Effects and mechanism of action of transcutaneous electrical acupuncture point stimulation in patients with abnormal semen parameters.

OBJECTIVE: To evaluate the effect of transcutaneous electrical acupuncture point stimulation (TEAS) on sperm parameters and the underlying molecular mechanisms. METHODS: A total of 121 patients diagnosed with oligozoospermia, asthenozoospermia or oligoasthenozoospermia were randomised into four groups (three treatment groups, one control): the TEAS groups were treated with 2 Hz (n=31), 100 Hz (n=31), or mock stimulation (n=29) at acupuncture points BL23, ST36, CV1 and CV4 for 2 months. The control group (n=30) was provided with lifestyle advice only. RESULTS: The changes in total sperm count and motility in the 2 Hz TEAS group were significantly greater than those in the mock group and the control group. The change in neutral α-glucosidase (NAG) and zinc levels in the 2 Hz group were significantly greater than those in the mock group and control group, and the changes in fructose levels of the 2 Hz group were significantly greater than those in the control group. Significant increases in calcium and integrin-binding protein 1 (CIB1) and reduction of cyclin-dependent kinase 1 b (CDK1) were also found after 2 Hz TEAS treatment. CONCLUSIONS: The present findings suggest that 2 Hz TEAS can improve sperm count and motility in patients with abnormal semen parameters, and is associated with increases in seminal plasma zinc, NAG and fructose. The upregulation of CIB1 and downregulation of CDK1 by TEAS may be associated with its positive effects on sperm motility and count. TRIAL REGISTRATION: http://www.chictr.org ; registration no. ChiCTR-TRC-11001775.

Effects of supplementation of Tris-egg yolk extender with royal jelly on chilled and frozen-thawed ram semen characteristics.

This study was conducted to examine the effect of supplementation of Tris-egg yolk extender with lyophilized royal jelly (RJ) on chilled and frozen-thawed ram semen parameters. Ejaculates were collected by artificial vagina from 4 mature rams, twice a week for 4 weeks. Only samples with motility of ≥70% were included, pooled and divided into four equal parts and then diluted in extenders with various concentrations of RJ (0, 1, 3 and 5%, vol/vol) to a final concentration of 200 × 106 sperm/mL and was incubated at 37 °C for 30 min and were subsequently evaluated. After equilibration of extended semen for 2 h at 4 °C, some semen samples were packed in 0.25 mL plastic straws. Then, the straws were frozen in the liquid nitrogen vapor phase for 15 min and stored at -196 °C in liquid nitrogen. The frozen straws were thawed in warm water (37 °C) for 30 s and evaluated; whereas, other semen samples were stored in the refrigerator (4 °C) up to 7 days. The chilled samples were kept in water bath (37 °C) for 5 min and then were evaluated. After dilution, the lowest and highest sperm total abnormality was recorded in 3 and 5% RJ supplemented groups, respectively (P < 0.05). The chilled sperm total motility and membrane integrity were significantly (P < 0.05) higher in 3% than those in 0% and 5% RJ supplemented groups. The chilled sperm progressive motility and viability was significantly (P < 0.05) higher in 1 and 3% than those in 0 and 5% RJ supplemented groups. The frozen-thawed sperm total motility, progressive motility, membrane integrity and viability were significantly higher in 3% RJ supplemented group (P < 0.05). In conclusion, supplementation of Tris-egg yolk extender with 3% lyophilized RJ had a protective effect on chilled and cryopreserved ram spermatozoa.

Effect of reduced glutathione (GSH) supplementation to Tris-egg yolk extender on chilled semen variables of dogs.

