Bovine Cervical Mucus Research Articles

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.

Efficacy and safety of a new vaginal contraceptive antimicrobial formulation containing high molecular weight poly(sodium 4-styrenesulfonate).

Host cell infection by sexually transmitted disease (STD)-causing microbes and fertilization by spermatozoa may have some mechanisms in common. If so, certain noncytotoxic agents could inhibit the functional activity of both organisms. High molecular mass poly(sodium 4-styrenesulfonate) (T-PSS) may be one of these compounds. T-PSS alone (1 mg/ml) or in a gel (2% or 5% T-PSS) completely prevented conception in the rabbit. Contraception was not due to sperm cytotoxicity or to an effect on sperm migration. However, T-PSS inhibited sperm hyaluronidase (IC(50) = 5.3 microg/ml) and acrosin (IC(50) = 0.3 microg/ml) and caused the loss of acrosomes from spermatozoa (85% maximal loss by 0.5 microg/ml). T-PSS (5% in gel) also reduced sperm penetration into bovine cervical mucus (73% inhibition by 1 mg gel/ml). T-PSS (5% in gel) inhibited human immunodeficiency virus (HIV; IC(50)= 16 microg gel/ml) and herpes simplex viruses (HSV-1 and HSV-2; IC(50) = 1.3 and 1.0 microg gel/ml, respectively). The drug showed high efficacy against a number of clinical isolates and laboratory strains. T-PSS (5% in gel) also inhibited Neisseria gonorrhea (IC(50) < 1.0 gel/ml) and Chlamydia trachomatis (IC(50) = 1.2 microg gel/ml) but had no effect on lactobacilli. These results imply that T-PSS is an effective functional inhibitor of both spermatozoa and certain STD-causing microbes. The noncytotoxic nature should make T-PSS safe for vaginal use. T-PSS was nonmutagenic in vitro and possessed an acute oral toxicity of >5 g/kg (rat). Gel with 10% T-PSS did not irritate the skin or penile mucosa (rabbit) and caused no dermal sensitization (guinea pig). Vaginal administration of the 5% T-PSS gel to the rabbit for 14 consecutive days caused no systemic toxicity and only mild (acceptable) vaginal irritation. T-PSS in gel form is worthy of clinical evaluation as a vaginal contraceptive HIV/STD preventative.

Concentration of calcium, zinc, magnesium, potassium and sodium in the bovine cervical mucus during normal oestrus and oestrus induced by progesterone and/or PGF(2alpha).

Ninety-two healthy Friesian cows were used to determine the concentration of calcium (Ca), zinc (Zn), magnesium (Mg), potassium (K) and sodium (Na) in bovine cervical mucus and to compare these concentrations in cervical mucus collected during spontaneous and induced oestrus. Cows were assigned to four groups: (1) no treatment (2) PRID for 12 days plus pregnant mare's serum gonadotrophin (PMSG; 1000 IU, i.m.) at the removal of the PRID (3) PGF(2alpha), two doses (6 mg, i.m.) 11 days apart, and (4) PRID for 7 days plus one dose of PGF(2alpha) (6 mg, i.m.) 24 h before removal of the PRID. The cows of the three induced-oestrus groups received two artificial inseminations (AI), whereas those in the spontaneous-oestrus group received only one. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. The results of this study can be summarized as follows: (1) viscosity was significantly lower (P<0.001) and crystallization was significantly (P<0.001) higher in the cervical mucus of cows in the spontaneous-oestrus group compared with cows in the induced-oestrus groups; (2) Na concentration was similar in all groups; (3) concentration of all the other elements in the spontaneous-oestrus group was significantly lower (P<0.05) than in the induced-oestrus groups; (4) there was significant positive correlation (P<0.001) between viscosity and all elements, and significant negative correlation (P<0.05-P<0.001) between crystallization and all elements.

Lactoferrin-binding proteins of Tritrichomonas foetus.

