Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of the vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple–dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37°C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.     

Evaluation of sperm acrosome reaction in the Asiatic elephant

This study focuses on the effect of chemicals on acrosome reaction in elephant spermatozoa. Semen was collected at the Washington Park Zoo in Portland, Oregon, from an 11-yr-old Asian elephant by artificial vagina (7 ejaculates) and transported to Mahidol University in Bangkok in extender at 4 to 5 degrees C within 24 to 28 h. A total of 500 x 10(6) sperm/mL was used for the control and for each of the 4 treatment groups: 1) cAMP (0.1 mM); 2) caffeine (0.1 mM); 3) Penicillamine hypotaurine and epinephine, PHE (penicillamine 2 mM, hypotaurine 1 mM, epinephine 1 mM); and 4) heparin (10 microg/mL) at 39 degrees C for 2 h. Aliquots were removed and the sperm viability, abnormal morphology, and acrosome status were evaluated by triple stain technique. Transmission electron microscopy (TEM) was used to observe changes of the sperm head membrane in all treatment groups. Trypan blue reliably stained dead spermatozoa, while rose Bengal stained only the spermatozoa with intact acrosomes. The concentration of dead sperm cells was similar in the 4 groups. The percentages of live acrosome-reacted spermatozoa in the control and in groups treated with caffeine, PHE, cAMP and heparin were 19.5 +/- 4.3, 38.1 +/- 4.0, 34.8 +/- 3.7, 29.8 +/- 0.8 and 28.0 +/- 4.2, respectively. The acrosome reaction rate was higher in the treatment groups than in the control (P<0.05). Caffeine and PHE caused significantly higher acrosome reaction of the sperm head than cAMP or heparin (P<0.05). The electron micrographs showed that the acrosome reaction occurred by the presence of apical vesiculation. The results indicated that 1) the triple stain technique allowed for evaluation of both viability and acrosome reaction simultaneously in elephant spermatozoa,2) acrosome reaction occurred at a high rate in all 3 treatment groups. 3) the effects of caffeine and PHE were significantly higher (P<0.05) than of cAMP and heparin, and 4) the data obtained from the triple stain technique corresponded to those from TEM.     

Vital staining and acrosomal evaluation of bovine sperm

Dried smears prepared from vitally stained sperm were evaluated as a method of simultaneously determining sperm viability and acrosomal morphology. A combination Fast Green FCF-Eosin B stain was used. The stained smears were examined at × 1, 250 using differential interference contrast microscopy (DIC). For comparison, the percentage of sperm with intact acrosomes was also determined from wet smears using DIC. Acrosomal morphology was not altered by the staining procedure, as the percentage of intact acrosomes was similar whether quantitated from wet or stained smears. Absence of eosinophilic staining in the acrosome was used as an indication of sperm viability. The percentage of sperm with unstained acrosomes was highly correlated with the percentage of intact acrosomes quantitated from stained smears. Thus, vital staining provided an indication of sperm viability comparable to acrosomal integrity, a highly reliable technique. The major advantages of using dried stained smears were more thorough examination of individual sperm without sperm activity interference, simultaneous evidence of sperm viability and morphology, and the opportunity to delay evaluation. In addition, diluting spermatozoa in complex or simple media with or without egg yolk or follicular fluid did not interfere with subsequent staining or acrosomal evaluation.     

Kinematic study on the effect of pH on bull sperm function

Since the mammalian spermatozoa became capable of motion, during the epididymal transit, the spermatozoon swims in a liquid medium and it is completely dependent on the environmental conditions. Some reports have suggested an influence of pH on sperm kinetic characteristics, but no study has objectively described how motility changes in a different environmental pH. In this study, we evaluated the effect of different environmental pHs (5.5, 6, 6.5, 7, 7.5, 8, and 8.5) on kinetic parameters, sperm viability, mitochondrial activity, and sperm morphology of bull semen immediately and 1 h after dilution. The results showed higher values for sperm motility characteristics, viability, and mitochondrial activity at pH 7 and 7.5. Values of pH lower than 6.5 and higher than 8 resulted in suboptimal motility, with a decrease in most parameters. At pH 8 and 8.5, a discrepancy between viability and total and progressive motility was found, with a significant amount of spermatozoa that were live but immotile. This reduction seemed related to a decrease in mitochondrial activity, possibly due to the increase in pH. The flow cytometric evaluation of sperm viability assessed by calcein AM was very consistent with the amount of spermatozoa with membrane integrity, evaluated in fluorescence by propidium iodide/SYBR-14 stain. Thus, the calcein AM stain could be used as viability stain instead the classic propidium iodide/SYBR-14 stain because this could allow the addiction of other functional stains without a overlapping of the fluorescent signal in the flow cytometer.     

