm parameters, 28 oligospermic men and 68 patients with azoospermia.

Results

Inhibin B and AMH concentrations varied considerably in the three groups, but were significantly higher in normospermic men (inhibin B: 714.36±522.66 ng/l, AMH: 97.08±135.15 pmol/l) than in oligospermic men (inhibin B: 417.5±386.9 ng/l, AMH: 62.02±93.33 pmol/l) and azoospermic men (59.61±2.65 ng/l et 13.12±31.94 pmol/l, respectively) (p<0.001). A significant correlation (p=0.0054) was observed between seminal inhibin B concentration and sperm production. Testicular biopsy allowed sperm retrieval in 11 out of 21 patients (52.3%). The predictive value of seminal inhibin B was analyzed using receiver operating characteristics (ROC) curve analysis. The best discriminating inhibin B concentration was 30 ng/l with an area under the curve (AUC) of 0.63.

Conclusion

This study confirms the correlation between seminal inhibin B and AMH concentrations and spermatogenesis. However, the significance of these two markers as predictors of the presence of testicular sperm in men with non-obstructive azoospermia is limited. This analysis shows that AMH and inhibin B, either alone or in combination with serum FSH, fail to predict the presence of sperm in men with non-obstructive azoospermia undergoing testicular sperm extraction.     

Cholesterol and phospholipid levels of washed and percoll gradient centrifuged mouse sperm: Presence of lipids possessing inhibitory effects on sperm motility

Levels of DNA, cholesterol, and phospholipids of mouse caudal epididymal and vas deferens sperm that were processed through simple washing and Percoll gradient centrifugation were measured. The DNA and cholesterol contents of washed sperm and Percoll gradient centrifuged (PGC) sperm (DNA = 3.6 ± 0.3 pg/sperm and 3.4 ± 0.3 pg/sperm, respectively; cholesterol = 0.219 ± 0.057 nmole/μg DNA and 0.224 ± 0.030 nmole/μg DNA, respectively, for washed and PGC sperm) were not significantly different from each other; however, the phospholipid level of PGC sperm was only one half of that of washed sperm (0.315 ± 0.071 nmole/μg DNA versus 0.720 ± 0.075 nmole/μg DNA, respectively). The presence of 0.3% bovine serum albumin (BSA) in the culture medium used in sperm washing did not change the cholesterol and phospholipid contents of washed sperm. Similarly, the cholesterol and phospholipid levels of washed sperm and PGC sperm that were further incubated in BSA-containing medium for 30 min remained the same. Interestingly, substantial amounts of lipids, as determined by the cholesterol and phospholipid levels, were released into the supernatants of the sperm washes, and sperm needed to be washed at least twice to ensure their stable levels of cholesterol and phospholipids. The lipid mixture in the first sperm wash supernatant was shown to have inhibitory effects on PGC sperm motility. © 1996 Wiley-Liss, Inc.     

Serum and semen levels of immunoreactive prolactin, LH and FSH in normospermic, oligospermic and azoospermic men with obstructive infertility prior to and after vasoepididymostomy.

Attempts were made to validate RIA for prolactin (PRL), LH and FSH in semen from normospermic, oligospermic and azoospermic subjects. The RIA used to measure PRL and LH in semen fulfilled the criteria of reliability, whereas low levels of FSH in semen precluded the validation of FSH assay in semen. Semen levels of PRL and LH were significantly (P < 0.05) higher than serum levels in all groups of subjects investigated. Semen levels of FSH in azoospermic men after vasoepididymostomy (VEA), were significantly (P < 0.05) higher compared to azoospermic men prior to surgery. Serum levels of PRL were significantly higher (P < 0.05) in normospermic men compared to oligospermic and azoospermic men prior to and after surgery. Semen levels of PRL in normospermic men were comparable with oligospermic and azoospermic subjects prior to and after surgery. Serum levels of LH in oligospermic and azoospermic men who did not undergo surgery and in men reporting oligospermia after VEA were comparable to normospermic subjects but in men showing azoospermia post surgically, serum LH levels were significantly (P < 0.005) elevated. Semen levels of LH in men reporting azoospermia before surgery and in subjects showing oligospermia or azoospermia post surgically were significantly lower (P < 0.05) compared to men with normal sperm count. Serum levels of FSH were significantly elevated (P < 0.05) compared to semen levels in oligospermic men prior to surgery but this increase was not seen in post VEA subjects. These results were discussed.     