Tritrichomonas foetus is a common, sexually transmitted, protozoan parasite of cattle. It has an essential requirement for iron, which it obtains from host lactoferrin. However, specific lactoferrin-binding protein receptors have not yet been identified in T. foetus. To differentiate specific and nonspecific binding of lactoferrin, lactoferrin affinity chromatography and Western blotting was used to identify metabolically or surface-labeled T. foetus lactoferrin-binding proteins. Bovine lactoferrin was shown to bind more efficiently than human lactoferrin, and each of these bound much better than bovine transferrin. This is relevant because T. foetus is both species-specific and only infects the mucosal surface of the reproductive tract, which has little transferrin. Whereas the majority of lactoferrin binding was specific, competitive inhibition studies showed that nonspecific, charge-related binding of lactoferrin to T. foetus may also be involved. In the presence of bovine cervical mucus, binding of lactoferrin to T. foetus was diminished, suggesting that mucus has an effect on lactoferrin binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface biotinylated proteins affinity-purified on lactoferrin-Sepharose showed biotinylated bands at Mr values of 22, 49, 55, 72, and 155 kDa. Because lactoferrin-binding proteins may be susceptible to digestion by T. foetus extracellular cysteine proteinases, it is suspected that the 155-kDa protein is the specific lactoferrin-binding protein and that the lower-Mr lactoferrin-binding molecules may be fragmentation products that contain the lactoferrin-binding site; however, other interpretations are clearly feasible. It is possible that there may be multiple proteins or multimers of the same protein. In summary, the data showed that binding of lactoferrin to T. foetus may be regulated by an interplay of specific receptor interactions as well as by hydrophobic and charge-related interactions.

Chemical properties of bovine cervical mucus during normal estrus and estrus induced by progesterone and/or PGF2α

Ninety-two Friesian cows were used to determine the chemical properties of cervical mucus during normal estrus and estrus induced by progesterone (P 4)-releasing intravaginal devices (PRID) and/or prostaglandin F2α. The animals were assigned to 4 groups (no treatment, a PRID for 12 days plus injection of 1000 IU PMSG at the removal of PRID, a double im injection of PGF 2α 11 days apart, or PRID for 7 days plus an im injection of PGF 2α 24 h before the removal of PRID). A number of cows with normal estrus exhibit three consecutive estrous cycles after delivery. Cows that had not shown estrus for 3 months after delivery had their ovaries palpated twice at 10-day intervals, to determine their ovarian activity. Then PRID and/or PGF2α was administered in cows that had a palpable corpus luteum in one of the two palpations (cyclic cows). A double artificial insemination (AI) was performed to the cows of the three induced-estrus groups, while the cows with normal estrus received only one AI. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. Additionally, samples of cervical mucus were collected from 20 cows during their first estrus after the induced one. The results are summarized as follows: 1) The biochemical properties of cervical mucus in the first three estrus periods after delivery were similar. 2) These properties were similar both in normal estrus after delivery and in the first estrus after an induced one. 3) Glucose and fructose concentrations for normal estrus were similar to those for induced estrus groups. 4) Total protein and cholesterol concentrations were significantly lower (P < 0.001) in normal than in induced estrus, while no difference was found among the induced estrus groups. 5) Pregnancy rates of the cows did not differ significantly among the normal and the induced-estrus groups. 6) The percentages of cows in the induced-estrus groups that produced cervical mucus with total protein and cholesterol concentrations similar to those for the normal estrus groups was very low.

Correlation between the spermatozoal characteristics and sperm penetration distance in polacrylamide gel and bovine cervical mucus

Correlation between the spermatozoal characteristics and the sperm penetration distance in polyacrylamide gel was assessed, utilizing frozen thawed semen samples obtained from 6 bulls, and it was compared with the correlation between sperm penetration in bovine cervical mucus and spermatozoal characteristics. In vitro sperm penetration tests were performed with mucus and gel. The sperm penetration in gel and mucus was significantly and positively correlated with post-thaw motility (r=0.81; r=0.89:P<0.01) and acrosome integrity (r=0.88; r=0.94:P<0.01). A significant negative correlation with abnormal spermatozoa (r=−0.84;r=0.83:P<0.01) was observed. Both sperm concentration and post-thaw live spermatozoa were not significantly correlated. A significant multiple regression between sperm penetration and the spermatozoal characteristics both in gel (R 2=0.87; F=40.47; P<0.01) and mucus (R 2=0.91; F=60.48; P<0.01) was observed. The major spermatozoal characteristics determining the capacity of spermatozoa to penetrate gel were post-thaw motility, percentage of abnormal spermatozoa and acrosome integrity. The acrosome integrity has a more significant contribution. The correlation established with sperm penetration in gel was very similar to that of sperm penetration in mucus. The utility of gel as a mucus substitute in in vitro sperm penetration tests was discussed.

