Sperm Hypo-osmotic Swelling Test Research Articles

Heparin binding proteins in seminal plasma of breeding buffalo bulls and their relation with semen freezability and in vivo fertility

The present study was conducted to determine the concentrations of heparin binding proteins (HBP) in seminal fluid of breeding buffalo bulls and establish their relationship with frozen-thawed semen characteristics and fertility. Both fresh and frozen semen were collected from same ejaculate of 30 breeding buffalo bulls. Based on HBP concentrations in seminal plasma obtained from affinity chromatography, all the bulls were divided into 2 groups, viz. high fertility bulls (HFB, ≥ 6.5 mg/ml; n=15) and low fertility bulls (LFB, < 6.5 mg/ml; n=15) for further comparisons. The frozen-thawed semen of same ejaculate was evaluated for per cent CASA-based sperm kinetics, viability, Hypo-osmotic swelling test (HOST), acrosome integrity, in vitro acrosome reaction and first service conception rate (FSCR). The overall HBP concentrations were significantly higher in seminal plasma (7.31±0.14 vs 5.33±0.19 mg/ml) and fresh (0.66±0.03 vs 0.59±0.02 mg/109 sperms) sperm extracts of HFB than LFB. The frozen-thawed semen characteristics, viz. total motility, per cent acrosome reaction and FSCR were also significantly higher in bulls with high fertility than in lower ones. No significant difference was observed in sperm viability, HOST and acrosome integrity in the 2 groups of bulls. Total 12 HBP bands in range of 11 to 135 kDa were recorded on SDS-PAGE. Higher levels of HBP in seminal plasma of HFB might be responsible for their better semen quality and fertility.

Expression study of fertility related genes with respect to sperm motility, HOST and acrosome integrity in bovine semen

The present study was conducted for molecular evaluation of bull semen and to study its relationship with different semen characteristics. Fresh semen samples were collected from six breeding bulls. A total of six ejaculates were collected from each of six healthy breeding Jersey bulls at 4 days interval. Immediately after collection, the samples were subjected to physio-morphological and biomolecular evaluation. Percentage of Hypo-osmotic Swelling Test (HOST) positive spermatozoa and acrosome-intact sperms increased with an increase in initial sperm motility. Expression of Chaperonin Containing T-Complex protein 1, subunit 8 (CCT8) gene was found to be negatively correlated with sperm motility, whereas the expression of Adenylate Kinase 1 (AK1) gene did not show any significant relationship with sperm motility.

Does natural honey act as an alternative to antibiotics in the semen extender for cryopreservation of crossbred ram semen?

Antibiotics are added to semen extenders to take care of heavy microbial load, however, their continuous use poses a constant threat of developing antibiotic resistance by the common microbes present in the semen. Our hypothesis was that natural honey, having antibacterial activity and rich in fructose could replace the use of antibiotics and fructose in the semen extender. Twenty-four ejaculates from six crossbred rams were obtained and extended with tris-based extender without (control) and with honey at 2.5% (T1), 5% (T2) and 7% (T3). Sperm quality was measured in terms of percentage sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa. The semen samples at post-thaw were also evaluated for total viable count (colony forming units/ml). At post-thaw, control exhibited significantly (P<0.05) higher sperm motility in comparison to T2 and T3. The percent of live sperm count, intact acrosome and HOST reacted spermatozoa were significantly higher (P<0.05) for control than all other treatment groups at post-thaw. Among treatment groups, T1 maintained significantly higher (P<0.05) percentage of live sperm count, intact acrosome and HOST reacted spermatozoa than T2 and T3. The total viable count at post-thaw was significantly lower (P<0.05) for control than all the treatment groups. In conclusion, honey cannot be used as an alternative to antibiotics to take care of heavy microbial load in semen, however, levels up to 2.5% may be supplemented to semen as an energy source.