Comparison of sperm viability with seminal plasma metal levels

This study compares the semen levels of lead (Pb), cadmium (Cd), and aluminum (Al) in relation to live sperm in semen samples from 64 apparently healthy men. The measured levels were separated into live sperm count tertiles (<25% [18 subjects], 25–50% [26 subjects], and >50% [20 subjects]). The mean ± SD for each group was calculated, and the difference between the means of the high and low tertiles were compared by ANOVA. Significant differences were observed between the high and low live sperm groups for Pb (p < 0.01) and Al (p < 0.05), but not Cd. Spearman’s rank correlation between sperm viability and the semen plasma metal levels showed a direct relation to Mg (p < 0.05). However, there was an inverse relation to lead (p < 0.001), cadmium (p < 0.01), and aluminum (p < 0.01). There was no significant correlation between Ca and Zn. Linear regression between the live sperm counts and semen level of the three metals show that metal levels were inversely correlated with the percentage of live sperm (p < 0.001, <0.01). Apparently, the presence of these metals in the environment and in seminal plasma exerts a toxic effect on sperm.     

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.     

Dietary omega-3 fatty acids from linseed oil improve quality of post-thaw but not fresh sperm in Holstein bulls

Docosahexaenoic acid (DHA), a member of the n-3 fatty acid family present in fish oil, has several positive effects on bovine sperm, including membrane integrity, motility and viability, as well as cold sensitivity. Our objective was to investigate effects of varying amounts of omega-3 fatty acids from linseed oil, administered orally, on quality of fresh and frozen-thawed bull sperm. Twenty fertile Holstein bulls (874 ± 45.38 kg) were randomly and equally assigned to four groups and received encapsulated (rumen-protected) fats for 12 weeks, as follows: group P, 300 g palm oil; group Pl, 200 g palm oil + 100 g linseed oil; group pL, 100 g palm oil + 200 g linseed oil; and group L, 300 g linseed oil. Sperm quality of fresh and frozen-thawed semen was evaluated by routine assays including sperm motion characteristics (CASA), membrane integrity (eosin-nigrosin), membrane activity (hypo-osmotic swelling test; HOST) and malondialdehyde (MDA) content. There were no significant differences among groups in semen volume, sperm concentration or sperm quality parameters in fresh semen. However, after freezing-thawing, total and progressive motility in group P (59.61 ± 1.95 and 40.19 ± 2.48%, respectively; LSM ± SEM) were lower (P < 0.05) than in groups Pl (66.06 ± 1.95 and 47.53 ± 2.48%), pL (65.67 ± 1.95 and 47.48 ± 2.48%) and L (65.36 ± 1.95 and 47.62 ± 2.48)%, with no significant differences among the latter three groups. Furthermore, membrane integrity (eosin-nigrosin) and activity (HOST) were lower (P < 0.05) in group P (55.79 ± 2.15 and 42.19 ± 2.17%) compared to groups Pl (62.73 ± 2.15 and 48.93 ± 2.17%), pL (64.06 ± 2.15 and 50.01 ± 2.17%) and L (64.47 ± 2.15 and 49.68 ± 2.17%), with no significant differences among the latter three. Furthermore, there were more (P < 0.05) morphologically abnormal sperm in group P (25.99 ± 1.62%) than in groups Pl, PL and L (21.55 ± 1.62, 21.69 ± 1.62 and 20.90 ± 1.62%). In conclusion, feeding Holstein bulls 100–300 g linseed oil daily improved sperm cryotolerance.     

