Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10(-4) dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.     

Mammalian sperm chemotaxis and its association with capacitation

Much progress has been made in recent years in establishing mammalian sperm chemotaxis and understanding sperm capacitation. Thus far, chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime of 50 min to 4 h) and they occur only once in the sperm's lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in humans) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the time during which capacitated spermatozoa are available in the female reproductive tract. The sperm chemoattractants have not been identified, but they appear to be heat‐stable peptides. Although the molecular mechanism and the in vivo location of sperm chemotaxis are not known, a number of possible mechanisms and locations are discussed. Dev. Genet. 25:87–94, 1999. © 1999 Wiley‐Liss, Inc.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.     

Study of apoptosis-related markers in ram spermatozoa

Certain features of capacitated or frozen-thawed spermatozoa have been considered to be an apoptosis-like phenomenon, and, it has been suggested that the presence of apoptotic sperm in seminal doses could be one of the reasons for poor fertility. The objective of this study was to determine whether phosphatidylserine (PS) translocation, caspase activity and DNA fragmentation, which are considered to be apoptotic markers in somatic cells, occur in ram sperm. Fresh ejaculates and sperm samples in different physiological state (cold-shocked, in vitro capacitated and acrosome-reacted (AR)) were compared. Simultaneous staining with 6-carboxifluorescein diacetate (6-CFDA) and Annexin V-Cy3.18 (AnnV) revealed four different sperm subpopulations in ejaculates. The main subpopulation was composed of viable cells without PS exposure (CFDA+/AnnV-). A total of 40.8% of sperm showed inverted PS, with two levels of alteration: CFDA+/AnnV+ in midpiece ("type I AnnV+"), and in acrosome and midpiece ("type II AnnV+"). The fewest subpopulation contained non-viable cells showing Annexin labelling in the entire cell (CFDA-/AnnV+). Labeling of caspases-3 and -7 by immunocytochemistry revealing different sperm subtypes depending on their localization in apical, equatorial, post-acrosomal regions and tail. The results obtained by western-blot showed, for the first time to our knowledge, that caspase-like proteins are present in fresh ram semen as both inactive and active forms. The proportion of sperm with fragmented DNA [terminal transferase-mediated dUDP nick end-labeling (TUNEL)-positive] were found rarely (2.7+/-0.5%) in all fresh ejaculates involved in this study. The analysis of total activity of both caspases by a fluorometric method showed a decrease in vitro capacitated and acrosome-reacted samples as well as in cryoinjured samples. However, the percentage of TUNEL-positive sperm demonstrating DNA fragmentation was significantly increased after in vitro induced capacitation and acrosome reaction, as well as after cold-shock although this augment was not significant. PS exposure is not totally dependent on caspases in ram spermatozoa as the addition of a caspase inhibitor prevented the increase in PS inversion due to incubation in capacitating conditions but not to the ionophore-induced acrosome reaction or cold-shock.     

Le chimiotactisme des spermatozoïdes a-t-il un rôle dans la fécondation?

Sperm chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime between 50 min and 4 h) and they occur only once in the sperm’s lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in man) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the duration of time over which capacitated spermatozoa would be available in the female reproductive tract. The sperm chemoattractants have not been identified but they appear to be heat-stable peptides. Thein vivo location of sperm chemotaxis is not known; a number of possible locations are discussed.     

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.     

Influence of caffeine on movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa

When added to frozen-thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between samples in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen-thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

肝素处理山羊精子体外获能的研究

系统研究了作用浓度、时间和温度以及输卵管上皮细胞和卵丘细胞对肝素处理山羊精子体外获能后的精子活力、质膜完整性、顶体完整率、获能比例及受精和卵裂的影响,为改善山羊精子体外获能效果和研究获能机理提供了必要的数据。主要实验结果如下：1、在获能液中添加5、10、25、50和100μg/mL肝素处理45min时,添加50和100μg/mL肝素精子获能比率最高（分别为55%和56%）,但添加100μg/mL肝素处理后顶体完整率明显（P<0.05）低于对照组。说明山羊精子获能的最佳肝素浓度为50μg/mL。2、肝素作用时间（0, 10, 20, 30, 45, 60 和120 min）的延长,获能精子比例逐渐提高。其中,肝素处理45～120 min各组的获能精子比例差异不显著（P>0.05）,处理120 min组的精子活力和质膜完整率显著低于其它各组。说明50μg/mL肝素处理精子获能的最佳时间是45～60 min。3、在42℃和38.5℃下处理时,获能精子比例显著高于15℃和37℃,但42℃处理后精子活力和顶体完整率显著低于其它温度。因此,385℃为山羊精子获能的最佳温度。4、与输卵管上皮细胞共培养获能精子比例显著高于对照组和卵丘细胞组,但精子活力、质膜完整率和顶体完整率差异不显著。输卵管上皮组的受精率（91.3％）和卵裂率（72.2％）显著高于对照组（81.2％,65.0％）。说明与输卵管上皮细胞共培养能显著提高肝素处理山羊精子体外获能的效果。     