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C. jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the "Simple" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the "Simple" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the "Simple" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the "Simple" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species.     

SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis

Abstract

Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue® fixative and SpermBlue® stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue® fixative or by adding 1 ml SpermBlue® fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue® stain or adding four drops of SpermBlue® stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX® or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue® staining process. SpermBlue® stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer® ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 × or 1000 × magnification for most of the species studied.     

The effects on boar sperm quality of dietary supplementation with omega-3 polyunsaturated fatty acids differ among porcine breeds

The present study was undertaken to shed light on the relationship between boar sperm quality and dietary supplementation with omega-3 polyunsaturated fatty acids, which has been reported inconsistently in the literature. With this aim, such effects were evaluated and compared among three different porcine breeds: Duroc, Large-White, and Pietrain. Animals were randomly separated into two groups and fed either with a control diet or with a diet supplemented with omega-3. Sperm quality of these boar (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) was assessed every week for a 26-week period. Supplementing boar's diet with omega-3 did not affect ejaculate volume, sperm concentration, sperm motility, sperm viability, and acrosome and mitochondrial sheath integrity. In contrast, supplemented diet positively affected both sperm morphology in Large-White and Pietrain breeds and the osmotic resistance of Pietrain spermatozoa. No effects were seen for the same sperm parameters in Duroc breed. These breed-differences in boar fed with the supplemented diet could explain the contradictions in literature and might be related with differences in the composition of plasma membrane among breeds reported by other authors. Because no harmful effects were observed in the three evaluated breeds, but positive effects in Large-White and Pietrain boar, we can conclude that omega-3 fatty acids may be added to boar's diet at the levels used in this study to improve their sperm quality. More research is, however, needed to determine how these fatty acids differently affect the morphology and the osmotic resistance of the spermatozoa in these breeds.     

Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae)

Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67+/-11.67% (mean+/-S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49+/-3.24%, intact acrosome was detected in 63.74+/-2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups-highly active and progressively motile spermatozoa with SMI of 62.75+/-0.38 and low activity and poorly progressive with SMI of 46.16+/-1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable.     

Effect of storage media and storage time on survival of spermatozoa recovered from canine and feline epididymides

The aim of this study was evaluate the survival ability of canine and feline spermatozoa maintained within epididymides stored at 4 degrees C for 24, 48 or 72 h in sterile isotonic saline solution (SAL) or a Tris-egg yolk (TEY) storage medium. Fifteen domestic dogs and 15 cats were neutered and their testes were placed in TEY or SAL and stored at 4 degrees C for either 24, 48 or 72 h. Sperm samples were obtained by cutting the cauda epididymides into a Tris extender and were evaluated for motility, velocity, viability, plasma membrane integrity, and acrosome morphology. In dogs, there were no significant differences between storage media for motility, plasma membrane integrity, viability and velocity. However, dog sperm stored in TEY had better acrosome morphology compared to sperm stored in SAL (P < 0.05). Dog sperm recovered at 72 h had a reduction in all parameters studied compared to those recovered at 24 h (P < 0.05). In cats, sperm recovered from epididymides stored in TEY had higher motility, plasma membrane integrity and velocity at all times compared to those stored in SAL (P < 0.05). Cat sperm recovered at 72 h had reduced motility, acrosome morphology, viability and velocity compared to those recovered at 24 h (P < 0.05). The addition of TEY to canine epididymal sperm, thus, had a better protective effect than SAL only on acrosome morphology. In cats, in contrast, TEY had a better protective effect than SAL on all epididymal sperm parameters studied. In both species, sperm recovered at 72 h had a significant reduction in all parameters studied compared to those recovered at 24 h.     

Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single-wavelength fluorescence microscopy.

The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen-thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC-conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome-intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI-neg/PSA-neg--viable, physiologic acrosome reacted (AR); group II, PI-neg/PSA-pos--viable, non-AR; group III, PI-pos/PSA-neg--nonviable, non-AR; group IV, PI-pos/PSA-neg--nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen-thawed sperm may undergo premature break-down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.     

Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen

Cryopreservation imposes irreversible damage to sperm membranes, such as swelling and disruption of plasma and acrosome membranes, changes in membrane fluidity, altered influx of calcium, and changes in enzyme activity. Morphological integrity of the sperm plasma membrane has been widely studied using different techniques, including exposure of spermatozoa to hypoosmotic solutions (provides information concerning the biochemical activity of the sperm tail membrane), supravital test using eosin stain (yields information regarding sperm head membrane integrity), and Trypan-blue Giemsa stain (TBG; reveals both sperm plasma membrane and acrosome integrity). The objective of this study was to combine these tests in order to provide information about the integrity of the whole sperm surface, as well as acrosome status, and determine if the results of these tests were associated with sperm in vitro fertilizing ability. Stepwise regression analyses yielded a model in which fertility (maintain variable) was expressed as a combination of the results of different spermatological parameters (independent variables). The results of a test combining supravital eosin staining of samples previously submitted to hypoosmotic swelling test (STHOS) accounted for the greatest proportion of variation in fertilization rates (78%). Inclusion of the results of dual staining with TBG increased the proportion of variation in fertility rate that could be accounted for to 82%. Therefore, sperm plasma membrane integrity and function, and acrosome integrity can be considered important variables for normal sperm function and STHOST and TBG could be used for the prognosis of the potential fertility of bovine semen samples used for IVF or AI.     

Staining of sturgeon spermatozoa with trypsin inhibitor from soybean, Alexa Fluor® 488 conjugate for visualization of sturgeon acrosome

Trypsin-like activity, similar to acrosin, is present in sturgeon spermatozoa and can be a potential target for trypsin inhibitors. The objective of this work was to use a fluorescent soybean trypsin inhibitor (SBTI) conjugate with the Alexa Fluor^® 488 dye for visualization of the sturgeon acrosome. After incubation with SBTI-Alexa, a strong signal was observed both in the acrosome and midpiece or implantation fossa region. We have also found that SBTI-Alexa staining can be combined with PI viability test. Detailed examination of staining pattern revealed that SBTI-Alexa can stain either acrosome or whole sperm. Staining of whole sperm correlated with dead staining ( r ² = 0.94, P < 0.01). However, in fresh semen most cells (93–97%) were not stained with SBTI-Alexa, probably due to intact acrosomes. Further studies should test if SBTI-Alexa can be applied to monitor the acrosome status during the acrosome reaction and cryopreservation.     

Influence of anaesthetic drugs on the epididymal sperm quality in domestic cats

The present study investigated the effect of different anaesthetic agents commonly used in cats on the fresh and frozen-thawed epididymal sperm. Seventeen male domestic cats were castrated using pentobarbital, ketamine HCl or isoflurane. Sperm samples were recovered from epididymides and evaluated before and after freezing, determining the vigor, motility, morphology, acrosome status, sperm viability and functional membrane integrity. Fresh epididymal sperm was influenced by the drugs used, noting that motility features, i.e. vigor (p≤0.05) and progressive motility (p≤0.05), were higher for the inhalation anaesthetic while the others did not showed statistical differences. In frozen-thawed sperm samples, cats treated with barbiturics showed lower values for acrosome status (p≤0.05) and integrity and functionality of membrane (p≤0.05 and p≤0.01, respectively) than in the others groups. Results suggested that drugs used for castration in cats could affect the sperm quality and this should be considered when implementing sperm cryopreservation in the feline.     