Human Semen Refrigeration at + 4 °C: Bio-kinetic Characteristics

The aim of our study was to evaluate the bio-kinetic characteristics of human semen refrigerated for different periods and to compare the effects of refrigeration at +4 °C against cryopreservation of human sperm at −196 °C. Semen was obtained from 30 male partners of infertile couples (infertile subjects) with the following semen profile: sperm count ≥10 × 10⁶/ml; progressive motility ≥20%; atypical forms <70% and white blood cells <1.0 × 10⁶/ml. Fifteen normospermic subjects were also selected as controls (control subjects). The following tests were carried out on basal, refrigerated and cryopreserved sperm: a) sperm kinetic properties (by Superimposed Image Analysis System); b) the Hypoosmotic Viability Test (HVT) (combined Hypoosmotic Swelling and Viability Test). The results of the study showed that the percentage recovery of kinetic properties and of HVT were optimum for up to 48 h. After refrigeration for 72 h, a drastic decrease in straight motility recovery was observed. No significant differences were observed between cryopreservation and refrigeration at +4 °C for 48 h for motility or HVT recoveries in samples from control subjects. However, in infertile subjects, a significant decrease in straight progressive motility and HVT recoveries was observed in cryopreserved samples compared to those refrigerated for 48 h. Neither refrigeration nor cryopreservation led to the growth of pathogenic bacteria in any of the cases studied. Based on the above results, refrigeration could represent a useful alternative to the cryopreservation method.     

Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation

In human in vitro fertilization (I.V.F.), it was first assumed that all the embryos obtained had the same developmental potential whatever the quality of sperm. However, this has not been confirmed. We have used the coculture technique and determined the blastocyst formation rate in three groups of patients: group 1: patients with normal sperm count (>20 × 10⁶/ml), motility (>30%), and morphology (>50%); group 2: patients treated by I.V.F. with frozen donor sperm; group 3: patients with severely impaired sperm quality (<3 × 10⁶ forward motile and morphologically normal spermatozoa per ml). In group 1, we found a strong correlation between cleavage rate and blastocyst formation rate (P < 0.0001) with a blastocyst formation rate comprised between 40% and 50%. This was not true for the two other groups for which the overall number of blastocysts obtained and the number of patients having at least one blastocyst were severely reduced (P < 0.0001). These data are discussed in terms of DNA quality, timing of formation of the pronuclei, and delays in cell cycles at the time of genomic activation. These observations lead to a new approach to the study of fertilizing ability of poor quality sperm. It may help in the decision as to whether couples treated for male infertility should be excluded from I.V.F. protocols. © 1994 Wiley-Liss, Inc.     

Exposure by males to light emitted from media devices at night is linked with decline of sperm quality and correlated with sleep quality measures

ABSTRACT

The last several decades have been characterized by the widespread usage of digital devices, especially smartphones. At the same time, there have been reports of both decline in sleep duration and quality and male fertility decline. The aim of this study was to assess the relationship between evening exposure to the light-emitting screens of digital media devices and measures of both sleep and sperm quality. Semen samples were obtained from 116 men undergoing fertility evaluation for the following sperm variables: volume (mL), pH, sperm concentration (million/mL), motility percentage (progressive% + non-progressive motility%), and total sperm count. Exposure to the screens of electronic devices and sleep habits was obtained by means of a questionnaire. Smartphone and tablet usage in the evening and after bedtime was negatively correlated with sperm motility (?0.392; ?0.369; p < .05), sperm progressive motility (?0.322; ?0.299; p < .05), and sperm concentration (?0.169; p < .05), and positively correlated with the percentage of immotile sperm (0.382; 0.344; p < .05). In addition, sleep duration was positively correlated with sperm total and progressive motility (0.249; 0.233; p < .05) and negatively correlated with semen pH (?0.349; p < .05). A significant negative correlation was observed between subjective sleepiness and total and progressive motility (?0.264; p < .05) as well as total motile sperm number (?0.173; p < .05). The results of this study support a link between evening and post-bedtime exposure to light-emitting digital media screens and sperm quality. Further research is required to establish the proposed causative link and may lead to the future development of relevant therapeutic and lifestyle interventions.     