Effect of Different Glycerol Levels on Quality of Frozen Semen of Mizo Local Boar

A total of 24 ejaculates were obtained from 3 Mizo local boars (Zovawk), were used by split sample technique for evaluating the effect of four glycerol levels on quality of semen extended with Lactose egg yolk glycerol (LEYG) extender at equilibration and after freezing. The sperm motility at equilibration and after freezing was significantly higher (P<0.01) for 3 percent glycerol levels, than for 2 and 4 percent glycerol levels. The live sperm at equilibration and after freezing was significantly higher (P<0.01) for 3 percent glycerol levels than for 2 and 4 percent glycerol levels, and for 2 percent than for 3 percent glycerol level. The Hypo Osmotic Sperm Swelling Test (HOSST) reacted spermatozoa at equilibration and after freezing was significantly (P<0.01) higher for 3 percent glycerol levels, than for 2 and 4 percent glycerol levels, and for 2 percent than for 3 percent glycerol level. The acrosomal integrity at equilibration and after freezing was significantly (P<0.01) higher for 3 percent glycerol levels, than for 2 and 4 percent glycerol levels, and for 2 percent than for 3 percent glycerol level. In conclusion, preservation of boar semen in LEYG extender using 3 percent glycerol found to be superior.

Effect of vitamin e on the quality of frozen buck semen

A total of 40 ejaculates collected from five Beetal bucks twice weekly were used to study the effect of 0 (control), 2, 5 and 7 mM vitamin E on the quality of frozen semen by split sample technique. The semen extended using Tris was filled in French mini straws (0.25 ml) and sealed with polyvinyl alcohol powder of different colours for identification of semen of different bucks processed with different concentrations of vitamin E. The mean per cent, sperm motility, live sperm, live intact acrosome and hypo-osmotic sperm swelling test (HOST)-reacted sperm were significantly (P<0.05) higher in the extender with 2 mM vitamin E (63.75±0.56, 64.29±0.38, 45.24±0.42 and 52.89±0.23, respectively) than that with 5, 7 mM vitamin E and the control. The release of alanine transaminase and aspartate transaminase from post-thaw spermatozoa was also the lowest (35.67±0.31 and 215.08±0.41 U/L, respectively) in the extender containing 2 mM vitamin E.

Evaluation of subgroups of the human sperm hypoosmotic swelling test in normozoospermic male cases with recurrent fertilization failure: a prospective case-controlled study

To compare subgroups of the human sperm hypoosmotic swelling test subgroups in both recurrent fertilization negative infertile cases with normal semen analysis and fertilization positive controls. This was a prospective case-controlled study performed with normospermic 33 previously fertile male (secondary infertility) and 41 infertile men who had undergone two or three unsuccessful in vitro fertilization attempts. HOS test was investigated in 4 subgroups including HOS 1, HOS 2, HOS 3, and HOS 4 according to the degree of sperm tail swelling and compared between the two groups. Four subgroups were compared and statistical significance was demonstrated in HOS 1, HOS 3 and HOS 4 tests (p < 0.001) in fertile and infertile men. Highest HOS 1 and lowest HOS 4 grades were determined in Group A. However, no statistical significance was determined between two groups in HOS 2 test which was minimal swelling in sperm tails. HOS 1, HOS 3 and HOS 4 subgroups of HOS test are reliable and useful methods providing important information regarding the sperm function. A high HOS test 1 grade plus a low HOS test 4 grade should suggest a fertility problem, despite a normal semen analysis. HOS test subgroups provide additional information in normospermic cases with unexplained infertility.

Effect of Smoking on Sperm Vitality, DNA Integrity, Seminal Oxidative Stress, Zinc in Fertile Men

To assess the effect of smoking on sperm vitality, sperm DNA integrity, semen reactive oxygen species, and zinc levels in fertile men. One-hundred sixty men were investigated. They were divided into 2 equal groups: healthy fertile nonsmokers and healthy fertile smokers. They were subjected to history taking, clinical examination, and semen analysis. In their semen, sperm hypo-osmotic swelling test, sperm DNA fragmentation test, seminal reactive oxygen species, and zinc were assessed. Compared with fertile nonsmokers, fertile smokers were significantly associated with lower hypo-osmotic swelling test and seminal zinc levels and significantly associated with higher sperm DNA fragmentation percent and seminal reactive oxygen species levels. Smoking (cigarettes/day and duration) has detrimental effects on sperm motility, viability, DNA fragmentation, seminal zinc levels, and semen reactive oxygen species levels, even in fertile men, and it is directly correlated with cigarette quantity and smoking duration.

Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities

Ram semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the semen. The aim of the present work was to study the effect of two anti-oxidants, namely, glutamine and an amino acid solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rams were evaluated and pooled at 37 °C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 °C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0 ± 4.4%) and hypo-osmotic swelling test (HOST) (64.1 ± 5.5%), when compared to glutamine (2.5 mM) and BME (13 and 26%) ( P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4 ± 0.9 kU/g protein), compared to the other treatments ( P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control ( P < 0.05). Findings showed that glutamine (5 mM) supplementation in semen extenders, was of greater benefit to frozen–thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen–thawed ram sperm is used.