Physical properties of bovine cervical mucus during normal and induced (progesterone and/or PGF2α) estrus

Ninety two Friesian cows were used to determine physical properties of cervical mucus collected during normal estrus and estrus induced. Estrus was induced using either progesterone (P 4) releasing intravaginal devices (PRID) and/or prostaglandin F2α (PGF2α). The animals were assigned to 4 groups (no treatment, a PRID for 12 days plus an injection of 1000 IU PMSG at the removal of the PRID, a double injection of 3 mL PGF2α 11 days apart, and a PRID for 7 days plus an injection of PGF2α 24 h before the removal of PRID). A number of cows with normal estrus exhibited three consecutive estrus cycles after calving. Cows that had not shown estrus for three months after calving had their reproductive system palpated twice at 10-day intervals, to determine their ovarian activity. Then PRID and/or PGF2α was administered to cows that were found to have a palpable corpus luteum in one of two palpations (cycling cows). The cows of the three induced estrous groups were artificially inseminated (AI) twice, while those with normal estrus received only a single AI. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. Additionally, samples of cervical mucus were collected from 20 cows at their first estrus after the induced estrus. The results are summarized as follows: 1) The physical properties of cervical mucus were similar in the first three normal consecutive estrus cycles after calving. 2) The physical properties of cervical mucus in normal estrus after calving were similar to those in the first estrus after an induced estrus. 3) The pH values for normal estrus were similar to those for induced estrus. 4) Viscosity of cervical mucus in the normal estrous group was significantly lower than that in the induced estrus. Furthermore, significant differences were noticed among the three induced estrous groups. 5) Spinnbarkeit, crystallization and receptivity of cervical mucus (penetration test) were significantly higher in the normal estrous group than in the induced estrous groups, while no difference was detected among induced estrus groups. 6) Pregnancy rates in physical properties of cervical mucus at the time of the second AI were similar to those for normal estrus but this suggestion needs further investigation. On the other hand, at the time of the first AI very few cows in the induced estrus groups produced cervical mucus similar to that of cows with normal estrus. The physical properties that differed the most were viscosity, spinnbarkeit, and the penetration test. Other authors found that cows do not exhibit estrus at the same determined time (11,26). On the basis of this finding physical properties of cervical mucus could be used as an indicator for the time of estrus manifestation, in order to increase pregnancy rates by applying AI at an optimal time. In summary, the results from the present study showed that the physical properties of cervical mucus in the first three consecutive normal estrous periods after calving are similar; that the first estrus after an induced estrus is a normal estrus; that viscosity, spinnbarkeit, crystallization and receptivity of cervical mucus (penetration test) are significantly different for normal than for induced-estrus groups at the time of first AI; and a small percentage of cows with induced estrus produce cervical mucus with parameters of viscosity, spinnbarkeit and receptivity similar to those found in normal estrus. These properties could be used as indicators for the quality of cervical mucus produced by the cows with induced estrus.

Inhibition of hamster sperm acrosomal enzyme by gossypol is closely associated with the decrease in fertilization capacity

To elucidate the mechanism of sterility induced by gossypol, we studied the relationship between the activities of acrosomal enzymes and their fertilizing capacity in the hamster. The results showed that the ability of spermatozoa to penetrate into bovine cervical mucus, hyperactivated motility (HAM) and fertility in vivo were significantly inhibited when spermatozoa were exposed to gossypol (2.5 μg–60 μg/mL) for 15 min in vitro. Also, following administration of gossypol (12.5 mg/kg/day) for 6 weeks, sperm motility, HAM and rate of fertilization in vitro by the hamster cauda epididymal spermatozoa were significantly decreased and the extracts of testis delayed dispersion of the cumulus oophorus cells, suggesting that hyaluronidase and other acrosomal enzymes might be inhibited by gossypol. In addition, acrosin and arylsulfatase activities were also markedly inhibited. These data show that the inhibition of acrosin and arylsulfatase activities is the main cause of gossypol-induced infertility. The inhibition was dependent upon gossypol dose and the duration of administration. Thus, the assay of acrosin and arylsulfatase activities may provide a useful tool for monitoring sterility induced by gossypol.

Effects of cryopreservation on penetration ability of human spermatozoa into bovine cervical mucus.

Cryopreserved sperm exhibit lower fertilizing capacity in comparison to fresh sperm, partly due to effects of glycerol as the common cryoprotectant medium. Since standard semen analysis is not a good predictive method to assess sperm fertilizing capacity, functional tests like cervical mucus penetration may provide more useful information. A total of 24 semen samples were examined before and after cryopreservation for sperm parameters as well as number and motility of penetrated sperm into bovine cervical mucus (BCM) as an alternative for human cervical mucus. Freezing and thawing procedures have negative effects on sperm penetration into cervical mucus. No significant relation was noticed between sperm motility percentage or its penetration into BCM before and after cryopreservation, which denotes the variability in resistance of sperm to damaging effects of freezing.