Hereditary haemochromatosis gene (HFE) H63D mutation shows an association with abnormal sperm motility

The aim of this study was to screen infertile men for HFE H63D mutation in correlation with clinical characteristics of infertile men (sperm concentration, sperm motility, morphology, testicular volume, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and total Testosterone levels) and find out if the HFE H63D mutation has an effect on male infertility. After excluding hormonal treatment, any scrotal pathology, having any systemic diseases such as diabetes mellitus, sickle cell anemia and microdeletions of the Y chromosome, a total of 148 infertile men with age range between 17 and 52-years-old (average age 29.6 ± 7.2) were enrolled into the study. Our analysis indicates that the mean FSH levels are significantly higher (6.3 ± 4.6 mIU/ml, P = 0.03), whereas sperm motility is significantly lower (36.6 ± 28.1%, P = 0.01) in the infertile men with the HFE H63D mutation compared with subjects lacking this mutation. Comparison of allele frequencies of the infertile men with Ts < 50% versus the infertile men with Ts > 50% revealed a significant difference as expected (P = 0.001, OR = 0.14, %95 CI = 0.04–0.44). Comparison of allele frequencies of infertile men with abnormal sperm motility versus infertile men with normal sperm motility revealed a highly significant difference (P = 0.005, OR = 3.11, %95 CI = 1.41–6.86). Thus, the HFE H63D mutation seems to be an important risk factor for impaired sperm motility and is clinically associated with male infertility.     

In vitro-cultured bovine granulosa and oviductal cells secrete sperm motility-maintaining factor(s)

Since bovine cumulus oophorous and oviductual cell cultures are known to support and maintain frozen-thawed bovine sperm viability and motility for extended time periods, we investigated whether granulosa cell (GC)- and oviductual cell (OC)-conditioned media have similar effects. GC and OC were cultured for 3 days in TCM-199 medium supplemented with 10% fetal calf serum. At that time, the supernatant was discarded from GC and the monolayers were covered with Sp-TALP medium containing 6 mg/ml bovine serum albumin, while the OC were recovered by centrifugation and transferred to culture bottles containing Sp-TALP. Two days later, GC-conditioned and OC-conditioned Sp-TALP were recovered and dialyzed, and their retentates were lyophilized. Bovine follicular fluid (BFF) was also dialyzed, and its retentate was lyophilized. When sperm were incubated in GC- or OC-conditioned media, motility remained above 62% and 42% at 6 hr and 30 hr, respectively, and motility was higher than that of the control both at 6 hr (39%; P < 0.001) and at 30 hr (9%; P < 0.0001). Similarly, when sperm were incubated in the lyophilized retentates of GC- and OC-conditioned media and in BFF at a dose of 0.1, 0.5, or 1.0 mg/ml, the motility rates were higher both at 6 hr (P < 0.05) and at 30 hr (P < 0.01) compared to the control. The increase in motility was dose dependent; a 1.0 mg/ml dose improved (P < 0.05) motility compared to a 0.1 mg mg/ml dose. Heat treatment of the retentates of GC, OC, and BFF at 55°C for 30 min did not destroy their ability to support and maintain motility. However, heating at 100°C for 5 min destroyed their ability to support motility. Molecular sieving of retentates on Sephancryl S-300 yielded fractions that were highly effective (P < 0.01) in enhancing and maintaining motility compared to the other fractions. In conclusion, GC and OC secrete nondialyzable, heat-labile factor(s), which support and maintain sperm viability and motility for up to 30 hr. © 1994 Wiley-Liss, Inc.     

