Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5 degrees C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5 degrees C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5 degrees C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.     

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley-Liss, Inc.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.     

Use of atomic force microscopy for morphological and morphometric analyses of acrosome intact and acrosome-reacted human sperm

The objective of this study was to use atomic force microscopy (AFM), with submicron resolution, for morphophologic and morphometric analyses of acrosome intact and acrosome-reacted human sperm heads. A mixed population of acrosome intact and reacted sperm was produced by treating capacitated sperm with A23187, which induced the acrosome reaction in approximately 50% of total sperm population. This A23187-treated sperm suspension was then plated onto a coverslip and acrosome reacted sperm were preidentified by their specific staining with rhodamine-conjugated Concanavalin A. The sperm coverslip was then air-dried and scanned by a Nanoscope IIIa atomic force microscope, using the contact mode. Top and side view images processed through the illuminate mode revealed three dimensional sperm head contour, with the highest point situated in the head posterior in both acrosome intact and acrosome reacted sperm. Maximum height, length, and width measured in 50 acrosome intact and 50 acrosome-reacted sperm were the same in both populations. However, head length at half maximum height was significantly decreased in acrosome reacted sperm (2.99 +/- 0.24 microm vs. 3.56 +/- 0.32 microm of acrosome intact sperm), due to the sudden change of the height contour from the maximum peak to the anterior tip of acrosome-reacted sperm. Our results described here can therefore be used to differentiate acrosome intact and reacted sperm from each other. This would allow future studies on subcellular changes, related to the acrosome reaction, at the submicron resolution level under more physiological conditions, since AFM does not require fixing or staining of the samples.     

Detection of cooling-induced membrane changes in the response of boar sperm to capacitating conditions

There is a need for methods of rapid and sensitive sperm function assessment. As spermatozoa are not able to fertilize an oocyte before having undergone a series of complex physiological changes collectively called capacitation, it is logical to assess sperm function under fertilizing conditions in vitro. In this study, the responsiveness of sperm to capacitating conditions in vitro was monitored by changes in sperm response to ionophore and by changes in the amount of intracellular calcium ions in stored boar semen. Boar semen was diluted at 32 and 20 degrees C and stored for 24 and 72 h at 16 and 10 degrees C. Ionophore-induced changes and increased intracellular calcium ion content in boar spermatozoa were recorded by flow cytometry and found to progress as a function of time during incubation under capacitating conditions. All responsiveness parameters (increases in proportions of membrane-defective spermatozoa, acrosome-reacted spermatozoa, and cells with high intracellular calcium levels) were shown to be sensitive to subtle physiological changes occurring at low storage temperatures. The initial levels of sperm with a high calcium content were higher in semen stored at 10 degrees C, but the accumulation of internal calcium was lower than in semen stored at 16 degrees C. The loss of membrane integrity and increase in the proportion of acrosome-reacted cells were higher in semen stored at 10 degrees C. Dilution at 20 degrees C had no negative effect on membrane integrity or responsiveness to capacitating conditions. There was no significant difference between semen stored for 24 and 72 h in terms of membrane integrity, acrosome reaction, and intracellular calcium after capacitation treatment. However, dynamics of cell death and acrosome reaction in response to capacitating conditions were somewhat accelerated after 72 h storage, especially in semen stored at 10 degrees C. It can be concluded that the simultaneous use of the sperm membrane responsiveness and kinetic parameters is a sensitive tool for the detection of storage-related membrane changes in boar semen.     

Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida

Mammalian sperm must be acrosome reacted before penetrating the zona pellucida. In some species the sperm undergo the acrosome reaction before binding to the zona pellucida and in other species only acrosome intact sperm can initiate binding to the zona. In this study we addressed the question of acrosomal status and sperm-zona binding with human gametes. Sperm acrosome reactions were induced by treatment with human follicular fluid or N-(6-amino-hexyl)-5-chloro-naphthalene sulfonamide (W-7). The sperm suspensions, containing various percentages of acrosome-reacted sperm, were then incubated with human oocytes for 1 min. The acrosomal status of the sperm population bound to the zona was similar to the acrosomal status of the population of sperm in suspension (R2 = 0.77), regardless of the treatment to induce acrosome reactions. Our interpretation of these results is that both acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. However, we reported earlier (N. L. Cross, P. Morales, J. W. Overstreet, and F. W. Hanson, 1988, Biol. Reprod. 38, 235-244) that the human zona pellucida is able to induce acrosome reactions. Thus, to exclude the possibility that sperm had undergone the acrosome reaction on the zona within 1 min of binding, sperm were suspended in a nominally calcium-free Tyrode's medium (0 Ca-mTyr) before incubation with oocytes (this medium was supplemented with SrCl2 and spermine to support sperm motility and zona binding). In 0 Ca-mTyr, the proportion of acrosome-reacted sperm on the zona was still highly correlated with the proportion of reacted sperm in suspension, indicating that the sperm were reacted before binding. Evidence that 0 Ca-mTyr effectively inhibited acrosome reactions induced by the zona pellucida was derived from experiments in which sperm were treated with human follicular fluid or control medium and the suspensions were diluted with either 0 Ca-mTyr or control medium.4+ Human oocytes were added for 1 min (pulse) at which time some oocytes were fixed and other oocytes were transferred to sperm-free medium and incubated for 35 min (chase) before fixation. Sperm diluted in control medium, pretreated with either human follicular fluid or control medium, showed a similar increase (40%) in the percentage of acrosome reactions among the zona-bound sperm after the chase. Sperm diluted in 0 Ca-mTyr did not show an increase in the percentage of acrosome-reacted sperm on the zona pellucida after the chase.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa

The effect of heparin (5 IU), caffeine (5 mM) and calcium-ionophore A23187 (0.1 mM) on motility and in vitro induction of the acrosome reaction in glass wool filtered frozen-thawed bull and goat semen was studied. The motile spermatozoa fraction was obtained after glass wool filtration of frozen-thawed semen. The seminal plasma was removed from filtered semen by centrifugation, and the sperm pellet was resuspended in Sperm-TALP medium. Samples of treated and untreated control semen of both species were incubated at 37 degrees C. At 1, 15 and 30 min of incubation the proportions of progressively motile and acrosome-reacted spermatozoa were assessed. Trypan blue and Giemsa stain was used to differentiate live and dead spermatozoa having undergone acrosome reaction. Glass wool filtration enhanced the proportion of motile spermatozoa from 43% to 62% in the bovine and from 41% to 60% in the caprine. Whereas the effect of incubation with caffeine, heparin and calcium-ionophore on spermatozoan motility was negligible, the treatment of semen with calcium-ionophore resulted in a significantly improved percentage of live spermatozoa with true acrosome reaction at all stages of incubation, both in the bovine and the caprine.     

Cholesterol-loaded cyclodextrin is efficient in preserving sperm quality of cryopreserved ram semen with low freezability

Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-β-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability.     

Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.     

Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility

When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.     

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.     

In vitro penetration of hamster oocyte-cumulus complexes using physiological numbers of sperm

We have compared the ability of uncapacitated, capacitated acrosome intact, and acrosome-reacted hamster sperm to penetrate the cumulus and corona radiata of fresh hamster oocyte-cumulus complexes (OCC) in vitro. This was done using physiological numbers (1-20) of sperm so that cumulus and corona radiata cells did not disperse during challenge. Uncapacitated sperm did not penetrate to the zona pellucida surface; most (74%) uncapacitated sperm bound to cumulus cells at the periphery of the OCC. Capacitated acrosome-intact sperm penetrated to the zona pellucida surface; a significant percentage of these sperm arrived at the zona pellucida without showing evidence of initiating an acrosome reaction. Most capacitated acrosome-reacted sperm did not enter the extracellular matrix between cumulus and corona radiata cells; those which did penetrated to the zona surface with difficulty, if at all. These results suggest that the changes which occur in the sperm surface during capacitation are more important than the acrosome reaction in enabling hamster sperm to penetrate the cumulus and corona radiata. The effects of gold sodium thiomalate (GST) and polyphloretin phosphate (PPP) (inhibitors of hyaluronidase) on penetration of the OCC by capacitated sperm were also examined. Both synthetic inhibitors blocked sperm penetration to the zona pellucida, but the effective concentrations of inhibitors were far in excess of what was needed to block hyaluronidase activity. Reasons for concluding that the action of these inhibitors is nonspecific are discussed. These data show that hamster sperm with intact acrosomes can penetrate the cumulus and corona radiata cell layers of fresh OCC in vitro and support the hypothesis that the acrosome reaction occurs on the zona pellucida surface.     

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.     

Induction of acrosome reactions by the human zona pellucida

We have used two approaches to test the ability of the human zona pellucida to induce acrosome reactions in human sperm. First, nonviable human oocytes were incubated for 1 min in a suspension of capacitated sperm (of which fewer than 5% were acrosome-reacted) to allow binding of about 200 sperm per oocyte. Some of the oocytes were fixed immediately, and the remainder were fixed after a further 1-h incubation without free-swimming sperm. As determined by light microscopy, sperm on the zona were only 3 +/- 2% (avg. +/- SD) acrosome-reacted at 1 min, and the incidence increased to 46 +/- 15% during the next hour. Electron microscopy confirmed that most sperm on the zona at 1 min were acrosome-intact. A few sperm were in an early stage of the acrosome reaction. Acrosome reactions occurring on the zona during the subsequent hour appeared to be morphologically normal. Second, treatment of sperm in suspension with acid-disaggregated zonae (2 to 4 zonae/microliter) increased the incidence of acrosome-reacted sperm from 3 +/- 1% to 24 +/- 4%. We conclude that the human zona pellucida, or material intimately associated with it, can induce acrosome reactions in human sperm.     