Cryopreservation effects on penetration of human sperm into bovine cervical mucus

Cryopreservation effects on penetration of human sperm into bovine cervical mucus

CORRELATION OF THE BOVINE CERVICAL MUCUS PENETRATION TEST WITH HUMAN SPERM CHARACTERISTICS IN 1406 EJACULATES

The bovine cervical mucus penetration test (BCMPT) was performed to determine its usefulness in screening the ability of sperm to successfully penetrate mucus in vitro. Ejaculates were obtained by masturbation from patients attending an infertility clinic. Routine semen analysis was performed using a microcomputerized multiple-exposure photography system. The BCMPT was performed. Overall, the average penetration of the mucus was 38 +/- 0.46 mm. Of the 1,406 ejaculates analyzed, 244 (17%) displayed a negative result (0-20 mm), 291 (21%) a questionable result (21-30 mm), and 871 (62%) a positive result (>30 mm). A highly significant (p < .001) correlation between mucus penetration distance and sperm MD (r = 0.541), MI (r = 0.484), count (r = 0.475), motility (r = 0.448), velocity (r = 0.400) and morphology (r = 0.369) was observed. Overall, the finding of an abnormal semen parameter resulted in a 34 +/- 5% accurate prediction of a negative or questionable BCMPT (<30 mm), while a normal semen parameter resulted in a 90 +/- 4% accurate prediction of a positive BCMPT (>30 mm). Sperm MD showed the strongest positive predictive value (98%), while morphology showed the greatest negative predictive value (50%). Of the 1,406 samples, 25 +/- 2% of the samples with normal semen parameters displayed a negative BCMPT. Conversely, 6 +/- 2% of samples with abnormal parameters showed a positive BCMPT. The BCMPT successfully identifies a significant subpopulation of patients as having an inadequate penetration of mucus with otherwise normal semen characteristics.

Factors influencing nonoxynol-9 permeation and bioactivity in cervical mucus

The effects of delivery gel pH and osmolarity on both the mass transport and `biodiffusion' of the spermicide nonoxynol-9 (N9) in bovine cervical mucus were evaluated. Delivery gels were calcium chloride crosslinked alginate containing 3% N9, and were manufactured over a pH range of 3.4 to 5.9 and an osmolarity range of 300 to 900 mosmol. Mass transfer parameters (diffusion coefficients and total drug loading) were determined using a new UV spectrophotometric technique while biodiffusion (the diffusion distance into mucus at which sperm are killed) was assessed using the Double Ended Test. It was found that delivery gel pH had a significant effect on spermicidal efficacy of the alginate–N9 system; biodiffusion increased with decreasing pH. Actual N9 diffusion into mucus was found to be influenced by both the delivery gel pH and osmolarity. At high N9 concentration (near the gel/mucus interface), mass transport tended to decrease with decreasing pH at the highest osmolarity. At low concentration, mass transport tended to decrease with increasing osmolarity and decrease with increasing pH at the highest osmolarity. The difference between low and high concentration behavior can be attributed to N9 micelle formation. These findings are interpreted in the context of the design of intravaginal drug delivery vehicles for spermicides.

Sialic Acid-Specific Lectin-Mediated Adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.

Pentoxifylline stimulates various sperm motion parameters and cervical mucus penetrability in patients with asthenozoospermia.

Pentoxifylline (PTX) was incubated in vitro with human spermatozoa to examine its effects on sperm motility characteristics and bovine cervical mucus penetrability (BCMP). Sperm motion parameters were assessed by computer-assisted motion analysis (CASA) using HTM-IVOS and BCMP was evaluated using the Penetrak kit. In vitro incubation with PTX (1 mg ml-1; 3.6 mM, 30 min) did not significantly change percentage motility, average path velocity (VAP), straight-line velocity (VSL) or beat cross frequency (BCF) of spermatozoa from normozoospermic or asthenozoospermic samples. However, it significantly increased curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and hyperactivated motility (HA), and significantly decreased linearity (LIN) of spermatozoa from both samples. Pentoxifylline was found to increase BCMP scores for spermatozoa from asthenozoospermic samples, but did not affect scores for spermatozoa from normozoospermic samples. Bovine cervical mucus penetrability (BCMP) was found to be positively and significantly correlated with the percentage motility of both non-PTX-treated and PTX-treated spermatozoa for asthenozoospermic samples. These results demonstrated that PTX enhanced several motion sperm parameters as well as BCMP in asthenozoospermic samples and suggest a potential use of the methylxanthine in infertile patients with motility defects undergoing artificial insemination.