Ascorbic Acid, Catalase and Chlorpromazine Reduce Cryopreservation-induced Damages to Crossbred Bull Spermatozoa

The present study evaluated the effectiveness of ascorbic acid, catalase, chlorpromazine and their combinations in reducing the cryodamages to crossbred bull (Bos taurus x Bos indicus) spermatozoa. A total of 32 ejaculates (eight each from four bulls) were diluted in Tris-citric acid-fructose-egg yolk-glycerol extender. Each ejaculate was split into six parts (five treatment and one control). Treatment groups included 10 mm ascorbic acid, 0.1 mm chlorpromazine, 200 IU/ml catalase, 10 mm ascorbic acid + 0.1 mm chlorpromazine or 200 IU/ml catalase + 0.1 mm chlorpromazine in the extender. Fluorescent probes (Fluorescein isothiocyanate--Pisum sativum agglutinin + Propidium iodide) were used for the assessment of spermatozoa viability and acrosomal status. The proportion of acrosome intact live (AIL), acrosome intact dead, acrosome reacted live and acrosome reacted dead sperm was assessed in fresh, equilibrated and frozen-thawed semen. The functional status of the sperm was assessed using hypo-osmotic sperm swelling test (HOSST). Activities of acrosin and hyaluronidase enzyme were also determined. Lipid peroxidation level was assayed based on the melonaldehyde (MDA) production. In cryopreserved semen, the values of AIL spermatozoa, HOSST response, hyaluronidase and acrosin activity were reduced by 53%, 47%, 34% and 54%, respectively from their initial values in fresh semen. However, MDA level was threefold higher in the frozen-thawed sperm compared with fresh sperm. Significant (p < 0.05) improvement in motility, viability, HOSST response, retention of hyaluonidase and acrosin and reduction in MDA was recorded in ascorbic acid, catalase, ascorbic acid + chlorpromazine and catalase + chlorpromazine incorporated groups. The percentage of AIL sperm was significantly (p < 0.05) higher in ascorbic acid, catalase and ascorbic acid + chlorpromazine incorporated groups compared with the control. Chlorpromazine alone did not improve the post-thaw semen quality but when combined with either ascorbic acid or catalase, improvement in semen quality was noticed. It was inferred that incorporation of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender improved the post-thaw semen quality in crossbred bulls.

Effect of Oviductal Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah Buffaloes

The present study was undertaken to elucidate the effect of non-luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non-luteal based on ovarian morphology. Non-luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10,000 g at 4 degrees C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at -20 degrees C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris-citrate-egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non-luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5 degrees C, filled in 0.5 ml French straws, exposed to LN(2) vapour, plunged into LN(2) and then stored at -196 degrees C. The equilibrated and frozen-thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo-osmotic sperm swelling test (HOST). In frozen-thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non-luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze-thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen-thawed semen. It was inferred that incorporation of non-luteal whole oviductal fluid proteins improved the sperm quality in frozen-thawed semen in Murrah buffaloes.

Preservation of boar semen at 18 °C induces lipid peroxidation and apoptosis like changes in spermatozoa

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (Δ Ψ m) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 °C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, Δ Ψ m, and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of Δ Ψ m was done using 3,3′-dihexyloxacarbocyanine iodide [DiOC 6(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly ( p < 0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10 9 sperm) were low in sperm (99.83 ± 2.69) and seminal plasma (191.98 ± 11.58), which gradually increased up to the 96 h of storage. Highest negative correlation ( r value) was observed between MDA levels and sperm motility (−0.97), live percent (−0.97), acrosomal integrity (−0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (−0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage ( r = 0.98). There was a significant ( p < 0.05) increase in the sperm cells with low Δ Ψ m from 0 to 96 h of preservation. Before preservation, 14.85 ± 4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00 ± 6.25%. The apoptotic sperm population was 8.33 ± 2.31% in fresh semen, while this population was 25.19 ± 4.25% at 96 h of preservation and the difference was significant ( p < 0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 °C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.