One reason for reduced longevity of chilled dog semen is oxidative stress. The antioxidant glutathione (GSH) improves viability of frozen-thawed dog sperm, but its effect on chilled dog semen has not been investigated. An experiment consisting of two parts was performed: Sperm rich fractions, SRF, were split, diluted with a Tris-egg yolk (TEY) extender containing 0, 5 or 10 mM GSH and stored at 4 °C for 10 days (Part 1; n = 19) or 4 days (Part 2; n = 11), respectively. For Part 1 of the study, percentage (%) of motile, viable, morphologically abnormal spermatozoa and % acrosomal deviations were assessed on days 0, 1, 2, 4 and 10 after dilution. For % sperm motility, samples from all three aliquots of each SRF (0/5/10 mM GSH) were pipetted simultaneously and analysed in a randomised order (time point of analysis, TPA). In Part 2 of the study, motility analysis was performed during 4 days storage and samples were analysed immediately after pipetting (part 2). Most investigated parameters were affected by storage time. For motility variables, there was an effect of GSH identified for circular, CM (ANOVA, Part 1: P = 0.05, Part 2: P < 0.0001) and local motility, LM (ANOVA, Part 2: P = 0.004). Furthermore, there was a trend for an interaction between time and sperm treatment for CM (Part 2: P = 0.077). In conclusion, in the present study there was not an overall positive effect of GSH addition (5/10 mM) on sperm motility in chilled dog semen samples that were characterised to be of good quality during 4- and 10-days of storage.

Free gossypol supplementation frequency and reproductive toxicity in young bulls.

The aim of the present study was to analyze seminal quality of young bulls subjected to different frequencies of gossypol supplementation. Forty-eight Nellore bulls, with 19 months of age and weighing 357.8 ±â€¯7.2 kg, were used in this study. Animals were fed with 10.5 kg of standard supplement containing free-gossypol from whole cottonseed (WCS) at the following frequency: 3x/week (G3x), 5x/week (G5x) or 7x/week (G7x - Control). Additionally, a negative control was provided, and the treated animals received only mineral supplement (MM) ad libtum. The experiment lasted for 84 days and semen was collected at the beginning and at the end for analysis and cryopreservation. Fresh semen was used for initial analysis and plasma membrane integrity and sperm morphology were also determined. General motility using computer assisted sperm analysis (CASA), plasma and acrosomal membranes integrity, mitochondrial activity, and induced oxidative stress were assessed in post-thawed semen. The study design was completely randomized. Parametric data were analyzed by ANOVA and non-parametric data by the Wilcoxon test, using the statistical program SAS. Level of significance was set at 5%. Supplementation with WCS, regardless the frequency, increased total (P = .009) and head (P = .005) defects in comparison to animals receiving only forage and mineral supplement. Infrequent supplementation, particularly 5 times in the week (G5X), increased head (P = .026) and midpiece (P = .014) abnormalities. Sperm motility in fresh semen was lower in animals that received daily supplementation than those supplemented on alternate days (P = .021). Additionally, animals supplemented daily showed lower percentage of spermatozoa with intact acrosome compared to those supplemented on alternate days (P = .005). Thus, regardless the frequency of supplementation, free-gossypol supplementation affects sperm quality. Although the amount of free gossypol supplied weekly was the same among treatments, daily supplementation compromised sperm kinetics, differently from infrequent supplementation that led to sperm defects developed during spermatogenesis.

Effectiveness of antibiotic treatment in infertile patients with sterile leukocytospermia induced by tobacco use.

