Bovine Cervical Mucus Penetration Test Research Articles

Effect of Superoxide dismutase on cryopreservation of mithun semen

The present study was designed to assess the effect of superoxide dismutase (SOD) on post thaw semen quality parameters (SQPs), kinetic profiles and seminal biochemical profiles in mithun. A total of 25 ejaculates were selected and each was split into four equal aliquots after dilution with Tris-citrate-glycerol (TCG) extender such as Group I: control, Group II, III and IV contained 50, 100 and 150 U/ml of SOD, respectively. Cryopreserved and thawed samples were analysed for their motility parameters (progressive forward and in bovine cervical mucus penetration test [BCMPT]), kinetic and velocity parameters by computer assisted sperm analyser (CASA), viability, sperm morphological and nuclear abnormalities, acrosomal, plasma membrane and nuclear integrities and sperm enzymatic leakage and biochemical (sperm cholesterol and antioxidant and oxidative stress) profiles. Study revealed a significant (p <0.05) enhancement in viability, sperm morphological and nuclear normalities, acrosome integrity, motility (progressive and BCMPT), sperm cholesterol content and reduction in leakage of intracellular enzymes in Group III. Moreover, intactness of acrosome and biochemical membranes were protected significantly (p < 0.05) in addition to significant (p < 0.05) improvement in kinetic and velocity profiles in extender containing 100 U/mL SOD. These results clearly suggest that however the cryopreservation of mithun’s spermatozoa in TCG was comparable with other species and inclusion of 100 U/mL SOD holds a clear advantage over control or 50 or 150 U/mL SOD. It can be concluded that SOD supplementation in semen extender can be effectively utilized to reduce the oxidative stress and improved the antioxidant profiles with cascading beneficial effects on cryopreserved semen quality parameters in mithun.

Effect of trehalose on post thaw semen quality profiles, sperm kinetic profiles and antioxidant and oxidative stress profiles in Mithun

Present study was designed to assess the effect of trehalose on post thaw semen quality parameters (SQPs), sperm velocity and kinetic profiles, antioxidant and oxidative stress profiles and sperm cholesterol efflux in mithun. A total of 25 ejaculates were selected based on the biophysical parameters and each sample was split into four equal aliquots after dilution with the Tris-citrate-glycerol (TCG) extender such as Group I: control, Group II, III and IV: 50, 75 and 100 mM of trehalose, respectively. Cryopreserved and thawed samples were analysed for their motility parameters (progressive forward and in bovine cervical mucus penetration test [BCMPT]), kinetic and velocity parameters by computer assisted sperm analyser (CASA), viability, sperm morphological and nuclear abnormalities, acrosomal integrity, plasma membrane integrity and nuclear integrity and sperm intra-cellular enzymatic leakage and biochemical (sperm cholesterol, antioxidants and malondialdehyde) profiles. Study revealed a significant enhancement in viability, sperm morphological and nuclear normalities, acrosome integrity, motility, sperm cholesterol content and reduction in leakage of intracellular enzymes in Group II. Moreover, intactness of acrosome and biochemical membranes were protected significantly in addition to significant improvement in kinetic and velocity profiles in extender containing 50 mM trehalose. These results clearly indicated that however the cryopreservation of mithun’s spermatozoa in TCG was comparable with other species, inclusion of 50 mM trehalose holds a clear advantage over control or 75 or 100 mM trehalose. It can be concluded from the present study that trehalose supplementation in semen extender can be effectively utilized to reduce the oxidative stress and improve the antioxidant profiles with cascading beneficial effects on cryopreserved semen quality parameters in mithun.

Glutathione in semen extender modulates post thaw semen quality profiles, and antioxidant and oxidative stress profiles in mithun