The physiology of sperm recovered from the human cervix: acrosomal status and response to inducers of the acrosome reaction

Cervical mucus was collected from 35 women after artificial insemination. Mucus collections were performed at 1 h, 1 day, 2 days, or 3 days following insemination. Sperm viability was greater than 80% at all recovery times as assessed by exclusion of the supravital dye Hoechst 33258. Virtually 100% of the viable sperm were acrosome-intact at all times as assessed with a fluorescein isothiocyanate-conjugated pea lectin. Sperm were recovered from the mucus after migration into the Biggers, Whittin, and Whittingham medium in vitro. Sperm did not undergo the acrosome reaction in response to human follicular fluid immediately after migration from the mucus but did respond to this agonist after 6 h of incubation in vitro. Sperm recovered at all times after insemination had the same pattern of response to follicular fluid. Sperm that penetrated a column of cervical mucus in vitro also responded to follicular fluid with an increase in acrosome reactions after migration from the mucus and incubation for 6 h in vitro. Unlike the sperm that migrated from cervical mucus, sperm that were separated from semen by Percoll density centrifugation did not undergo the acrosome reaction when challenged with follicular fluid after 6 h but did respond after 24 h incubation. Sperm that migrated from cervical mucus had a similar increase in acrosome reactions after 6 h incubation, regardless of whether the acrosome reaction agonist was follicular fluid or disaggregated human zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo fertility of rams in relation to sperm-zona pellucida binding and sperm-zona pellucida penetration of ovine oocytes

Sperm-zona pellucida (zona) binding and sperm-zona penetration have been suggested for use as in vitro bioassays of fertility since both are essential steps in the fertilization process. The correlations of sperm-zona binding and sperm-zona penetration with the in vivo fertility of sheep were investigated in this study. In vivo fertility was estimated from a heterospermic insemination trial using cryopreserved ram semen. Neither zona binding, zona penetration nor the ability to undergo an acrosome reaction was significantly correlated with the in vivo fertility of the rams (P = 0.78, P = 0.66, and P = 0.85, respectively). These results suggest that the zona binding and zona penetration bioassays may not be useful estimators for assessing cryopreserved ram sperm fertility.     

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.     

Evaluation of seasonal variations of semen freezability in Leccese ram

The experiment was carried out in Southern Italy (41 degrees N latitude) to examine the effects of seasonal variations of semen freezability in Leccese ram. Semen from five rams, collected every 2 weeks for a whole year, was frozen in straws, using a system based on Tris-fructose egg yolk as extender to constitute semen doses of 100x10(6) spermatozoa. Post-thaw survival and acrosomal status of cells were assessed by dual staining by Hoechst 33258 and FITC-PSA. Three different forms of fluorescence distribution were displayed indicating sperm without acrosome (unstained cells), sperm with damaged acrosome (cells with incomplete fluorescence over the head), sperm with widespread fluorescence (cells completely fluorescent). Motility and kinetic rating at thawing and after 1 and 3h incubation (37 degrees C) were also assessed.Semen frozen in summer and autumn, corresponding to the breeding season, showed the highest (P<0.01) post-thaw survival of spermatozoa (41.7%) and the lowest (P<0.01) incidence of spermatozoa with damaged acrosome. The positive influence of the summer-autumn period was expressed also on motility and kinetic rating of spermatozoa at thawing. The integrity of the acrosomal membrane was positively correlated (P<0.01) with sperm viability before processing (r=0.32) and after thawing (r=0.51).In conclusion, the results show that season exerts a significant influence on semen freezability in Leccese ram, with the best performance occurring the summer and autumn period, corresponding to the reproductive season in temperate zones.     

Sperm output and hormone concentrations in Finn and Dorset rams exposed to long- and short-day lighting

Seasonal changes in photoperiod have a substantial effect on sexual behavior and reproduction in rams. Little information is available on sperm output from high libido versus average libido rams subjected to intensive semen collection while being exposed to controlled short versus long photoperiods. Six Finn and six Dorset rams were compared in a reversal design, which allowed rams of both breeds to be exposed to 8 h versus 16 h of light. During each of two 84-d periods rams were subjected twice to an initial depletion of epididymal sperm reserves by collecting up to 26 ejaculates of semen in 3 d, followed by up to 10 ejaculates per day, 1, 3, 5, and 7 d after the initial depletion. A total of 2673 semen samples were collected. Nearly twice as many ejaculates (63.6% of the total) were obtained from Finn rams as from Dorset rams during both the initial and subsequent 3-d sperm depletion periods. This difference in libido was associated with obtaining 33.6 +/- 3.1 x 10(9) sperm from Finn rams versus 10.0 +/- 2.2 x 10(9) sperm from Dorset rams during the initial depletion period (P<0.05). Changes in photoperiod did not affect sperm output (P>0.05) in Finn rams, but may have affected Dorset rams. With 16 h of light, prolactin was significantly (P<0.05) increased in both breeds, particularly in Finn rams. Testosterone in both breeds followed an endogenous rhythm, not affected by the change in controlled photoperiods.     

Comparison of TNC and standard extender on post-thaw quality and in vivo fertility of Thai native chicken sperm

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.