Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.     

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.     

Assessment of boar sperm viability using a combination of two fluorophores

A combination of the fluorophore probes, calcein acetylmethyl ester (CAM) and ethidium homodimer (EH), were used to assess viability of ejaculated boar spermatozoa. Both CAM and EH have been used as indicators of biosynthetic activity and membrane integrity in monolayer cell cultures, with CAM shown to permeate and undergo enzymatic cleavage in viable monolayer cells giving the cell a green fluorescence, and EH penetrating only membrane damaged cells giving cells a red fluorescence. To determine if these fluorophores can be used to assess boar sperm viability, ejaculates from 10 boars were divided into 3 test groups (cytotoxic-treated, swim-up and washed), utilizing a split-ejaculate technique; each group consisted of both a probe-treated and control sample. Sample viability was ascertained in the control groups by visual estimation of the percentage motile spermatozoa, whereas the number of spermatozoa showing green (CAM = viable) or red (EH = non-viable) fluorescence were quantitated for each of the probe-treated groups using a fluorsecein or rhodamine filter, respectively. All spermatozoa exposed to the combined probes had an uptake of one or both fluorophores. The cytotoxic-treated group exhibited 0% gross motility, with 100% of the sperm heads showing red fluorescence. In the swim-up group, no difference was detected (P > 0.05) between control gross motility and the percentage of completely green fluorescing spermatozoa (85% vs. 86.6%, respectively). In the washed group, a significant difference (P = 0.039) was detected between gross motility estimates and the percentage of calcein-green fluorescent spermatozoa (57% vs. 60%, respectively). This study demonstrated that 1) CAM fluoresces only viable sperm, giving off a green fluorescence, 2) EH fluoresces in only non-viable sperm, giving off a red fluorescence, 3) visual estimation of motile sperm can approximate a semen sample's viability, but is not as precise as fluorophore determination, and 4) sperm incubation with the fluorophore combination CAM and EH provided an accurate technique for the objective assessment of boar sperm viability via their distinct fluorescent patterns in boar sperm.     

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.     

Viability and apoptosis in spermatozoa of transgenic rabbits

The aim of our study was to compare the viability of sperm cells from transgenic (mWAP-hFVIII gene) or non-transgenic (normal) rabbit males as assessed by viability (SYBR-14/PI) and apoptosis (annexin V) tests. These results were evaluated using female conception rates following insemination with the respective sperm samples. No significant differences were found in concentration and motility between transgenic and non-transgenic spermatozoa. Spermatozoa from both transgenic (63.05 ± 20.05%) or non-transgenic (65.75 ± 22.15%) males, stained with SYBR-14 (green), were found to be morphologically normal. In both groups, the highest proportion of annexin V-positive sperm staining was found in the post-acrosomal part of the sperm head (8.66 and 27.53%). The percentage of sperm that stained with SYBR-14/PI or with annexin V/DAPI was correlated with liveborn in transgenic rabbits (R2 = 0.6118 and R2 = 0.2187, respectively) or non-transgenic rabbits (R2 = 0.671 and R2 = 0.3579, respectively). These data indicate that there was no difference in the viability of rabbit transgenic and non-transgenic spermatozoa when determined by both fluorescence assays.     

Effect on turkey spermatozoa of a frothy fluid derived from the cloaca of a male turkey

The influence on turkey spermatozoa of a frothy fluid derived from the cloacal region of a male turkey was investigated. The frothy fluid was collected from the turkey tom during mounting, and semen for the experiment was obtained from the ductus deferens removed after necropsy. Spermatozoa diluted with frothy fluid were examined for motility, viability, and fertilizing capacity and compared with semen diluted with phosphate buffer or undiluted ductal semen. The life-span of spermatozoa suspended in frothy fluid was slightly prolonged during in vitro storage as compared with the undiluted semen or the semen diluted with phosphate buffer; however, a rapid increase of the number of deformed spermatozoa during storage was observed in the semen diluted with frothy fluid. The fertilizing ability of spermatozoa was not influenced by the dilution with frothy fluid when the diluted spermatozoa were inseminated intravaginally immediately after the dilution. On the contrary, when spermatozoa suspended in frothy fluid were preserved at 0 C for 24 h, their fertilizing capacity decreased drastically, probably due to the increased number of abnormal spermatozoa during in vitro preservation.     

Effects of castration on thiol status in rat spermatozoa and epididymal fluid

Mammalian spermatozoa gain their fertilizing ability as they mature in the epididymis, a process which is accompanied by oxidation of sperm protein thiols. Since sperm maturation is dependent upon normal androgenic support to the epididymis, the present work was designed to study the effects of castration on thiol status. Spermatozoa and epididymal fluid were isolated from the epididymides of male rats 5 days after castration or after 11 daily injections of the antiandrogen, cyproterone acetate. Spermatozoa and epididymal fluid were labeled with the fluorescent thiol labeling agent monobromobimane. Intact spermatozoa were evaluated by fluorescence microscopy, protein thiols were analyzed by electrophoresis, and fertilizing ability was examined after insemination of sperm suspension into the uterine horns of immature superovulated female rats. We found that both treatments resulted in an increase in cauda sperm thiols as shown by increased fluorescence in the intact spermatozoa. Protamines and nonbasic proteins were found to have increased levels of reactive thiols. The protein profiles of epididymal fluid from castrated rats were different from those of the controls, and the fluorescence patterns corresponded to the protein profiles. Our results indicate that testosterone withdrawal leads to inhibition of sperm thiol oxidation. Mol. Reprod. Dev. 47:295–301, 1997. © 1997 Wiley-Liss, Inc.     