Our study investigated whether antibiotic therapy in infertile tobacco users with sterile leukocytospermia and a history of former sexual transmitted disease (STD) has an advantage over cessation of smoking alone. For this study, 80 male infertile smokers were divided into two subgroups. The first group numbered 40 patients treated with a two week course of doxycycline, 100 mg, twice daily, while the second group simply ceased smoking. A control group of 20 patients with chronic abacterial prostatitis (CPPS III a) were treated with the same antibiotic for two weeks. Patients from the first group exhibited significantly higher values in semen volume, total motility, and progressive motility of spermatozoa, when compared with the second group, two weeks post treatment. The latter two measures persisted during the 12 week follow up period. In both groups of patients, the correlation coefficient between the number of leukocytes and progressive motility was determined as statistically significant. However, in the control group the correlation was not statistically significant; however progressive motility, total motility, and vitality were significantly improved after a two week course of antibiotics. Our study implies that antibiotic treatment is perhaps effective in treating sterile leukocytospermia in smokers with a former STD and is a more effective treatment option in improving sperm parameters over smoking cessation alone. ABBREVIATIONS: STD: sexual transmitted disease; CPPS IIIa: chronic abacterial prostatitis; PSA: prostatic specific antigen; EPS: expressed prostatic secretion; WBC: white blood count; LUTS: lower urinary tract symptoms; PPMT: pre-massage and post-massage urine test; VAS: visual analogue scale.

Effect of docosahexanoic acid on quality of frozen-thawed bull semen in BioXcell extender.

This study was conducted to investigate the effect of docosahexanoic acid (DHA) supplementation in BioXcell extender on the quality of frozen-thawed bull semen. Twenty-four ejaculates were collected from three bulls (eight from each bull). Ejaculates with motility ≥70% and normal morphology ≥80% were extended into BioXcell extender to which 0 (control), 3, 5, 10 or 15ngmL-1 DHA was added. The supplemented semen samples were incubated at 37°C for 15min for DHA uptake by spermatozoa. Later, samples were cooled for 2h at 5°C and packaged into 0.25-mL straws, frozen in liquid nitrogen for 24h and subsequently thawed for evaluation. Results are presented as percentages ± s.e.m. Supplementation with DHA at 3ngmL-1 significantly improved sperm functional parameters including sperm motility, normal morphology, viability, acrosome integrity and membrane integrity when compared with other supplemented groups and the control. Lipid peroxidation increased as the incorporation of DHA supplementation increased. In conclusion, 3ngmL-1 concentration of DHA resulted in superior quality of frozen-thawed bull spermatozoa and is suggested as the optimum level of DHA to be added into BioXcell extender.

The effects of tomato juice on male infertility.

BACKGROUND AND OBJECTIVES: This study aimed to investigate the effects of tomato juice consumption on seminal plasma lycopene levels and sperm parameters in infertile men. METHODS AND STUDY DESIGN: Subjects were male infertility patients with poor sperm concentration (<20×10 6/mL) and/or motility (<50%). Following a fourweek observation period, subjects were randomly assigned among three groups: a tomato juice group, an antioxidant group, and a control group. The subjects in the tomato juice group and the antioxidant group daily consumed one can of tomato juice (containing 30 mg of lycopene) or one antioxidant capsule (containing vitamin C 600 mg, vitamin E 200 mg, and glutathione 300 mg), respectively, for 12 weeks (feeding period). Seminal plasma lycopene levels and sperm parameters were measured every 6 weeks during the feeding period. RESULTS: Forty-four patients completed the study (control group: 12, antioxidant group: 15, tomato juice group: 17). In the tomato juice group, plasma lycopene level was significantly increased at the 12th week of the feeding period. Moreover, a decrease in seminal plasma white blood cells and an increase in sperm motility in the tomato juice group were statistically significant at the 12th and 6th weeks, respectively, compared to the control group. In the antioxidant capsule group, no significant improvement was observed in semen parameters. CONCLUSIONS: In conclusion, regular consumption of tomato juice seems to improve sperm motility in infertile patients. This is the first report to show that commercially available food, such as tomato juice, might be beneficial for male infertility.

Protective effects of l-carnitine on astheno- and normozoospermic human semen samples during cryopreservation.

