Cyto-immunological studies of guinea pig sperm antigens

Summary Antigenic localization in guinea pig epididymal sperm and testicular imprints as well as in viable, motile guinea pig epididymal sperm was studied by means of fluorescent labelled antibody techniques. Globulins from rabbits and chickens immunized with guinea pig epididymal sperm were used in the direct procedure while sera from sheep and fowl injected with rabbit globulins were used in the indirect procedure. The main findings were: 1) spermatozoa from the distal portion of the epididymis displayed brilliant fluorescent acrosomes and less intensely stained midpieces and principal pieces when treated as dried smears in both the direct and indirect methods; 2) testicular spermatozoa were similarly stained but whereas in epididymal spermatozoa the whole acrosome stained intensely, the testicular spermatozoal acrosome displayed intense fluorescence of the inner acrosome; 3) protoplasmic droplets fluoresced strongly; 4) cross-reactivity was observed between human and guinea pig sperm but not between rat and guinea pig sperm, indicating an antigenic relationship between human and guinea pig but not between guinea pig and rat; 5) treatment of viable, motile guinea pig spermatozoa with fluorescent globulins resulted in agglutination and immobilization as well as formation of antigen-antibody aggregates adherent to the cell membrane of the head, midpiece and principal piece; the formation of such fluorescent aggregates in the medium surrounding the treated motile sperm was indicative of leaching of antigenic material from the sperm cells.This investigation was supported by funds from United States Public Health Service grant HE-05798-03, The Ford Foundation and National Science Foundation.     

The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation

It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.     

The use of lectins to characterize and separate living canine spermatozoa: A preliminary study

This study was designed as a preliminary attempt to develop a methodology for relating the glucidic structure of the sperm membrane to sperm morphology. Differences in plasma membrane glycoconjugates between motile and nonmotile spermatozoa were studied by using 7 lectins. Fresh spermatozoa from 3 dogs were analyzed by fluorescein isothiocyanate (FITC) labeled lectins. The binding of lectins to the sperm membrane and the capability of the lectins to agglutinate spermatozoa were estimated semi-quantitatively by observation with either an epifluorescence or a phase contrast microscope, respectively. All the lectins tested bound to non motile spermatozoa, with Helix pomatia , Pisum sativum and Arachis hypogaea showing intense fluorescence, Triticum vulgare and Glycine maxima showing moderate fluorescence, and Phaseolus vulgaris and Phytolacca americana showing low fluorescence. However, Helix pomatia . and Triticum vulgare also bound to rapid and slow moving spermatozoa, and were the only 2 lectins that induced sperm agglutination. These results suggest that lectins could be a possible tool for characterizing and separating spermatozoa with different rates of motility.     

The Frequency of Heteroploidy in Sperm of Patients with Infertility

The incidence of heteroploid spermatozoa was studied by mono- and dual-colored fluorescence in situ hybridization in semen samples from donors and patients with normal and impaired spermatogenesis. The frequency of heteroploid sperm in the ejaculate was linearly and inversely correlated with sperm parameters (sperm concentration in the ejaculate and proportion of motile and morphologically normal spermatozoa). The level of heterploidy was the most significant in the semen samples from patients with oligoasthenoteratospermia and oligoasthenospermia.     

The frequency of heteroploidy in spermatozoa of patients with infertility

The incidence of heteroploid spermatozoa was studied by mono- and dual-colored fluorescence in situ hybridization in semen samples from donors and patients with normal and impaired spermatogenesis. The frequency of heteroploid sperm in the ejaculate was linearly and inversely correlated with sperm parameters (sperm concentration in the ejaculate and proportion of motile and morphologically normal spermatozoa). The level of heterploidy was the most significant in the semen samples from patients with oligoasthenoteratospermia and oligoasthenospermia.     

Evaluation of acrosomal status of bovine spermatozoa using concanavalin a lectin

We report here that fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) specifically labels the acrosomal region of acrosome-reacted bovine spermatozoa. This labeling is found to be useful in evaluating the acrosome status of bovine spermatozoa. When fresh bovine spermatozoa that had been fixed with 4% formaldehyde, smeared on glass slides and then air-dried were stained by FITC-ConA, weak fluorescence was observed on the acrosomal region, although almost all the spermatozoa appeared to be acrosome-intact. However, when fresh sperm suspensions were incubated with FITC-ConA and then mounted on glass slides, no fluorescence was observed on the acrosomal region. Therefore, in the ensuing experiments, both the fixation and the FITC-ConA staining of spermatozoa were done in suspension. When ethanol-treated spermatozoa, whose outer membrane may be permeabilized, were stained with FITC-ConA, the fluorescence was extensively observed on the inner acrosomal region. This fluorescence was inhibited in the presence of 0.2 M D-mannose, a competitive sugar, suggesting that FITC-ConA binds specifically to glycocomponents on the inner acrosomal membrane. We next tried to stain fresh or frozen-thawed spermatozoa from 3 different bulls that had been treated with the calcium ionophore A23187, which is known to induce acrosome reaction of bovine spermatozoa, with FITC-ConA. A significant correlation between the percentage of ConA-labeled spermatozoa and that of rose bengal stained negative ones at various time points during A23187 incubation was achieved. Furthermore, suitability of dual staining to distinguish between physiological acrosome reaction (acrosome-lost and live) and degenerative acrosomal loss (acrosome-lost and dead) using FITC-ConA and Hoechst bis-benzimide 33258 (H258) supravital stain was also confirmed. From these results, it was concluded that the FITC-ConA labeling procedure is a feasible and reliable method for the assessment of physiological acrosome reaction of bovine spermatozoa.     