Present study was designed to assess the effect of glutathione (GSH) on post thaw semen quality parameters (SQPs), sperm kinetic profiles, antioxidant and oxidative stress profiles and sperm cholesterol efflux in mithun. Ejaculates (25) were selected based on biophysical parameters for the present experiment. Each sample was split into four equal aliquots after dilution with the Tris-citrate-glycerol (TCG) extender, viz. Group I (control), Group II, III and IV contained 5, 10 and 15 mM of GSH, respectively. Cryopreserved and thawed samples were analysed for their motility parameters (progressive forward and in bovine cervical mucus penetration test [BCMPT]), kinetic and velocity parameters by computer assisted sperm analyser (CASA), viability, sperm and nuclear abnormalities, acrosomal integrity, plasma membrane and nuclear integrities, sperm enzymatic leakage and biochemical (sperm cholesterol and antioxidant and oxidative stress) profiles. Study revealed a significant enhancement in viability, sperm and nuclear normalities, acrosomal integrity, motility (progressive and in cervical mucus), sperm cholesterol content and reduction in leakage of intracellular enzymes in Group III. Moreover, intactness of acrosome and biochemical membranes were protected significantly in addition to significant improvement in kinetic and velocity profiles in extender containing 10 mM GSH. These results clearly indicate that though the cryopreservation of mithun’s spermatozoa in TCG was comparable with other species, inclusion of 10 mM GSH holds a clear advantage over control and 5 or 15 mM GSH. The study concludes that GSH supplementation in semen extender can be effectively utilized to reduce the oxidative stress and improve the antioxidant profiles with cascading beneficial effects on cryopreserved semen quality parameters in mithun bull.

Taurine in semen extender modulates post-thaw semen quality, sperm kinematics and oxidative stress status in mithun (Bos frontalis) spermatozoa

Objective: To assess the effect of taurine on post-thaw semen quality parameters, sperm kinematics, antioxidant and oxidative stress profiles and sperm cholesterol efflux in mithun (Bos frontalis). Methods: A total of 50 ejaculates (n=25 samples) were selected based on biophysical parameters. Each sample was split into four equal aliquots after dilution with the Tris-citrate-glycerol extender. Group I, II, III and IV contained 0 mM (the control), 25 mM, 50 mM and 100 mM of taurine, respectively. Frozen-thawed samples were analysed for motility parameters (progressive forward and in bovine cervical mucus penetration test), kinetic and velocity parameters by computer-assisted sperm analyzer, viability, sperm and nuclear abnormalities, acrosome integrity, plasma membrane and nuclear integrities, sperm enzymatic leakage and biochemical (sperm cholesterol and oxidative stress) profiles. Results: The extender containing 50 mM taurine led to a significant enhancement in viability, acrosomal integrity, plasma membrane integrity, motility (progressive and in cervical mucus), and sperm cholesterol content and notably reduced sperm morphological and nuclear abnormalities, and leakage of intracellular enzymes compared to other taurine treated and untreated control groups (P<0.05). Moreover, in addition to significant improvement in kinetic and velocity profiles, 50 mM taurine protected the integrity of acrosome and biochemical membranes than in the untreated control and other taurine treated groups. Inclusion of 50 mM taurine held a clear advantage over the control or 25 mM or 100 mM taurine in cryopreservation of mithun semen. Conclusions: Taurine (50 mM) supplementation in semen extender can be effectively utilized to reduce oxidative stress and improve post-thaw semen quality in mithun.

Flaxseed oil modulates semen production and its quality profiles, freezability, testicular biometrics and endocrinological profiles in mithun

Mithun (Bos frontalis) is a unique domestic free range bovine species of North Eastern Hilly (NEH) regions of India. Effect of feed supplementation of Flaxseed oil (FSO) on semen production and its quality profiles, freezability, oxidative stress, apoptotic sperm percentage and subsequently on endocrinological profiles & scrotal and testicular biometrics in different seasons was studied in mithun. The experimental animals were divided into two groups, Gr I: Control (n = 3) and Gr II: Treatment (n = 3; Flaxseed oil @ 150 mL/day). FSO was supplemented through oral drench in the morning hours just before concentrate feeding. A total of 80 semen samples (n = 80; 20 semen samples from each season; each 10 semen samples from control and treatment groups per season) were collected, not more than twice per week in winter, spring, autumn and summer seasons. Semen quality profiles (SQPs) such as volume, sperm concentration, motility (forward progressive and total), motility & velocity profiles by computer assisted sperm analyser (CASA), viability, total sperm abnormality, acrosome integrity, plasma membrane & nuclear abnormality and apoptotic sperm percentage were estimated in fresh semen. Along with SQPs measured in fresh semen, motility in estrus bovine cervical mucus (bovine cervical mucus penetration test; BCMPT) and mitochondrial membrane potential (MMP) by JC-1 stain were determined in the post-thawed semen samples. Biochemical profiles (aspartate aminotransferase; AST, alanine aminotransferase; ALT, total cholesterol; CHO), antioxidant profiles (superoxide dismutase; SOD, catalase; CAT, glutathione; GSH, total antioxidant capacity; TAC) and oxidative stress profile (malondialdehyde; MDA) were estimated in fresh semen whereas AST, ALT, lactate dehydrogenase (LDH), TAC and MDA were estimated in the frozen thawed semen samples. Endocrinological profiles such as follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, cortisol and thyroxin and scrotal circumference (SC) & testicular biometrics were measured in both groups in different seasons. Result revealed a significant (p < 0.05) improvement in motility (total & forward progressive, motility & velocity by CASA and vanguard distance in cervical mucus), viability, intactness of acrosome & plasma membrane, MMP, antioxidant profiles and reduction in total sperm and nuclear abnormalities, reduction in leakage of intracellular enzymes and reduction in oxidative stress profile and reduction of apoptotic sperm percentage were observed in FSO supplemented than in un-supplemented control group accordingly in fresh and post thawed semen samples. Blood FSH, LH, testosterone and thyroxin concentration were significantly (p < 0.05) increased and cortisol concentration was significantly (p < 0.05) decreased in FSO supplemented group than in unsupplemented control group. Similarly, SC and testicular biometrics were increased significantly (p < 0.05) in supplemented than unsupplemented group for different seasons and significantly (p < 0.05) higher in winter and spring than in summer season in the experimental groups. It can be concluded from the study that supplementation of FSO can effectively be utilized to improve the antioxidant profiles, reduction of oxidative stress with cascading beneficial effects on SQPs and fertility status of the mithun bull.