Chilled storage of Asian seabass,Lates calcarifer Bloch semen: Effects of ions,extenders and storage periods on sperm quality and fertilization ability

The development of a chilled storage protocol of fish sperm requires an understanding of sperm biology and function as the activation/inhibition of fish sperm is greatly affected by several environmental factors. This study aimed to determine the effects of ionic and non-ionic solutions, extender types (Ringer's solution, Ca-F HBSS solution, HBSS solution, He and Wood solution, Saline solution, and Modified Cortland solution), and chilled-storage period on sperm quality and fertilization ability of Asian seabass, Lates calcarifer semen. Regulation of Asian seabass sperm motility was dependent on the osmolality of both ionic and non-ionic activation media. The threshold levels on the initiation of sperm motility were detected in KCl (>100 mM), NaCl (>50 mM), CaCl₂ (>50 mM), glucose (>300 mM), and mannitol (>100 mM) solutions. Relatively high percentages of sperm motility (>80%) were observed when activated with KCl, NaCl, CaCl₂, glucose, and mannitol solutions at above 700, 600, 350, 1,000, and 1,000 mM, respectively. Ringer's solution was the most optimal extender for chilled storage of Asian seabass semen at 2–4°C supported by the retention of sperm motility and viability for 6 days. Semen diluted in Ringer's solution and chilled-stored for 2 days exhibited acceptable fertilization (66.1% ± 6.2%) and hatching (56.4% ± 2.9%) rates. This report, for the first time, describes the ionicity and non-ionicity effects on the motility of Asian seabass sperm.     

The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm

Adding cryopreservation media with reduced glutathione (GSH) has previously been shown to maintain the motility, membrane integrity and fertilizing ability of frozen-thawed boar sperm, although the effects of GSH on good (GFE) and poor freezability (PFE) ejaculates rely upon the intrinsic ejaculate freezability. The resilience to withstand freeze-thawing procedures has previously been related to the existence of a specific distribution of motile sperm subpopulations, which differs between GFE and PFE. Thus, the main aim of this study was to determine whether the addition of GSH to freezing media has any impact on the distribution of motile sperm subpopulations in GFE and PFE. With this purpose, 18 GFE and 13 PFE were cryopreserved with or without 2 mM GSH. Sperm quality and motile subpopulations were evaluated at 30 min and 4 h post-thawing. Three subpopulations were identified and the percentages of spermatozoa belonging to the fastest and most linear subpopulation, which was referred as ‘SP1’, decreased over post-thawing time. Good freezability ejaculates that were cryopreserved in the presence of 2 mM exhibited a significantly higher percentage of spermatozoa belonging to SP1 than the other combinations of treatment and freezability both at 30 min (mean ± SEM: GFE-C: 16.6 ± 0.4; GFE-GSH 27.7 ± 0.6) and 4 h post-thawing (GFE-C: 7.8 ± 0.2 vs. GFE-GSH: 16.7 ± 0.4). In conclusion, the positive effect of GSH on the motility of frozen-thawed sperm is related to a specific sperm subpopulation (SP1), which could coincide with the fertile sperm one.     

An examination on composition of spermatozoa obtained from pre-operative and post-operative varicocele patients

Varicocele is one of the main and surgically repairable causes of male infertility, which arises from dilatation and torsion of the testicular veins in the plexus pampiniformis. In this study, we examined semen samples from 40 patients diagnosed with varicocele between the ages of 15 and 30 years, according to WHO criteria (pre-operatively, and at 3, 6, and 12 months post-operatively). The mean spermatozoa concentration was found to be 45.25?±?34.83 million/ml pre-operatively, while the mean post-operative concentration was 48.85?±?35.73 million/ml at three months, 51.72?±?32.82 million/ml at six months, and 49.63?±?28.05 million/ml at 12 months (P?>?0.05). The mean rate of A?+?B motile spermatozoa was 35.5?±?14.71% pre-operatively, 42.65?±?16.80% at three months, 43?±?13.52%at six months and 44?±?14.76 percent at 12 months post-operatively (P?<?0.05). The mean Kruger morphology score was 3.15?±?3.0% pre-operatively, and 3.20?±?2.83% at three months, 2.97?±?2.61%at six months and 3.27?±?2.50%at 12 months post-operatively (P?>?0.05). The nucleus, acrosomal cap, mitochondrial structure and microtubules of the tail of the spermatozoa were examined under electron microscopy. The mean DNA fragmentation index (DFI%) of the spermatozoa was 20.57?±?4.60% pre-operatively, and post-operatively at 17.27?±?3.65% at three months, 15.5?±?3.23% at six months and 15.3?±?3.63% at 12 months (P?<?0.001).The findings suggest that despite the increased count and motility, as well as the improved DNA fractures observed post-operatively in the spermatozoa of varicocele patients, the morphology rates remain intact.     