This study was conducted to determine the effects of l-carnitine (LC), as an antioxidant, in preventing spermatozoa damage during the freezing-thawing process in both astheno- and normozoospermic human semen samples. Seventy semen samples (37 asthenozoospermic and 33 normozoospermic) were involved in this study. Cryopreservation medium supplemented with 1.0 g/l LC was mixed with semen at a ratio of 1:1 (v/v). Controls were cryopreserved with freezing medium only. Assessment of motility, viability (VIA), mitochondrial membrane potential (MMP) and DNA fragmentation index (DFI) were performed on aliquots of fresh semen, frozen-thawed control and frozen-thawed LC treated samples. Supplementation of the cryopreservation medium with LC induced a significant improvement in post-thaw sperm parameters in both the asthenozoospermic and normozoospermic semen samples, compared with those of the control, regarding sperm fast forward motility, forward motility, total motility and VIA. LC showed better protective effects towards asthenozoospermia for DFI (F = 115.85, P < 0.01) and VIA (F = 67.14, P < 0.01) than did normozoospermic semen samples. We conclude that supplementation with LC prior to the cryopreservation process reduced spermatozoa cryodamage in both asthenozoospermic and normozoospermic semen samples. LC had better protective effects for asthenozoospermic human semen samples. Future research should focus on the molecular mechanism for and the different protective effects of LC between asthenozoospermic and normozoospermic semen samples during cryopreservation.

Lutein, Trolox, ascorbic acid and combination of Trolox with ascorbic acid can improve boar semen quality during cryopreservation.

BACKGROUND: Due to pour quality of cryopreserved boar semen, artificial innsemination with frozen-thawed semen is quite limited. Developing protocols of boar semen cryopreservation represents a priority but also a challange. OBJECTIVE: The goal of the present study was to evaluate the antioxidant potential of lutein, Trolox, ascorbic acid, and certain combinations of Trolox with ascorbic acid on boar semen cryopreservation procedure. MATERIALS AND TMETHODS: Antioxidants were added to lactose-egg yolk extender, containing a final concentration of 3% glycerol and 0.5% Equex-STM. Semen of six boars was cryopreserved using straw-freezing procedure. After cryopreservation semen was thawed and evaluated for motility, normal apical ridge (NAR), hypo-osmotic swelling test (HOST) and DNA fragmentation index (DFI). Data were analyzed by one-way ANOVA. RESULTS: The results showed better motility after thawing at the concentration of 10 µM lutein, 200 µM Trolox, 200 µM ascorbic acid and 400-200 µM Trolox and ascorbic acid. The supplementation on boar freezing extender with 10 µM lutein increased post-thawed motility, NAR and HOST values (P < 0.01), and decrease DFI (P < 0.05) in comparison with control group. Similar results were obtained using 400-200 µM Trolox and ascorbic acid, with better results in the case of DFI (P < 0.01). In comparison with the control group, a concentration of 200 µM Trolox and 200 µM ascorbic acid provided significant differences (P < 0.01) of motility and NAR. CONCLUSION: The analysis of sperm characteristics showed that lutein and the mix between Trolox and ascorbic acid used in boar semen cryopreservation can improve the quality of spermatozoa.

Sage (Salvia officinalis) and fennel (Foeniculum vulgare) improve cryopreserved boar epididymal semen quality study.

The aim of this study was to evaluate the effect of fennel and sage extracts and the influence of the egg yolk source (fresh or pasteurized) on the success of freezing boar epididymal spermatozoa. In experiment 1, epididymal sperm was recovered by flushing and cryopreserved in a lactose-egg yolk solution supplemented with various concentrations (10, 5 and 2.5 g/L) of sage or fennel. Sperm quality was evaluated (motility, viability, HOST and acrosome integrity) at 0 h and 2 h after thawing. Fennel 10 g/L and sage 5 g/L and control (no extracts) were selected for experiment 2 which also compared fresh or pasteurized egg yolk in the freezing extender and measured DNA integrity of the frozen sperm. Results showed that the interaction between fennel and sage antioxidants with fresh egg yolk significantly improved post thaw sperm quality and protected boar epididymal spermatozoa from cryopreservation damage as a result of oxidative stress.

Antioxidative effects of cysteamine, hyaluronan and fetuin on post-thaw semen quality, DNA integrity and oxidative stress parameters in the Brown Swiss bull.