Effect of different levels and sources of zinc supplementation on quantitative and qualitative semen attributes and serum testosterone level in crossbred cattle (Bos indicus×Bos taurus) bulls

An experiment was conducted on 16 crossbred bulls (about 2 years of age, 316.2+/-0.77 kg average body weight), divided into groups I, II, III and IV to study the effect of different levels of Zn supplementation from inorganic and organic sources on semen quality. The animals in the first 3 groups were supplemented with 0, 35 and 70 ppm Zn from Zn sulfate, respectively and the animals in-group IV were supplemented with 35 ppm Zn as Zn propionate. Semen collection and evaluation was done in the first month (to assess semen quality at the start of the experiment) and 7th, 8th and 9th month of experimental feeding to evaluate the effect of supplemental Zn on semen attributes. We gave 6 months for Zn feeding, so that 3 sperm cycles of spermatogenesis had passed and the collected semen reflected the complete effect of Zn supplementation. Six ejaculates from each bull were collected and evaluated for semen quantitative (ejaculate volume, sperm concentration and sperm number per ejaculate) and qualitative characteristics (semen pH, mass motility, individual motility, sperm livability percent and abnormal sperm percent, percent intact acrosome, bovine cervical mucus penetration test, hypo-osmotic sperm swelling test) and activity of seminal plasma enzymes i.e., alkaline phosphatase, acid phosphatase, GOT and GPT. Testosterone level in the blood serum of crossbred bulls was also estimated. Mean values of semen quantitative and qualitative characteristics at the start of the experiment were statistically non significant (P > 0.05) in all the crossbred cattle bulls, however, there were statistically significant differences among the bulls of different groups after 6 months of zinc supplementation. Mean ejaculate volume (mL) was 2.37, 4.70, 5.86 and 6.38, respectively in groups I to IV, indicating a statistically significant (P < 0.05) higher semen volume in Zn-supplemented groups as compared to the control group of bulls. Similarly, sperm concentration (million.mL(-1)), live sperm (%) and motility (%) were significantly (P < 0.01) higher in Zn-supplemented groups as compared to the control group. The results of BCMPT and HOSST revealed a significant improvement in sperm functional ability in all the groups supplemented with Zn as compared to the control group. The activity of alkaline and acid phosphatase in seminal plasma was significantly (P < 0.05) higher in the Zn-supplemented groups, whereas GOT and GPT activities in seminal plasma were significantly (P < 0.05) lower in the Zn propionate supplemented group as compared to the control group. Testosterone concentration (ng.mL(-1)) in blood serum was significantly higher in animals of groups III and IV, as compared to control group. It may be concluded that Zn supplementation either in the inorganic or organic form in the diet of crossbred bulls improved the qualitative and quantitative attributes of semen; however, the number of sperm per ejaculate, mass motility and semen fertility test like bovine cervical mucus penetration was significantly higher in bulls given Zn in an organic form (Zn propionate) as compared to an inorganic form (Zn sulfate).

Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes

A study was undertaken to find out the effect of addition of oviductal proteins on sperm functions and lipid peroxidation (LPO) levels in buffaloes. Oviductal flushings were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle), centrifuged (3000 rpm; 30 min), filtered (0.2 μm) and frozen at −20 °C. The proteins in pooled nonluteal and luteal oviductal fluid were precipitated overnight using ammonium sulphate, centrifuged (10,000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored frozen at −20 °C. Six pooled good quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was split into three parts and extended in Tris-Egg yolk-Citrate extender (20% egg yolk: 7% glycerol), so that final dilution yielded approximately 60 million sperm cells/ml and cryopreserved in 0.5 ml French straws (30 million sperm cells per straw) in LN 2 (−196 °C). Before freezing, the nonluteal and luteal oviductal proteins (NLOP &LOP) were incorporated at the concentration of 1 mg/ml of extended semen. The equilibrated and frozen thawed (37 °C for 30 s) semen was evaluated for motility, viability and acrosomal integrity, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides these tests, LPO level was assessed in sperm and seminal plasma in equilibrated and frozen thawed semen. Results revealed that addition of oviductal proteins to semen before freezing convey beneficial effect in terms of spermatozoan motility, viability and acrosomal integrity. Nonluteal oviductal proteins favored significantly ( P < 0.05) higher sperm penetration distance in cervical mucus (23.00 ± 1.15 mm) than the control group (15.00 ± 3.46 mm) in frozen thawed semen. Similarly, swollen sperm percentage was also significantly ( P < 0.05) higher in NLOP treated group than the LOP included and control groups. In frozen thawed spermatozoa, the LPO level was significantly ( P < 0.05) lower in NLOP added group than the LOP added and control group. It was inferred that incorporation of oviductal proteins in extender before freezing reduced the lipid peroxidation levels in buffalo spermatozoa during cryopreservation and thereby improved the post-thaw semen quality.