Exogenous cholesterol modulates oxidative stress and freezability of mithun spermatozoa

Effect of cholesterol loaded cyclodextrin (CLC) on improvement of semen quality parameters (SQPs) and deduction of oxidative stresses following cryopreservation in mithun was explored. Total 50 ejaculates were selected out of 105 collected based on preliminary SQPs. Sperm was treated with 1 mg (Gr II) and 2 mg (Gr III) of CLC/ 120×106 spermatozoa and without CLC served as control (Gr I). Diluted semen samples were cryopreserved at ultralow temperature. Frozen thawed samples were evaluated for motility (progressive forward [FPM]; in bovine cervical mucus penetration test [BCMPT] and in computer assisted sperm analyzer [CASA]), viability, total sperm and nuclear abnormality, intactness of plasma membrane and acrosome, intracellular enzymatic leakage and oxidative profile (Malondialdehyde; MDA). Result revealed a significant improvement in motility (FPM, BCMPT and CASA), viability, acrosomal integrity, cholesterol content and reduction of sperm and nuclear abnormalities, leakage of intracellular enzymes and oxidative stressors in 1 mg CLC treated group as compared to control. Moreover, intactness of acrosome and biochemical membrane was protected significantly in extender containing 1 mg CLC. Hence inclusion of mithun spermatozoa with CLC (1 mg/120×106) prior to freezing improved the survivability in cryopreservation. The results clearly indicated the beneficial effects of CLC supplementation on freezability by reducing cryodamage and protecting the spermatozoa integrity.

Effects of low-density lipoproteins as additive on quality parameters and oxidative stress following cryopreservation of mithun (Bos frontalis) spermatozoa.

Artificial breeding of mithun poses several challenges including lack of standard protocol for cryopreservation of spermatozoa. This is further complicated by harmful effects of hen's egg yolk (EY) as additive in extender. Purified low-density lipoproteins (LDL) extracted from EY have been shown as beneficial over EY extender for long-term semen storage in several species. This investigation explored use of LDL versus EY on semen quality and oxidative stress following freezing-thawing of spermatozoa. A total of 25 of 50 ejaculates based on biophysical parameters were selected for the experiment. After diluting with the Tris-citrate-glycerol (TCG) extender, each sample was split into three equal aliquots: Group I, control, EY; Group II and Group III contained 8% and 10% purified LDL, respectively. Frozen-thawed samples were evaluated for motility parameters (progressive, and in the bovine cervical mucus penetration test [BCMPT]), viability, sperm and nuclear abnormality, acrosome integrity, and enzymatic (leakage of intracellular contents) and biochemical (oxidative stress) profiles and in vitro fertility (IVF) assay. Study revealed a significant (p<.05) improvement in viability, sperm and nuclear abnormality, acrosome integrity, motility (progressive and in cervical mucus), cholesterol content, and reduction in the leakage of intracellular enzymes in Group II. Moreover, intactness of acrosome and biochemical membranes was protected significantly (p<.05) in addition to significant (p<.05) improvement in binding per cent and binding index in IVF assay in extender containing 8% LDL. These results demonstrate that although cryopreservation of mithun's spermatozoa in EY was comparable with other species, addition of 8% LDL holds a clear advantage over EY or 10% LDL.