The aim of this study was to compare the effectiveness of antioxidants including cysteamine (2.5, 7.5 mm), hyaluronan (0.25, 1 mg ml(-1) ) and fetuin (5, 10 mg ml(-1) ) in the freezing of Brown Swiss bull semen. The best percentages of CASA motilities were achieved with 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine. For sperm morphology, 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine had better protective effects (P < 0.001). The results of hypo-osmotic swelling test showed that the percentage values of membrane integrity in all the groups, excluding that supplemented with 5 mg ml(-1) of fetuin, were higher than those of the control group (P < 0.001). Results obtained for the DNA damage of sperm cells demonstrated that 0.25 mg ml(-1) of hyaluronan, and 2.5 and 7.5 mm of cysteamine led to lower rates of spermatozoa with damaged DNA, compared with the control group (P < 0.001). The maintenance of superoxide dismutase and glutathione peroxidase antioxidant activities following freeze-thawing with 2.5 and 7.5 mm of cysteamine and 10 mg ml(-1) of fetuin was demonstrated to be at a higher level in comparison with the control group (P < 0.001). Malondialdehyde formation was found to be lower in the groups supplemented with 0.25 mg ml(-1) of hyaluronan and 7.5 mm of cysteamine after the freeze-thawing process (P < 0.001).

Supplementation of soybean lecithin-based semen extender by antioxidants: complementary flowcytometric study on post-thawed ram spermatozoa.

The purpose of the current study was to evaluate the effects of cysteine (C) and glutathione (G) on the post-thawed ram sperm quality. Collected semen samples from four mature rams were diluted with five soybean lecithin (SL)-based extenders containing: no antioxidant (SL-0), 5 mM cysteine (SL-C5), 10 mM cysteine (SL-C10), 5 mM glutathione (SL-G5) and 10 mM glutathione (SL-G10). After freeze-thawing process, motion and velocity parameters, plasma membrane integrity and functionality, morphological abnormality, lipid peroxidation, acrosomal status, mitochondria activity, and apoptosis status of post-thawed ram spermatozoa were assessed. The results showed that SL-C10 increased the total motility and plasma membrane integrity (p < 0.05) of post-thawed ram spermatozoa (55.86 ± 1.37 and 60.57 ± 1.34 %) compared to other extenders. Progressive motility was significantly higher in SL-C10 (24.71 ± 1.13 %) compared to SL-0 (20 ± 1.13 %) and SL-G10 (15 ± 1.13 %). Mitochondrial activity was significantly higher in SL-C10 (56.83 ± 2.29 %) compared to SL-G10 (38.75 ± 2.29 %). Capacitation and acrosomal status, lipid peroxidation, and the percentage of dead spermatozoa were not affected by different extenders. The percentage of live spermatozoa was higher in SL-C10 (56.33 ± 1.35 %) compared to other extenders. Also, SL-C10 resulted in a lower percentage of apoptotic spermatozoa (14.17 ± 0.53 %) compared to other extenders. The results of this study showed that supplementation of SL-based ram semen extender with 10 mM cysteine resulted in an improved quality of post-thawed ram spermatozoa.

Effect of pasteurized egg and Rosmarinus officinalis supplementation on quality of cryopreserved ram semen.

The aim was to assess the in vitro effect of pasteurized egg (PE) and rosemary (Rosmarinus officinalis) on frozen-thawed ram semen. Ejaculates from three mature rams of the Rasa Aragonesa breed were cryopreserved using a 2-step dilution method (Fraction 1: F1; Fraction 2: F2). In Experiment 1, semen was frozen in egg yolk (EY) or PE extenders. After thawing, similar results were obtained in terms of total and progressive motility, viability, hypo-osmotic swelling test (HOST) and acrosome integrity after 2 h incubation. In Experiment 2, addition of rosemary to F1, F2 or both fractions to EY extenders was evaluated. Rosemary in F1 decreased progressive motility (p = 0.013) after 2 h incubation. Finally, PE can be used as a substitute for EY to reduce hygienic risks in extenders and is easier to standardize. Supplementation of EY extender with rosemary in F1 reduced progressive motility. Rosemary supplementation in F2 does not affect semen quality.