Viability and Acrosome Staining of Bull, Boar and Rabbit Spermatozoa

A practical and reliable staining procedure was developed to distinguish the viability and acrosomal status of bull, boar and rabbit spermatozoa. The first stain with trypan blue or Congo red is rapid and avoids artifacts. This stain is precipitated by neutral red during the 2 min required for fixation. The precipitate gives a high contrast black color, resistant to the subsequent rinsings and persists during the time required for staining the acrosome with Giemsa. Ten classes of spermatozoa are distinguished (live or dead with intact acrosomes, loose acrosomes, damaged acrosomes, no acrosome, or with no acrosome and no postacrosomal ring). The intact acrosomes are purple, the loose acrosomes are dark lavender and the damaged acrosomes are pale lavender. The anterior part of the head of live spermatozoa with no acrosome is white or light pink and the same area of dead spermatozoa is white or pale gray. The postacrosomal ring is red. The postacrosomal area of the head of live spermatozoa is white or light pink and the same part of dead spermatozoa is black, dark violet or gray. The procedure did not give satisfactory results for stallion spermatozoa.     

Sperm head structure of a murid rodent from Southern Africa: The red veld rat Aethomys chrysophilus

The morphology of spermatozoa from the red veld rat, Aethomys chrysophilus, of Southern Africa is described; two very different types were found, which came from animals from two separate, as-yet-undescribed, species. In individuals from South Africa the sperm head had a somewhat disc-shaped nucleus and a large acrosome with a huge apical segment that, during epididymal transit, changed in form from initially projecting anteriorly to a highly complex structure that was flexed caudad and lay alongside part of the rest of the sperm head. In addition, the chromatin generally appeared to be not fully condensed. Spermatozoa from animals collected in Malawi were very different in morphology and had a head with a typical apical hook, a perforatorium, fully condensed chromatin, and a 4-μm-long ventral spur. Its sperm tail was also significantly longer. The time of divergence of these two groups of animals from a common ancestor is not known, but the present results show that a considerable morphological change in the sperm nucleus, acrosome, and subacrosomal space can evolve even between two, presumably closely related, species.     

Characterization of epididymal spermatozoa motility rate, morphology and longevity of springbok (Antidorcas marsupialis), impala (Aepyceros melampus) and blesbok (Damaliscus dorcus phillipsi): pre- and post-cryopreservation in South Africa

Cryopreservation of antelope epididymal spermatozoa could play a vital role in future breeding by developing a successful protocol for cryo-conserving them. The aim of this study was to characterize morphology, motility rates and longevity of epididymal spermatozoa from springbok, impala and blesbok. Cauda epididymal spermatozoa were collected post-mortem from both testicles of free-ranging springbok (n=18), impala (n=21) and blesbok (n=21), and divided into two groups (pre- and post-cryopreservation). Spermatozoa were cryopreserved in Biladyl supplemented with 20% egg yolk and 7% glycerol under field conditions. Pre-freeze and post-thaw sperm quality was evaluated. The longevity of thawed spermatozoa was evaluated under culture conditions that support domestic cattle in vitro fertilization. There was a significant difference between pre-freeze and post-thaw sperm motility index (SMI) (p<0.05), plasma membrane integrity (p<0.05) and acrosome integrity (p<0.05) for all species. Post-thaw SMI and plasma membrane integrity were comparable between species (p>0.05). The effects of cryopreservation on sperm cell morphology differed between species and between specific abnormal morphology. Blesbok had the least abnormalities in post-thaw spermatozoa. Cryopreservation substantially reduced the survivability and motility rates of antelope species. Blesbok spermatozoa tolerated cryopreservation and thawing process better than impala and springbok. The antelope cauda epididymal sperm maintained viability and acrosome integrity for at least 4h following incubation under conditions that support domestic cattle in vitro fertilization (IVF) with a decline in longevity over time across species however; species responded differently over time in terms of plasma membrane integrity and acrosome integrity. The antelope species may have different in vitro culture requirements, indicating differences in sperm physiology between the species. This research could contribute species-specific protocol development for IVF thus promoting ex-situ conservation strategies of African antelope species in South Africa.     