Separation of motile spermatozoa from frozen-thawed buffalo semen: swim-up vs filtration procedures

Three experiments were conducted to maximize the recovery rate of motile spermatozoa from frozen-thawed buffalo semen. In Experiment 1, the swim-up of motile spermatozoa was performed in the presence or absence of HEPES in TALP medium and CO2 in the environment. The recovery rate of motile spermatozoa in TALP medium (control), TALP + HEPES + CO2, TALP + HEPES and TALP + CO2 was 15, 18, 12 and 10%, respectively (P > 0.05), with sperm motility at 87, 89, 90 and 90%, respectively (P > 0.05). In Experiment 2, the pH of TALP medium was adjusted to 7.0, 7.5, 8.0, 8.5 and 9.0, and swim-up procedure was performed in the presence of HEPES and CO2. The recovery rate of motile spermatozoa at different pH was 14, 20, 24, 27 and 16%, respectively (P < 0.05). Motility of separated spermatozoa was 88, 91, 90, 89 and 90%, respectively (P > 0.05). In Experiment 3, the efficiency of ion-exchange filtration and Swim-up procedure in separating motile spermatozoa from frozen-thawed buffalo semen was compared. The recovery rate of motile spermatozoa was 95% in filtration procedure and 33% in swim-up procedure (P < 0.005). In all experiments, normal acrosomes did not vary due to treatments (P > 0.05). In conclusion, HEPES and CO2 had no significant effect on swim-up of buffalo spermatozoa. The pH 8.5 of TALP improved the recovery rate of motile spermatozoa in swim-up procedure. The ion-exchange filtration was found superior to swim-up procedure in harvesting maximum number of motile spermatozoa from frozen-thawed buffalo semen (95 vs 33%; P < 0.001).     

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.     

Effects of holding time during cooling and of type of package on plasma membrane integrity, motility and in vitro oocyte penetration ability of frozen-thawed boar spermatozoa

The effect of a prolonged holding time (HT) during cooling on plasma membrane integrity (PMI), motility and in vitro oocyte penetration ability of boar spermatozoa frozen-thawed in different types of package was investigated. Boar semen was frozen in a split-sample design using 3 different HTs (3, 10 and 20 h) during cooling and three different types of freezing package: Maxi-straws, Medium-straws and FlatPacks. Assessment of PMI (SYBR-14 and propidium iodide, fluorescence microscopy) and sperm motility (visually and with CASA) was done during cooling (at 32 degrees C, 15 degrees C, 5 degrees C) and post-thaw (PT). The in vitro oocyte penetration ability of the spermatozoa was tested only PT, using a homologous in vitro penetration assay (hIVP). During cooling the HTs used had no significant (p<0.05) effect on either PMI or percentage of motile spermatozoa Post-thaw PMI was significantly higher (p<0.05) for 10 h and 20 h HT compared with 3 h, and the percentage of motile spermatozoa decreased significantly with 20 h HT as opposed to 3 h and 10 h. Regarding the freezing packages, the FlatPacks and Maxi-straws yielded significantly more PMI than did the Medium-straws (p<0.05). Post-thaw motility was significantly higher for FlatPacks than for straws, in terms of both percentage motile spermatozoa, and sperm velocity and lateral head displacement (LHD). The hIVP did not show any significant differences among the HTs, although FlatPacks yielded a significantly higher penetration rate and more spermatozoa per penetrated oocyte (p<0.05) than did the straws. Changes in motility patterns, toward a more circular motility during cooling and PT, could be noticed where individual spermatozoa showed a capacitation-like motility pattern. The changes were more obvious with 10-h and 20-h HTs than with 3-h HT.     

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.     

Motility characteristics and membrane integrity of cryopreserved human spermatozoa.

The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.     