Urinary phytoestrogen levels related to idiopathic male infertility in Chinese men.

Phytoestrogens (PEs) are naturally occurring chemical constituents of certain plants. The internal PE exposures, mainly from diet, vary among different populations and in different regions due to various eating habits. To investigate the potential relationship between urinary PE levels and idiopathic male infertility and semen quality in Chinese adult males, 608 idiopathic infertile men and 469 fertile controls were recruited by eligibility screening procedures. Individual exposure to PEs was measured using UPLC-MS/MS as spot urinary concentrations of 6 PEs (daidzein, DAI; equol, EQU; genistein, GEN; naringenin, NAR; coumestrol, COU; and secoisolariciresinol, SEC), which were adjusted with urinary creatinine (CR). Semen quality was assessed by sperm concentration, number per ejaculum and motility. We found that exposures to DAI, GEN and SEC were significantly associated with idiopathic male infertility (P-value for trend=0.036; 0.002; and 0.0001, respectively), while these exposures had stronger association with infertile subjects with at least one abnormal semen parameter than those with all normal semen parameters. Exposures to DAI, GEN and SEC were also related to idiopathic male infertility with abnormal sperm concentration, number per ejaculum and motility (P-value for trend<0.05), while these exposures had stronger association with the infertile men with abnormal sperm number per ejaculum. These findings provide the evidence that PE exposures are related to male reproductive function and raise a public health concern because that exposure to PEs is ubiquitous in China.

Effects of combined antioxidant supplementation on human sperm motility and morphology during sperm manipulation in vitro.

OBJECTIVE: To evaluate the effect of antioxidant combinations on human sperm motility and morphologic normality during preparation and in vitro incubation to improve sperm quality for assisted reproductive technology (ART). DESIGN: Prospective study. SETTING: Laboratory. PATIENT(S): Six fertile males, 21 to 25 years of age, after 3 days of sexual abstinence. INTERVENTION(S): Sperm retrieved from patients with normal fertility prepared and incubated in vitro in medium supplemented with taurine, cysteine, and/or glutathione. MAIN OUTCOME MEASURE(S): Motility indices, including motility (MOT), average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), and morphologic normality. RESULT(S): Taurine supplementation during sperm preparation had no significant effects on motility or morphologic normality. However, when sperm was incubated for 24 hours under taurine-supplemented conditions, the rates of reduction in MOT, VAP, VCL, and VSL were statistically significantly decreased compared with taurine-free conditions. Morphologic normality was maintained, regardless of taurine treatment. The optimal taurine concentration for improving sperm motility and morphologic normality during preparation and in vitro incubation was 1 mM. Subsequently, combined treatment with 1 mM taurine, 1 mM cysteine, and 1 mM glutathione induced statistically significant synergistic effects on MOT, VAP, VCL, and VSL without any alteration of morphologic normality. CONCLUSION(S): Combined treatment with taurine, cysteine, and glutathione statistically significantly ameliorated the reduction in sperm motility during in vitro manipulation of human sperm.

Effect of dietary selenium on boar sperm quality.