Effect of buffalo seminal plasma heparin binding protein (HBP) on freezability and in vitro fertility of buffalo cauda spermatozoa

The study was conducted to assess the effect of heparin binding seminal plasma proteins (HBP) on freezability and in vitro fertilizing ability of buffalo cauda epididymal spermatozoa. Spermatozoal motility, viability and acrosomal integrity at prefreeze and post-thaw stages were studied. The in vitro fertilizing ability of spermatozoa was assessed by the application of two tests, i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic sperm swelling test (HOST). HBP isolated from buffalo seminal plasma and maintained in the laboratory were used for the study. Twelve pairs of epididymis from adult buffaloes slaughtered at the local abattoir were used for the study. The results indicated that HBP addition improved the progressive motility, BCMPT and HOST response at prefreeze level. HBP at a concentration of 40 μg/ml showed better results than HBP at a concentration of 80 μg/ml. However, subjecting the HBP treated spermatozoa to cryopreservation resulted in significant reduction of motility, viability, acrosomal integrity and response to BCMPT and HOST in the HBP treated groups when compared to those in control group. The deleterious effect of HBP was found to be concentration dependent with the higher concentration causing higher post-thaw damage.

Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 μm) and frozen at −20 °C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at −20 °C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris–egg yolk–citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN 2 (−196 °C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1 mg/ml of extended semen. The equilibrated and frozen-thawed (37 °C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 °C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher ( P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher ( P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher ( P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher ( P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.

Sperm MTT viability assay: a new method for evaluation of human sperm viability.

MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is commonly used as a cell proliferation assay. The objective of this study was to evaluate the capability of MTT assay to discriminate between viable and nonviable sperms and compare it sefficiency with E&N (eosin and nigrosin) and HOST (hypo-osmotic swelling test). MTT assay was modified to obtain optimal result for assessment of sperm viability. After standardization of method, MTT, E&N, and HOST were carried out simultaneously on 57 semen samples from patients referring to Isfahan Fertility and Infertility Center. The correlation coefficient between these tests and sperm motility was calculated using the SPSS statistical program. Specificity and sensitivity of each test was also obtained. The optimal conditions for sperm MTT viability assay were 2 h after addition of sperm to MTT in HAM'S F10 + 25 mM HEPES + 10% HSA at 37 degrees C and pH 7.4-7.45. Inter- and intra-assay coefficients of variations were 9 and 7%, respectively. The sensitivity and specificity for sperm MTT viability assay, E&N, and HOST were 97,98, and 99%, and 100, 100, and 83% respectively. High significant correlations were obtained between sperm MTT viability assay, E&N, HOST and motility. Sperm MTT viability assay can be used as a diagnostic test for discrimination of viable sperms from sperm population.

Detection of chromosomal abnormalities by fluorescent in-situ hybridization in immotile viable spermatozoa determined by hypo-osmotic sperm swelling test.

If randomly selected immotile spermatozoa are used for intracytoplasmic sperm injection (ICSI), pregnancy rates are significantly decreased. The hypo-osmotic swelling test (HOST) is the only method available to detect the viable, but immotile spermatozoa for ICSI. However, evidence is still lacking for the chromosomal abnormalities for the normal-looking, but immotile spermatozoa positive for HOST. Sperm samples from 20 infertile men with normal chromosomal constitution were obtained. After Percoll separation, morphologically normal but immotile spermatozoa were transported individually into HOST solution for 1 min using micropipettes. Cells that showed tail curling with swelling in HOST were then transferred back into human tubal fluid solution to allow reversal of swelling. These sperm cells were fixed and processed for the multi-colour fluorescence in-situ hybridization (FISH) for chromosomes X, Y and 18. The same FISH procedure was applied for the motile spermatozoa from the same cohort, which formed the control group. The average aneuploidy rates were 1.70 and 1.54% in 1000 HOST positive immotile and motile spermatozoa respectively detected by FISH for each patient. Our results indicate that morphologically normal, immotile but viable spermatozoa have an aneuploidy rate similar to that of normal motile spermatozoa.

Effect of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human sperm motility, viability and motion parameters.