Cryopreservation of very low numbers of spermatozoa from male patients undergoing infertility treatment using agarose capsules

This study tried to cryopreserve low numbers of spermatozoa from men undergoing infertility treatments by inserting into agarose capsules. The capsules were transferred into a drop of cryoprotectant solution and injected 3–4 motile spermatozoa that were selected by the swim-up method by conventional intracytoplasmic sperm injection. These capsules were put on a Cryotop^® and frozen in liquid nitrogen vapor, and then submerged into liquid nitrogen and subsequently thawed and recovered. The motile spermatozoa in the capsules were counted. Eventually, we cryopreserved 2142 motile spermatozoa in 702 agarose capsules from 26 male patients and 1356 (63%) spermatozoa maintained their motility after thawing. The spermatozoa motility rates after thawing (MRAT) ranged from 20.0% (5/25) to 95.1% (58/61) among patients. The median MRAT was 68.3% (interquartile range 46.1–75.7). The total number of motile spermatozoa collected by swim-up method strongly correlated with MRAT (r = 0.746). It was possible to cryopreserve spermatozoa from male patients undergoing infertility treatment using agarose capsules. However, there were wide differences in MRAT among patients. It seems the spermatozoa from semen where there were many motile spermatozoa may have higher freezing resistance. Further studies using this method in cryptozoospermic semen, testicular and epididymal spermatozoa are required.     

In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll

Frozen-thawed epididymal spermatozoa from four boars were separated through a Percoll gradient, and motility characteristics and in vitro fertility were assessed. Percoll-separated spermatozoa had a significantly higher percentage of motile and progressively motile spermatozoa than those that were not separated (P < 0.0001). However, there were no clear differences in other motility parameters between Percoll-separated and un-separated spermatozoa. Furthermore, sperm agglutination was decreased by Percoll separation (P < 0.05). The effects of Percoll separation on in vitro fertility of spermatozoa differed among boars. In addition, there were large differences in fertility between sperm samples in vitro. Sperm samples, which indicate highly motile and progressively motile, did not always show high in vitro fertility. Furthermore, there was no distinct pattern between fertility in vitro and motility parameters. There was no difference in fertility in vitro between Percoll-separated and un-separated spermatozoa from two of the four boars. However, in vitro fertility of Percoll-separated spermatozoa was higher than that of un-separated spermatozoa from the other two boars.     

The use of transmigration and spermac stain to evaluate epididymal cat spermatozoa

Epididymal spermatozoa of domestic cats were diluted with TEST medium and frozen. The parameters - estimated percentage of motile spermatozoa, concentration of spermatozoa, cell morphology and transmigration rate (TMR) - were evaluated before freezing and after thawing. Transmigration is a new method to measure the percentage of spermatozoa that consistently move forward, and has not been investigated with cat spermatozoa until now. Estimated percentage of motile spermatozoa averaged 65%, TMR was 76%, concentration of spermatozoa was 30,000 microl(-1) and the incidence of morphologically abnormal spermatozoa averaged 58% before freezing. After thawing, the estimated number of motile spermatozoa declined by 22%, but TMR remained at 76%. The TMR did not correlate with estimated motility but mostly was higher than the latter, which is postulated to be caused by the mobilizing effect of the countercurrent in the transmigration apparature. The estimated percentage of motile cells in the target chamber of the transmigration apparature was improved by using phosphate-buffered saline (PBS) as transmigration medium. Morphology was assessed both after fixation of spermatozoa in Hancock solution and after staining of smears with Spermac. Spermac did not stain all protoplasmic droplets but proved to be more suitable for the routine examination of acrosomal morphology after thawing.     

The effects of rapid cooling (cold shock) of ram semen, photoperiod, and egg yolk in diluents on the survival of spermatozoa before and after freezing

The effects of rapid cooling of semen (cold shock) from 30 degrees C to various temperatures above 0 degrees C on survival of ram spermatozoa suspended in diluents with or without egg yolk were assessed before and after freezing. Rapid cooling of extended semen from 30 to 15 degrees C had little or no effect on spermatozoa survival before or after freezing. Rapid cooling of extended semen from 30 degrees C to 10, 5, or 0 degrees C was accompanied by a progressive decrease in percentage of motile spermatozoa and percentage of intact acrosomes before freezing and a decrease in percentage of motile spermatozoa and after freezing. The ability of spermatozoa motile after cold shock to survive freezing and thawing, evaluated as cryosurvival, was not significantly (P greater than 0.05) affected by the temperature to which semen was cooled. The addition of egg yolk to the initial extender had a beneficial effect on percentage of motile spermatozoa particularly after rapid cooling of semen to 10 and 5 degrees C. Although egg yolk had little effect before freezing on semen rapidly cooled to temperatures above 15 degrees C and therefore not actually cold shocked, it substantially improved the subsequent survival of spermatozoa after freezing and thawing. Percentage of motile spermatozoa after cooling and after freezing was generally higher when the semen was collected during a decreasing photoperiod than during an increasing photoperiod.     