Evaluation of the sperm migration capacity of crossbred dairy cattle bull semen vis-à-vis field fertility trials

Crossbred dairy cattle bulls (6) were subjected to semen evaluation study twice a week for sperm migration capacity in bovine cervical mucus (BCM) and poly-acrylamide gel (PAG). Cervical mucus from respective cows subsequently inseminated with same semen, was tested for sperm penetration test (SPT). PAG was prepared according to standard procedures and stored at 4°C till further use. Different grades of PAG (2% and 3%) were examined for transparency, spinbarkeit, pH and visible gelification before using for sperm migration assay. Field fertility trials based on pregnancy outcome by assigning 10 cows/bull were conducted simultaneously to BCM penetration test (BCMPT) and PAG-SPT. 2% PAG was better in assessing sperm penetration distance than 3% PAG, however, the values differed nonsignificantly. Sperm migration in BCM was better than sperm migration in PAG, and differences were significant for semen of respective bulls. The pregnancy outcome from the bull (FC-1207) having maximum sperm penetration distance (SPD) in BCM (fresh semen, 38.63 ± 0.26 mm/20 min; extended semen, 37.39 ± 0.19 mm/20min; frozen semen, 36.85 ± 0.21 mm/20min), 2% PAG (fresh, 37.44 ± 0.54 mm/20min; extended, 36.60 ± 0.34 mm/20 min; frozen, 35.89 ± 0.44 mm/ 20min) were around 58.53%, whereas, bull showing lower values of SPD in BCM and 2% PAG had lower pregnancy outcome. Six bulls under study indicated a lot of individual variation for SPD and pregnancy outcome, highlighting lower pregnancy outcome for bulls showing less SPD of sperm from fresh and frozen semen. Positive correlations were exhibited between different penetration tests and pregnancy outcome as well as between BCMPT and PAG-SPT. PAG can thus substitute for bovine cervical mucus and for assessing the sperm migration ability of various bulls used for breeding programme beforehand. Positive correlations between these tests and pregnancy outcome based on field fertility trials indicates importance of these tests for evaluating the fertility of bulls particularly with PAG-SPT before subjecting them to field evaluation.

Penetration ability of human spermatozoa into standardized bovine cervical mucus (Penetrak) in patients with normal and pathological semen samples.

Semen samples from 89 men were analyzed and evaluated with a standardized bovine cervical mucus penetration test (Penetrak). 29 patients had a normozoospermia, 22 patients a teratozoospermia and 38 patients an oligozoospermia of various degree. The most important parameters of semen analysis were compared with sperm mucus penetration. The sperm mucus penetration test correlated best with progressive motility (r = 0.6758), followed by total motility (r = 0.6302), sperm count (r = 0.6190), and % normal spermatozoa (r = 0.5493) (p less than 0.01 for each correlation coefficient). In the group of patients with normozoospermia 6 cases had been detected with insufficient sperm mucus penetration and in both groups with subfertility 15 cases with adequate sperm mucus penetration. 8 semen samples with normal sperm count and normal sperm motility were used for IVF. Only 6 patients with normal sperm penetration (greater than 30 mm/90') the spermatozoa fertilized the ova, but not from two patients, who had a sperm mucus penetration of less than 30 mm/90'. The study demonstrated that standardized bovine mucus penetration test detects disturbances of sperm motility which may not be discovered by conventional semen analysis.

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).

Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa

Seventy semen ejaculates were obtained from 14 Murrah buffalo bulls and were subjected to plasma separation immediately after collection by centrifugation at 2000 rpm for 20 min and stored in liquid nitrogen until analysis. In the seminal plasma the total protein concentration were estimated and the heparin and gelatin binding (HB and GB) proteins were isolated using heparin and gelatin affinity column chromatography. The molecular weight of individual isolated HB and GB protein was determined by SDS–PAGE analysis. Buffalo bull spermatozoa was collected from cauda epididymis under aseptic conditions and was used for the in vitro fertility tests (i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST)). The heparin and gelatin binding buffalo seminal plasma proteins were used in six concentrations i.e. 10, 20, 30, 40, 50 and 60 μg/ml to test their effect on in vitro fertility assessment of cauda epididymal spermatozoa. The overall mean values of total protein, HB and GB proteins were recorded as 29 ± 2.7, 2.61 and 0.2 mg/ml, respectively. Eighteen total protein bands were observed in the range of 12–127 kDa. Eight major HB proteins were isolated in the range of 13–71 kDa. Seven major GB proteins were isolated in the range of 13–61 kDa in the buffalo seminal plasma. The mean penetration distance (mm) travelled by the buffalo cauda spermatozoa was maximum in HB proteins (26.9 ± 0.6) followed by GB proteins (25.4 ± 0.6) and control (21.2 ± 1.4). The difference in BCMPT values between protein treated and control group was significant ( P < 0.05). Almost similar trend in the effect of protein on values of HOST percentage in both HB and GB proteins treated semen samples were recorded (66.4 ± 0.65 and 66.1 ± 0.6, respectively). The difference in HOST values between proteins treated and control group (50.4 ± 2.0) was significant ( P < 0.05). The present results indicate that among the isolated proteins, 4 proteins were commonly seen in both the heparin and gelatin–sepharose affinity column chromatography, and the addition of buffalo seminal plasma proteins improved the in vitro sperm functions (40 μg/ml gave best results) of buffalo cauda spermatozoa.

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.

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.

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

The role of sperm creatine kinase in the assessment of male fertility

The lack of reliable methods to assess sperm fertilizing potential has been a long-standing problem for infertile couples and for their physicians. The most widely used tests, the measurements of sperm concentrations, motility, velocity and morphology in the ejaculate, are of limited utility. Indeed, following intrauterine insemination, a treatment that compensates for low motile sperm concentrations, there were no significant differences found in semen parameters among those who did or did not achieve pregnancies. Other available assays probing for selected sperm functions, such as membrane integrity, acrosome enzyme activity, bovine cervical mucus penetration test, zona-free hamster oocyte penetration test and sperm binding to various carbohydrates,10–13have all failed thus far to consistently predict male fertility. It became increasingly obvious that there was a need to identify cellular markers of sperm quality and fertilizing potential.

Computerized human sperm analysis

The recent introduction of various devices for computer-assisted sperm analysis allows us to have not only objective measurements of the seminal parameters but also to determine some characteristics of sperm motion such as curvilinear and linear velocity and amplitude of lateral head displacement that cannot be obtained by routine analysis. These new seminal parameters seem to be very useful in the diagnosis of male infertility and, among them, ALH seems to play a more important role; in one of our studies we found a significant correlation between this parameter and the results of the bovine cervical mucus penetration test (Penetrak). Furthermore it is possible to determine sperm subpopulations for each parameter and so it will be possible to express a more accurate judgement not only on the fertilizing capacity of semen but also to know the effects on spermatozoa of different activating and capacitating treatments.

Frozen bovine semen quality and bovine cervical mucus penetration test

Research has been carried out to test bovine cervical mucus penetration (penetration) as a means for evaluating frozen-thawed bovine semen. A commercially available cervical mucus penetration test kit (the kit) was used. A total of 158 previously frozen semen samples collected from 61 bulls were thawed in a 37 C water-bath for 2 minutes. Four ways to estimate penetration were compared using the distance traveled during 90 minutes 1) at 21 C, or 2) at 37 C, by 3) the first solitary mobile spermatozoon, or by 4) the front of the mass of the mobile spermatozoa. Penetration was measured using phase contrast microscopy and a millimeter grid. Spermatozoal quality parameters (concentration, total motility, progressive motility, acrosome integrity, total sperm integrity and cytoplasmic droplets) were measured and the correlation to penetration was calculated. The best way to assay penetration with the kit was by measuring the penetration of the first solitary mobile spermatozoon at 37 C. Semen quality variability was significant (P < 0.05) relative to penetration. Linear correlations between penetration and acrosome integrity r=0.42 as well as between penetration and total sperm integrity r=0.53 were highly significant (P < 0.001). There was significant linear multiple regression between penetration and acrosome integrity (expressed as percentage and number) and total sperm integrity (expressed as percentage and number) (r=0.62; F=23.5147; P<0.0001). There was a significant difference between the average progressive motility of samples with penetration > 20 mm and samples with penetration ≤ 20 mm (30.5 vs 20.8; P ≤ 0.01). However there were semen samples ( N = 8 123 ; 6.5%) with normal progressive motility (29.94 ± 5.30) after thawing and low penetration (≤ 20 mm). There was no linear correlation between penetration and fertility (60 days non-return percentage). Therefore the penetration can distinguish samples of frozen-thawed bovine semen with not good or good progressive motility (> 20%), but it is not useful to define the fertility level of semen samples.