The primary objective of this research was to evaluate the effect of long-term dietary selenium supplementation of commercial swine diets on semen production and sperm quality. The dietary treatments were a non-supplemented basal diet or the basal diet supplemented with 0.3ppm selenium in either an organic or inorganic form. A secondary objective was to determine if there were any beneficial effects of dietary selenium supplementation on changes in sperm quality during storage of semen post collection. Boars were fed dietary treatments from weaning at 20.97±0.18 d of age until the study was terminated when they were 382.97±0.18 d of age. Boars (n=6 per treatment) were maintained on a 1 time per week collection frequency for 5 months. Immediately after this, boars were collected six times over a 4 day period. Ejaculates were extended in a commercially available, 5-day semen extender and evaluated on day 1 and 6 of storage post-collection. Boars fed the organic selenium had higher (P<0.01) plasma levels of selenium compared to control boars and similar levels to those supplemented with the inorganic form (P=0.18). Dietary treatment did not affect (P>0.2) volume, concentration, total sperm in the ejaculate, sperm motility, progressive motility, morphology, lipid peroxidation, or glutathione peroxidase activity. These results indicate that supplementing a basal diet with organic or inorganic selenium did not affect semen quantity or sperm quality in fresh ejaculates nor did it appear to have any beneficial latent effects in extended semen stored post collection.

Trehalose and glycerol have a dose-dependent synergistic effect on the post-thawing quality of ram semen cryopreserved in a soybean lecithin-based extender.

The objective of this study was to examine the interaction of different concentrations of trehalose [0 (T0), 50 (T50) or 100 (T100) mM] and glycerol [5% (G5) or 7% (G7)] on post-thawed quality of ram semen, cryopreserved in a soybean lecithin (SL)-based extender. Twenty-eight ejaculates were collected from four rams and diluted with six trehalose/glycerol combinations: T0G5, T50G5, T100G5, T0G7, T50G7, and T100G7. Sperm motility (CASA), membrane integrity (eosin/nigrosin) and functionality (HOST), abnormal forms, capacitation status (CTC), mitochondrial activity (rhodamine 123), apoptotic features (Annexin V/propidium iodide) and lipoperoxidation (malondialdehyde production) were evaluated after thawing. Extender T100G5 yielded the highest results for total and progressive motility, sperm velocity, normal morphology, functional membranes, active mitochondria and membrane integrity, with P<0.05 in general, except for T50G7 (P>0.05). The combinations T0G5, T0G7 and T100G7 yielded the lowest post-thaw quality. We could not detect significant changes in other kinematic parameters, capacitation status or lipoperoxidation. We conclude that, in our SL-based extender, a combination of 100 mM trehalose and 5% glycerol was the most adequate combination to achieving post-thawing quality in our soybean lecithin-based extender, and our results support that a synergistic effect among trehalose and glycerol exists. We suggest that other combinations could improve these results.

Can permeable super oxide dismutase mimetic agents improve the quality of frozen-thawed ram semen?

This study was carried out to assess the effects of MnTBAP, a cell permeable antioxidant, on motility, membrane integrity, capacitation status and in vitro fertilization ability of frozen-thawed ram semen. Fresh semen ejaculates were collected with artificial vagina from five rams, mixed and divided into five equal fractions, and diluted (1:20 v/v) with commercial extender, Bioxell®, containing 0 (control), 50, 100, 150 and 200 µM of MnTBAP. All diluted sperm suspensions were cooled to 5°C for 2h followed by transfer into 0.5 ml French straws before being stored in liquid nitrogen. The results showed that MnTBAP supplementation of extender improved ram semen quality in a dose-dependent manner. Accordingly, the extender supplemented with 150µM MnTBAP resulted in higher sperm motility and improved acrosomal membrane integrity compared to control. However, further supplementation (200µM) with MnTBAP not only did not improve the results but inversely affected motility and membrane integrity. The results of in vitro fertilization (IVF) indicated that the presence of MnTBAP in semen extender has a marginal beneficial effect on developmental competence of inseminated oocytes, though this improvement was not significant. In conclusion, this study demonstrated that semen extender supplemented with MnTBAP can reduce the oxidative stress provoked by freeze/thaw processes. Moreover, beneficial effect of 100 µM of MnTBAP on preservation of spermatozoa in a non-capacitated state post freezing, an important criterion for in vitro or in vivo fertilization, was observed. However, at 150 µM of MnTBAP, the harmful effects of cryopreservation on membrane integrity were decreased. Regarding to importance of non-capacitated spermatozoa during IVF or artificial insemination, the optimum MnTBAP concentration appears to be 100 µM for commercial ram semen extender tested here.