Comparative viability of bovine sperm frozen on a cryomicroscope or in straws

The accuracy and repeatability of freezing rates and effects of evaporation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as assessed by using combinations of fluorescent stains and flow cytometry, was used in evaluating protocols for freezing spermatozoa on the cryomicroscope. Semen was diluted in Test-yolk (20%) extender containing 7% glycerol and frozen in 0.5-ml straws, 0.25-ml straws (over liquid nitrogen for 8 min) or in a quartz crucible using a Linkam BCS 196 cryomicroscope. Thawed samples were diluted with Hepes buffered medium containing 0.1% bovine serum albumin (BSA) and stained with either carboxymethylfluorescein diacetate (CMFDA) or SYBR-14 each in combination with propidium iodide (PI). Flow cytometry analysis of the samples revealed 2 major populations: 1) spermatozoa with intense green fluorescence (stained with CMFDA or SYBR-14), which were classified as plasma membrane-intact and 2) spermatozoa with intense red fluorescence, (stained with PI), which were classified as plasma membrane-damaged. Samples frozen using the cryomicroscope contained 29 and 26 % plasma membrane-intact (PMI) sperm cells, as assessed by CMFDA and SYBR-14, respectively. Cryopreservation of spermatozoa in 0.5-ml straws resulted in 22 and 20% plasma membrane- intact sperm cells, while spermatozoa frozen in 0.25-ml straws resulted in 34 and 31% PMI sperm cells for CMFDA and SYBR-14, respectively. No significant difference was observed (P > 0.05) for PMI spermatozoa stained with either CMFDA or SYBR-14. In addition, the ability to recover spermatozoa after freezing on the cryomicroscope establishes the Linkam BCS 196 as a useful tool for the study of sperm cell cryopreservation.     

Effect of insemination dose on pregnancy rate in mares

Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected.     

Motility and penetration competence of frozen-thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing

The objective was to determine if exposure of spermatozoa to seminal plasma before freezing decreases its freezability, assessed by percentage motile cells (using computer-assisted semen analysis) and in vitro penetration ability (using in vitro fertilization and chlortetracycline fluorescence assessment). Ejaculated spermatozoa from miniature pigs were washed by centrifugation within 20 min after collection, then incubated in seminal plasma or modified Hulsenberg VIII diluents (mHM). When the spermatozoa were cryopreserved, spermatozoa incubated in seminal plasma before freezing had significantly lower post-thaw motility than spermatozoa incubated in mHM. The incubation of spermatozoa in seminal plasma also significantly prevented frozen-thawed spermatozoa from penetrating the oocytes. The second experiment, using unfrozen spermatozoa, was to determine if the incubation of spermatozoa with seminal plasma reduced penetration ability before freezing, resulting in a significantly lower penetration rate after freezing (compared with spermatozoa incubated without seminal plasma). The penetration competence of unfrozen spermatozoa was significantly decreased by incubation in seminal plasma, but no difference in motility was observed between spermatozoa exposed to seminal plasma versus mHM. We concluded that ejaculated seminal plasma contained some factor(s) that modified the sperm before freezing and reduced the freezability and post-thaw penetration competence of spermatozoa.     

Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season

In the spermatozoa of Asterias amurensis , patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate. The type II spermatozoa were also immotile and the respiratory rate remained quite low for about 5 min after the dilution. Thereafter, they spontaneously became motile and the respiratory rate increased. The type III spermatozoa became motile upon their dilution and exhibited high respiratory rate. Differences in the motility and respiratory rate of spermatozoa among batches probably result from degree of their maturation. In moving spermatozoa, the ADP and AMP levels increased at the expense of ATP. 2,4-Dinitrophenol elevated the respiratory rate only in immotile spermatozoa, which showed a high ATP level and quite low ADP level, but did not made them motile. Oligomycin inhibited the respiration of both motile and immotile spermatozoa. Probably, the respiratory rate is made low by a shortage of ADP in immotile spermatozoa and is enhanced by ADP production due to the initiation of their movement.     

Influence of butylated hydroxytoluene (BHT) on the viability of ram spermatozoa undergoing cold shock

Butylated hydroxytoluene (BHT) significantly reduced the degree of acrosome damage which occurred to ram spermatozoa during cold shock. A higher percentage of spermatozoa were motile after cold shock in the presence of BHT than in its absence, but it had little effect on the quality of motility or the percentage of cells which stained with eosin. A concentration of 2-4 mM-BHT provided the maximum response. No advantage was gained by using the solvent, dimethyl sulphoxide, as a vehicle to introduce BHT to the cells. An osmotic stress test after cold shock also failed to demonstrate any advantage of BHT. It is concluded that BHT provides little protection to the functional capacity of ram spermatozoa undergoing cold stress and is unlikely to be of benefit for the preservation of ram semen.