Spectrophotometric analysis of molecular transport in gels

An automated spectrophotometric method has been developed for analyzing molecular transport out from and into gels. A Beckman DU7500 diode-array UV-visible spectrophotometer with gel scanner was modified to accept and longitudinally scan a quartz diffusion cell, 0.3×10×40 mm. Molecules of interest are identified and concentrations quantitated via analysis of spectrophotometric absorbance peaks relative to background absorbance of the gel. Thus, concentration profiles are obtained as functions of both position and time. Test data are fitted to a diffusion model via nonlinear least-squares regression. Precision and accuracy of the method were assessed via analysis of several test molecules and gels: (1) 30 mg/ml nonoxynol-9 (N9), contained in 1% sodium alginate gel cross-linked with 2.5 mM calcium chloride, permeating standardized, reconstituted bovine cervical mucus (BCM); (2) 2.5 mg/ml sodium fluorescein, contained in and permeating 10 mg/ml and 100 mg/ml gelatin gels; and (3) 1.0 mg/ml sodium ganciclovir, contained in and permeating 10 mg/ml sodium hyaluronate gel. Diffusion coefficients for (1) and (3) were 3.8×10 −7 and 54.1×10 −7 cm 2/s, respectively. All measurements of diffusion coefficients, partition coefficients, and solute loads obtained in this study were highly repeatable (most C.V.'s<8%). The mean diffusion coefficient for (2) was within 3% of values predicted from theory for the 100 mg/ml gel; the mean partition coefficient for (3) was within 2% of the expected value. This new technique is simpler than many traditional ones in that it does not require labeling of test molecules nor changes in refractive index of target materials. It is particularly well-suited to situations in which the target material is a gel, because no stirring of the target is necessary.

Glutathione in spermatozoa and seminal plasma of infertile men.

Glutathione has a central role in the defence against oxidative damage; however, the data on glutathione concentrations in the semen of infertile men are limited. To expand this knowledge the glutathione content of the ejaculates and blood plasmas of 77 infertile men and 11 controls were analysed. The concentrations of total glutathione were assessed in spermatozoa, seminal plasma and blood plasma using a coupled spectrophotometric assay. In the spermatozoa of patients with oligozoospermia the glutathione concentrations (2.57+/-0.96 nmol/10[8] spermatozoa; mean +/- SD) were significantly lower than in the controls (3.49+/-0.87 nmol/10[8] spermatozoa; P +/- 0.03). The glutathione content of spermatozoa from patients with normozoospermia showed large variations (3.04+/-1.37 nmol/10[8] spermatozoa). An association between the intracellular glutathione content and the ability to penetrate bovine cervical mucus was observed (r = 0.2, P = 0.04). The intracellular glutathione concentrations correlated with the glutathione levels in seminal plasma (r = 0.46, P < 0.0001). There was no correlation between glutathione concentrations in blood plasma and those in seminal plasma or in spermatozoa. The glutathione concentrations in seminal plasmas did not differ between the different groups, however, correlated with the serum follicle stimulating hormone concentrations (r = 0.53, P = 0.005). This study demonstrated that intracellular glutathione levels of spermatozoa are decreased in certain populations of infertile men.

Correlation of the Bovine Cervical Mucus Penetration Test With Human Sperm Characteristics in 1406 Ejaculates

Correlation of the Bovine Cervical Mucus Penetration Test With Human Sperm Characteristics in 1406 Ejaculates

P-253 Correlation of the bovine cervical mucus penetration test with human sperm characteristics in 1406 ejaculates

P-253 Correlation of the bovine cervical mucus penetration test with human sperm characteristics in 1406 ejaculates

Sperm kinematics of cryopreserved normozoospermic specimens after artificial stimulation

Objectives Cryopreservation of semen is associated with reduced motility after thawing, resulting in a decreased pregnancy rate. Artificial stimulation of motility has been used in fresh semen samples. This study measured the effect of motility stimulants on various motion characteristics and other sperm functions using cryopreserved semen. Methods Frozen semen samples from healthy donors were thawed, and motility stimulants were added in vitro and incubated for 60 minutes. The percentages of motile spermatozoa in each specimen and other motion characteristics were measured. In addition, spermatozoon's viability, membrane integrity, and ability to penetrate bovine cervical mucus were studied after addition of stimulants. Results Percentage motility and all other motion characteristics improved after stimulation with pentoxifylline, caffeine, or 2-deoxyadenosine. Linearity did not significantly differ in the control samples after adding any of the stimulants. Viability, membrane integrity, and penetration ability did not improve significantly and were comparable with control values. Conclusions Pentoxifylline, caffeine, and 2-deoxyadenosine can stimulate sperm motility and other motion characteristics. This may be beneficial in the cryopreservation of sperm from normal donors and oligozoospermic patients for use in assisted reproduction.