Effects of the purified IGF-I complex on the capacitation and acrosome reaction of rabbit spermatozoa

A protein complex containing IGF-I, purified from rabbit seminal plasma, was used to investigate its effects on the capacitation and acrosome reaction of rabbit spermatozoa. Uncapacitated sperm (Pattern F), capacitated sperm (Pattern B), and acrosome-reacted sperm (Pattern AR) were determined by CTC staining, and the results were validated by PSA-staining. The addition of the IGF-I complex to the capacitative medium directed the spermatozoa to spontaneous acrosome reaction. On the other hand, IGF-I complex, added to capacitated spermatozoa, acted as inducer of the acrosome reaction. Results of IVF experiments showed high rates of fertilization with capacitated spermatozoa, acrosome-reacted by either A23187 or IGF I complex, whereas significantly lower rates were obtained with spermatozoa capacitated in the presence of IGF-I complex.     

Human sperm chemotaxis: is progesterone a chemoattractant?

Follicular fluid (FF) induces sperm chemotaxis in human spermatozoa. Progesterone also causes sperm accumulation. However, sperm accumulation can be caused by chemotaxis, chemokinesis, and trapping of various kinds. It has been suggested that progesterone also induces chemotaxis in human spermatozoa. In view of the physiological significance of sperm chemotaxis in human fertilization and its potential clinical implications, it is important to determine unequivocally whether chemotaxis is induced by progesterone and, if so, whether progesterone in FF is the chemoattractant. To resolve these questions we looked for characteristic changes in the direction of sperm swimming toward pure progesterone as well as toward FF before and after progesterone removal. Progesterone caused sperm accumulation and hyperactivation-like motility, but it caused very few changes in the direction of sperm swimming that are characteristic of chemotaxis. Removal of progesterone (and other steroids) from FF by charcoal treatment abolished the sperm hyperactivation-like motility but not sperm chemotaxis. These results suggest that while progesterone might be a weak chemoattractant, it is not the major chemoattractant in FF. Progesterone probably causes human sperm accumulation mainly by inducing hyperactivation-like motility and, as a consequence, sperm trapping.     

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.     

Identification of capacitation in boar spermatozoa by chlortetracycline staining

The functional status of boar spermatozoa undergoing capacitation in vitro was investigated. Two fluorescent stains were used: chlortetracycline (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozoa, while the second, based on the identification of capacitated spermatozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Spermatozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. After CTC staining, 3 different fluorescent patterns were observed: Pattern A with the fluorescence uniformly distributed on the sperm head, Pattern B with the fluorescence concentrated in the post-acrosomial region, and Pattern C with the fluorescence concentrated in the acrosomial region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluorescence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescence to basal values. Since only the capacitated spermatozoa are believed to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the acrosomial region identifies capacitated spermatozoa. The analysis of acrosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa displaying Pattern C, thus confirming that CTC staining is suitable for the detection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage on the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations in sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. While fresh semen showed a progressive increase in capacitated spermatozoa, ranging from low levels at the beginning of the culture to 46% at the end of incubation, the refrigerated semen had a relatively high percentage of capacitated spermatozoa at the beginning of culture, but this proportion increased only slightly during the following 90 to 180 min of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execution, it can be used routinely to identify the optimal capacitation time for different sperm samples.     

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.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.     

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode's complete medium (TCM; a capacitating medium), Ca2+-free TCM (TCM-Ca2+), or Ca2+ and NaHCO3-free TCM (Tyrode's basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3h period of incubation at 38 degrees C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern (r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa (r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.     

Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex

Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.