Plasma membrane translocation of phosphatidylserine in human spermatozoa

The purpose of the study was to examine the phosphatidylserine translocation in human spermatozoa membrane during capacitation. Material consisted of human semen from normozoospermic men. Spermatozoa were stained with fluorescein-labelled annexin V. The presence and distribution of annexin V binding sites were analysed using the fluorescence microscope. Within first 60 min afterejaculation, 5-39% viable annexin V-positive spermatozoa were detected. The annexin V binding sites were found mainly in the midpiece. After 4 to 8 h of incubation of spermatozoa in capacitation medium (BMI), the number of cells positively stained with annexin V increased. After capacitation, the localisations of phosphatidylserine was changed and the annexin V binding sites were found also in the acrosomal region but never in the equatorial area. The process of the phosphatidylserine translocation observed during our experiments may reflect changes of the plasma membrane occurring during capacitation or, less likely, apoptosis of spermatozoa.     

The rôle of the spermathecal gland of the boll weevil,Anthonomus grandis

Spermatozoa were relatively inactive and did not enter the spermatheca of female boll weevils, Anthonomus grandis, whose spermathecal glands were removed as teneral adults. However, these females were able to lay fertile eggs for a 2 week period. When the spermathecal gland was removed from older females, spermathecal filling occurred, and although the spermatozoa retained their fertilizing capacity for extended periods, spermathecal emptying did not occur. Spermatozoa gradually lost their motility and fertilizing capacity, indicating that spermathecal secretions are effective in very small amounts. Spermatozoa were not activated by any of the materials contained in the normal male ejaculate. These materials alone did not effect spermathecal filling nor were they capable of maintaining the fertilizing capacity of the spermatozoa for very long. Sperm economy is low with less than 1 fertile egg laid per 100 spermatozoa used.     

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.     

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14? fluorescent stain), mitochondrial activity (Mitotraker? fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer - CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.     

The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa

The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual thresholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32-113, 61-105, 48-104 and 29-91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56-94.59%, 93.92-97.02%, 76.14-97.76% and 79.45-83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.     

Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting

We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.     

Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic stress fluorescent staining and flow cytometry

A study was conducted to evaluate the integrity of turkey sperm plasma membrane subjected to various hypo-osmotic conditions, and to develop a test to determine the percentage of viable spermatozoa capable of withstanding hypo-osmotic stress after in vitro storage. Semen from 10 toms was collected and pooled twice weekly for 6 wk, and each trial was repeated 6 times. For Trial I, spermatozoa were subjected to varying osmotic solutions by suspension in 100, 80, 60, 40, 20 or 0% PBS in distilled water (297 to 19 mosm/kg H₂O) and stained to assess membrane integrity with Calcein-AM (CAL) and propidium iodide (PI). The CAL detected viable spermatozoa (green fluorescence) while the PI stained dead cells (red fluorescence). Spermatozoa were evaluated microscopically and by flow cytometry. The percentage of viable spermatozoa, as determined by flow cytometry, was not different from that in 100% PBS (76.4 ± 3.8) to 20% PBS (74.1 ± 3.5). Fewer viable spermatozoa, however, were detected in 0% PBS (61.1 ± 4.8, P < 0.05). The percentages of swollen tails observed for viable (green stained) spermatozoa were 0, 4.5, 6.5, 24.3, 50.5 and 100% for 100, 80, 60, 40, 20 and 0% PBS, respectively. Semen was also evaluated fresh or after 24 h in vitro storage at 5 °C in PBS or H₂O (Trial II). The percentage of viable spermatozoa was not different for fresh or in vitro-stored spermatozoa in PBS. For spermatozoa stored 24 h in vitro, the percentage of viable cells was lower in H₂O (48.0 ± 5.1) than in PBS (66.1 ± 5.6, P < 0.05). Subjecting in vitro-stored sperm cells to hypo-osmotic stress before fluorescent staining resulted in detection of labile spermatozoa not accounted for by staining alone, indicating that the turkey sperm membrane is more susceptible to damage after cold storage.     

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.     

Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay

BACKGROUND: Neutrophil subpopulations with enhanced oxidative reactivity have been described in a number of clinical and in vitro settings. In the dichlorofluorescin (DCFH) oxidation assay, it is essential to maintain cellular viability and plasma membrane integrity through all stages of sample preparation. The process of erythrocyte lysing is crucial because a number of commercial lysing reagents can increase leukocyte membrane permeability. METHODS: We assessed viability [propidium iodide (PI) method], DCFH oxidation, and CD11b expression of resting or in vitro-stimulated neutrophils exposed to six different red cell lysing procedures. RESULTS: Formaldehyde-containing reagents (Optilyse B, FACS Lyse, and Erythrolyse) but not hypotonic shock or ammonium chloride (NH(4)Cl) solutions rendered 91.4--99.8% of resting neutrophils PI positive, with concomitant reductions in dichlorofluorescein (DCF) fluorescence, suggesting efflux of the fluorochrome. However, when stimulated with N-formyl-methionyl-leucyl-phenylalanine or Yersinia enterocolitica and then treated with FACS Lyse or Erythrolyse, up to 69.9% of neutrophils remained PI negative and exhibited enhanced DCF fluorescence. CD11b expression of PI-positive and -negative neutrophils was comparable, suggesting that they were activated equally. CONCLUSIONS: FACS Lyse and Erythrolyse can modify neutrophil plasma membrane integrity, whereas hypotonic shock and NH(4)Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay.     

A comparative study of spermatozoal chromatin using acridine orange staining and flow cytometry

Spermatozoa obtained from fish (Clarias gariepinus), human (Homo sapiens), turkeys (Meleagris gallapova), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), and monkeys (Macaca fascicularis) were stained with acridine orange before measuring fluorescence by flow cytometry. These mature sperm from various species produced different intensities of fluorescence while displaying similar ratios of red/green fluorescence. Comparison of the green fluorescence values for the various species showed the sequence (descending order of fluorescence values) human, turkey, monkey, hamster, rat and fish. The DNA complement (as base pairs in the haploid genome) of the various species did not increase in direct proportion to the fluorescence values. This suggests that the DNA was not equally accessible to the dye in the different species tested. The similarity in ratios of red/green fluorescence suggests that the structure of DNA in the chromatin is similar in the different species but abnormal 'satellite' populations of cells that show higher red/green fluorescence ratios than the parent population have been found in sperm samples from monkeys and from some infertile men. Their high red fluorescence intensities were not caused by RNA because treatment with RNAse did not alter the red fluorescence. It is possible that these cells contain larger amounts of denatured (single stranded) DNA.     

The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm

The present study aimed to establish a sensitive in vitro assay to assess the binding capacity of cat spermatozoa. Cat oocytes and epididymal sperm cells were isolated from gonads and cultured for in vitro fertilization. Before fertilization, the sperm cells were incubated either in 10 microM green dye Fluo-3-AM or 10 microM orange dye CellTracker Orange CMTMR (Molecular Probes), respectively. After removing the dyes by washing, sperm cells stained with each dye were added to medium drops containing oocytes in various proportions and cultured for 16 h at 37 degrees C, 5% CO(2). The oocytes were examined using fluorescence microscopy. Sperm bound to oocytes, and stained with different colors, were counted. When fresh epididymal sperm were mixed in at a specific proportion, the number of sperm bound to the zona pellucida (ZP) of oocytes reflected the proportion of differently colored sperm in the medium. This indicated that neither dye influenced the binding capacity of cat sperm. Mixing fresh and cryopreserved sperm, however, resulted in a higher number of fresh sperm bound to the oocyte surface in comparison to frozen-thawed sperm. Also, the pre-incubation of cat sperm cells with ZP derived peptide reduced the sperm binding capacity by 40%. In conclusion, the presented sperm competition assay allows assessment of fertilizing capacity of cat spermatozoa in vitro when a mixture of two different populations is used. The applied supravital fluorescence dyes do not affect motility and binding capacity of sperm cells and were clearly distinguishable under fluorescence microscopy. We demonstrate that the assay can be used to study the impact of sperm treatment, such as cryopreservation or pre-incubation in bioactive peptides, on fertilizing capacity.     

Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa.

The effect of a partial deletion of Y chromosome on sperm fertilizing ability was investigated through an in vitro fertilization technique. Epididymal spermatozoa of a congenic line, B10.BR-Ydel, which is characterized by a high incidence of abnormal spermatozoa, revealed a significantly lower in vitro fertilization rate (22%) than that (79%) of its control strain (B10.BR/SgSn), which has a normal-sized Y chromosome. Incidence of capacitated spermatozoa as determined by chlortetracycline fluorescence assay was significantly lower in B10.BR-Ydel than in B10.BR/SgSn spermatozoa. The fertilization rate was significantly improved when B10.BR-Ydel spermatozoa were separated from the supernatant of sperm suspension by Percoll gradient centrifugation. A reconstitution experiment revealed that the B10.BR-Ydel spermatozoa were more sensitive to the inhibitory effect of the supernatant than B10.BR/SgSn spermatozoa. Spermatozoa from F1 (C57BL/6N male x B10.BR-Ydel female) males showed higher fertilization rates than those from F1 (B10.BR.Ydel male x C57BL/6N female) males. These observations suggest that not only the morphology but also the fertilizing ability of spermatozoa is directly related to partial deletion of Y chromosome.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.