Male genital tract infections and non-specific inflammatory conditions may be associated with unexplained infertility. Previous studies have shown the presence of cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the semen of infertile men. However, the mechanism of effect of these cytokines on human sperm function is still controversial. The present study was undertaken to investigate the in-vitro effects of TNF-alpha and IFN-gamma on human sperm motion, viability and the hypoosmotic swelling test (HOST). Washed spermatozoa from normal volunteers (n = 9) were incubated in the presence/absence of TNF-alpha (1 microgram/mL) plus IFN-gamma (0.1 microgram/mL). Sperm motility, viability, HOST, and video sequences were recorded at different time intervals (0, 30, 60 and 180 min). Sperm motion parameters were analysed using computer-assisted semen analysis. There was a time-dependent negative effect of TNF-alpha plus IFN-gamma on sperm motility, viability, HOST, and lateral-head displacement (ALH). The maximum decrease was observed between 60 and 180 min for sperm motility (50.8 +/- 5.6%), viability (52.8 +/- 4.0%), HOST (38 +/- 2%) and ALH (4.7 +/- 0.1 microns) compared to control samples (62.2 +/- 2.8, 62.4 +/- 2.9, 58 +/- 4, and 5.3 +/- 0.4, respectively; All p < 0.05). There was no significant effect on sperm straight-line velocity and mean linearity when compared to control. These data suggest that the common inflammatory cytokines TNF-alpha plus IFN-gamma have only partial detrimental effects on sperm motility, viability, membrane integrity and lateral head displacement, which may contribute to the poor fertilizing potential of human spermatozoa during inflammatory conditions.

Confocal scanning laser microscopy of morphometric human sperm parameters: correlation with acrosin profiles and fertilizing capacity

To develop quantitative criteria for assessing sperm morphology and to determine the correlation between the percentage of morphologically normal spermatozoa and the outcome of the sperm hypo-osmotic swelling test, sperm acrosin profile, and sperm capacity for fertilization. The maximal length and width of the sperm head, the length of the midpiece and principal piece of the sperm tail, and the ratio of the surface of the acrosomal region to the total surface of the head were determined in specimens obtained from a group of infertile men and a group of fertile men using a confocal scanning laser microscope. Group A consisted of 53 infertile men who were participating in an IVF program, and group B consisted of 98 fertile men. The mean +/- 2 SD of the morphometric parameters in group B was established as representing the lowest and highest normal values in both groups. A normal spermatozoon was defined as one with morphometric parameters within normal levels. The lowest percentage of morphologically normal spermatozoa, hypo-osmotic swelling test result, and acrosin activity in group B were also taken as the lowest normal values in group A. In vitro fertilization program at the Tottori University School of Medicine, Yonago, Japan. Sperm morphometric parameters, percentage of morphologically normal spermatozoa, hypo-osmotic swelling test, and acrosin activity. The length of the midpiece, ratio (x 100) of the surface of the acrosomal region to the total surface of the sperm head, percentage of morphologically normal spermatozoa, outcome of hypo-osmotic swelling test, and acrosin activity were significantly higher in group B than in group A. The maximal width of the head was significantly lower in group B than in group A. Strongly positive correlations were observed between percentage of morphologically normal spermatozoa or length of midpiece and the proportion of fertilized oocytes in group A and between ratio (x 100) of the surface of the acrosomal region to the total surface of the head and acrosin activity in groups A and B. Sperm morphology showed high positive and negative predictive values for acrosin activity (normal/abnormal) and fertility potential (present/absent). Using quantitative strict criteria, we found that sperm morphology was an important predictor of sperm fertilizing capacity. The confocal scanning laser microscope provided useful information about the sperm cytoskeleton and its importance in fertilization.

White blood cells in semen affect hyperactivation but not sperm membrane integrity in the head and tail regions

The presence of high numbers of peroxidase-positive PML in ejaculated semen significantly reduced sperm HA, an important step leading to sperm capacitation. Sperm membranes at both the head and tail regions, as assessed by the hypo-osmotic viability parameter and the hypo-osmotic sperm swelling test, respectively, were not affected by peroxidase-containing leukocytes. Sperm motility was not affected, but sperm curvilinear and straight line velocity parameters were reduced in the presence of high concentrations of leukocytes in the ejaculate. The results suggested that the effect of leukocytes on sperm was through a reduction in sperm hyperactive motility but not through alterations in the sperm head and tail membranes.