Influence of suckling status and type of birth on serum hormone profiles and return to estrus in early-postpartum spring-lambing ewes

Twenty-three mature, spring-lambing, fine-wool ewes of Debouillet × Rambouillet breeding were allotted at parturition to one of four treatments which were arranged in a 2 × 2 factorial design with groups representing number of lambs born (i.e., one or two) and suckling intensity (i.e., lambs were weaned at 2 d of age or lambs remained with dams). Beginning at 0900 h on Day 2, 9, 16, 23, and 30 post partum (PP), jugular blood samples were collected from each dam at hourly intervals for the ensuing 6 h. Additional jugular blood samples were collected daily (Days 2 through 30). Animals were observed twice daily for signs of estrus using vasectomized rams. Interval from parturition to estrus (mean ± SEM) was similar (P > 0.15) in ewes nursing their offspring (117 ± 6 d) and those that had their lambs removed (124 ± 6 d). Dams producing single lambs returned to estrus in 126 ± 5 d compared with 116 ± 5 d (P > 0.15) for ewes producing twins. Serum luteinizing hormone and progesterone were low (< 1.7 and 0.5 ng/ml, respectively) in all ewes during the first 30 d PP. Serum insulin did not differ (P > 0.10) between suckled dams and those that had their lambs removed, but ewes giving birth to single offspring had higher (P < 0.05) insulin levels on Days 16 and 30 PP (543 ± 73 and 578 ± 57 pg/ml, respectively) than did dams producing twin lambs (324 ± 73 and 361 ± 57 pg/ml, respectively). Serum growth hormone (GH) did not differ (P > 0.40) between suckling intensity groups on Day 2 PP; however, by Days 16, 23, and 30, ewes in the suckled group had more (P < 0.05) GH than did those producing single offspring (5.4 and 3.6 ± 0.4 ng/ml, respectively). Early removal of lambs in spring-lambing ewes did not shorten the interval from parturition to estrus.     

Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters

The aim of the current study was to evaluate the effects of cysteine and ergothioneine on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities. Semen samples from 5 mature Merino rams were used in the study. Semen samples, which were diluted with a Tris-based extender containing l-Cysteine and l-(+)-Ergothioneine and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for evaluation.Ergothioneine at doses of 2 and 4 mM increased percentages of subjective motility, VSL and VCL, compared to controls following the freeze-thawing (P < 0.001). Ergothioneine at three different doses led to higher rates of progressive motility and VAP, compared to control groups (P < 0.001). Cysteine and ergothioneine at three doses provided the higher rates of ALH, in comparison to no antioxidant group (P < 0.001). As regards CASA motility, supplementation with antioxidants did not provide any significant difference on the percentage of post-thaw sperm CASA motilities, in comparison to the control. In regards of sperm membrane integrity, only cysteine 1 mM provided a greater protective effect, compared to control (P < 0.001). Percentages of sperm with high mitochondrial activity were dramatically increased with cysteine at doses of 1 and 2 mM, compared to control (P < 0.05). No significant differences were observed in sperm acrosome integrities among groups. CAT activity was increased significantly only in cysteine1 mM compared to control group (P < 0.001). Cysteine at doses of 2 and 4 mM showed a tendency of increased activities of CAT when compared to control. But these increases were not statistically significant. Supplementation with antioxidants did not significantly affect activities of SOD and GPx. Findings of this study showed that ergothioneine supplementation in semen extenders, was of greater benefit to motility and motion characteristics of frozen-thawed ram sperm.     

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.