Factors affecting triple staining of human sperm

We have previously described a triple stain for evaluating normal acrosome reactions of human sperm. This procedure uses trypan blue to distinguish live and dead sperm, Bismarck brown to stain the sperm's postacrosomal region, and rose Bengal to stain the sperm's acrosome. We have recently found that batches of rose Bengal vary significantly in their ability to produce good staining of the acrosome in this procedure. This appears to be due to variations in the intrinsic pH of rose Bengal solutions and the presence of nondye contaminants in the stain. In this study, we have evaluated acrosomal staining using 6 batches of rose Bengal and report a method for achieving uniform staining quality with each batch. Solutions of rose Bengal (0.8%) are made up in 0.1 M Tris HCl (pH 2.3) buffer and adjusted to pH 5.3 if necessary. For most batches of rose Bengal this promotes precipitation of some of the dye and an unidentified contaminating crystal. The precipitate is removed by centrifugation, and the supernatants have been found to give good to excellent staining of the acrosomes for all batches tested. Solutions of both rose Bengal and Bismarck brown are stable for at least 5 days but their pH values should be monitored daily and adjusted to 5.3 and 1.8 respectively if drifting occurs. We have also observed some variation in the intensity of rose Bengal staining of the acrosome from donor to donor and recommend that staining times in rose Bengal be adjusted for each donor.     

Role of rabbit prostate granules on sperm viability and acrosome reaction evaluated with different methods

In the present study, the role of rabbit seminal granules was observed. Their influence on motility, capacitation and acrosome reaction, as well as the presence of apoptosis and the morphology of rabbit sperm, were compared in different conditions. Ejaculated sperm from five mature New Zealand White rabbit bucks during three series of collections were studied, comparing raw semen, Percoll-selected sperm and Percoll-selected sperm plus prostate granules. We observed sperm motility kinetic traits by computer-assisted sperm analyzer (CASA) analysis in each sample. Acrosome status was evaluated by FITC-labeled Pisum sativum Agglutinin staining and chlortetracycline fluorescence assay, phosphatidylserine translocation was determined by AnnexinV/Propidium iodide assay and sperm morphology was studied using transmission electron microscopy (TEM). All traits were observed after 30 min incubation at 37 °C in 5% CO₂. Data showed that sperm motility and viability markedly improved in the presence of prostate granules, whereas capacitation, acrosome reaction and phosphatidylserine translocation were lowered. TEM confirmed these results. In conclusion, the role of granules was confirmed in synchronizing sperm capacitation and acrosome reaction with egg availability; indeed, rabbit ovulation occurs only 6 to 10 h after mating.     

Factors Affecting Triple Staining of Human Sperm

We have previously described a triple stain for evaluating normal acrosome reactions of human sperm. This procedure uses trypan blue to distinguish live and dead sperm, Bismarck brown to stain the sperm's postacrosomal region, and rose Bengal to stain the sperm's acrosome. We have recently found that batches of rose Bengal vary significantly in their ability to produce good staining of the acrosome in this procedure. This appears to be due to variations in the intrinsic pH of rose Bengal solutions and the presence of nondye contaminants in the stain. In this study, we have evaluated acrosomal staining using 6 batches of rose Bengal and report a method for achieving uniform staining quality with each batch. Solutions of rose Bengal (0.8%) are made up in 0.1 M Tris HC1 (pH 2.3) buffer and adjusted to pH 5.3 if necessary. For most batches of rose Bengal this promotes precipitation of some of the dye and an unidentified contaminating crystal. The precipitate is removed by centrifugation, and the supernatants have been found to give good to excellent staining of the acrosomes for all batches tested. Solutions of both rose Bengal and Bismarck brown are stable for at least 5 days but their pH values should be monitored daily and adjusted to 5.3 and 1.8 respectively if drifting occurs. We have also observed some variation in the intensity of rose Bengal staining of the acrosome from donor to donor and recommend that staining times in rose Bengal be adjusted for each donor.     

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5 degrees C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.