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen.

The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P < 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P < 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P > 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P > 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen.

Qualidade espermática e perfil plasmático de lipídeos em garanhões suplementados com óleo de arroz semirefinado com alto teor de gama-orizanol / Spermatic quality and plasmatic lipids in stallions supplemented with rice bran oil semirefined containing high level of gamma-oryzanol

O gama-orizanol é uma substância natural contida no óleo de arroz, que por suas características hipocolesterolêmicas e antioxidantes, têm sido objeto de estudo em humanos e, mais recentemente, em equinos. Foram utilizados seis garanhões, de várias raças, com idade de 10±5,4 anos e peso inicial de 472,67±90,48 kg. Objetivou-se verificar os efeitos da suplementação da dieta com óleo de arroz semi-refinado, contendo 1,1% de gama-orizanol, sobre os níveis de lipídeos plasmáticos (colesterol total, HDL-C, LDL-C, VLDL-C e triglicérides), de testosterona e da qualidade espermática. Os garanhões foram divididos em dois grupos, recebendo em cada refeição 150 mL de óleo de soja ou de arroz durante 60 dias. Realizaram-se colheitas amostrais de sangue e sêmen a cada 15 dias do período experimental. O delineamento experimental foi inteiramente casualizado, com medidas repetidas no tempo, e as médias comparadas pelo teste F, considerando-se o nível de 5% de significância. Os valores médios de testosterona, colesterol total, HDL-C, LDL-C, VLDL-C e triglicérides foram respectivamente 75,93 ng/dL; 92,73; 61,47; 26,99 e 4,28 mg/dL para o tratamento com óleo de soja; e de 62,13 ng/dL; 110,20; 66,73; 38,44 e 5,02 mg/dL para o tratamento com óleo de arroz. Em relação à qualidade espermática, não foi observada diferença (p<0,05) entre os tratamentos nas variáveis: volume, motilidade, concentração e defeitos. Podemos concluir que a suplementação supracitada não influencia a qualidade espermática nem as concentrações plasmáticas de testosterona, VLDL-C, HDL-C e triglicérides, porém, pode elevar as concentrações plasmáticas de colesterol total e de LDL-C.

Gamma-oryzanol is a natural substance contained in rice bran oil that has been studied for years in humans due to its hypocholeterolemic and antioxidant properties, and in recent years in horses. Using six stallions from different breeds in a randomized experimental design, weighing 472.67±90.48 kg and aging 10±5.4 years, this study was aimed to evaluate the effects of supplementation with rice bran oil containing 1.1% of gamma-oryzanol in plasmatic lipids levels (total cholesterol, HDL-C, LDL-C, VLDL-C and triglycerides), testosterone and sperm quality. Stallions were divided in two groups, one receiving 150 mL of soybean oil, and the other 150 mL of rice bran oil twice a day, during 60 days. Blood and sperm samples were collected every 15 days of the trial period. Data obtained was processed, and means compared using F test, at 5% of confidence level. Mean values of total cholesterol, HDL-C, LDL-C, VLDL-C, triglycerides and testosterone were respectively 92.73; 61.47; 26.99; 4.28 mg/dL and 75.93 ng/dL in the soybean oil supplemented group, and 110.20; 66.73; 38.44; 5.02 mg/dL and 62.13 ng/dL in the rice bran oil one. No difference among treatments (p<0,05) were observed in sperm quality parameters volume, motility, concentration and defects. Supplementation of stallions with rice bran oil containing 1.1% of gamma-oryzanol do not promote any change in sperm quality, testosterone, HDL-C, VLDL-C or triglycerides plasmatic levels, but an increase in total cholesterol and LDL-C.