Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40° C is recommended for stallion semen.     

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 tail membrane integrity by light microscopy

During routine evaluation of trypan blue-Giemsa stained semen smears, sperm cells can be found with unstained heads and with stained tails. It was hypothesized that these cells were immotile and should not be considered as live. Sperm motility was determined in isoosmotic, and presumably isotonic trypan blue-stained wet preparations. Bull, ram and boar semen smears were stained with hypoosmotic trypan blue-Giemsa to compare the relationship between the percentage of stained sperm tails and the percentage of sperm tails remaining straight under hypoosmotic conditions. Actively moving spermatozoa with unstained heads, but with stained tails were never observed in wet preparations. The correlation coefficient found between the percentage of sperm with stained tails and the percentage with straight tails was 0.81, 0.94 and 0.85 for bull, ram and boar spermatozoa, respectively. Results of this study show that sperm cells with an intact head membrane, but a stained and presumably membrane-damaged tail are not motile. Therefore these cells should be included in the dead category rather than alive in the usual live-dead studies with vital stains.     

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.     

Simultaneous evaluation of viability and acrosome integrity of mouse spermatozoa using light microscopy

Determination of the percentage of live cells with intact acrosomes and no morphologic aberrations could be a practical index of semen quality. We applied viability and acrosome staining techniques, originally described for bull, boar and rabbit sperm, to mouse spermatozoa. The viability stain was either trypan blue or Congo red. The stain was precipitated by neutral red in the fixative. The acrosome was stained by Giemsa. Sperm morphology, including cytoplasmic droplets, could be evaluated as well. The staining method described here is a useful routine tool for simultaneous evaluation of the plasma membrane integrity of different sperm subdomains, the status of the acrosome, and cellular morphology.     

Morphometry of stallion spermatozoa by computer-assisted image analysis

Metric measurements of stallion spermatozoal heads were determined for live, unfixed spermatozoa and for Feulgen-stained spermatozoa by videomicroscopy and computerized image analysis. Two ejaculates were collected from each of five stallions of normal fertility. Air-dried semen smears were Feulgen-stained, and live, unfixed spermatozoa were examined as wet-mount preparations. For Feulgen-stained spermatozoa, videoimages (x3850) were captured, and sperm heads were detected via image segmentation and particle analysis. For live, unfixed spermatozoa, phase contrast videoimages (x3850) were measured to determine width and length of the sperm head. For Feulgen-stained spermatozoa, there were significant effects (P < 0.001) of stallion and ejaculate on measured parameters of area, circumference, and the length and width of the sperm head. For live, unfixed spermatozoa, there were significant effects of stallion on length and width and of ejaculate on length of the sperm heads. There was a very poor correlation between length and width of sperm heads between Feulgen-stained and live, unfixed spermatozoa. Two indices of sperm shape (oval factor and aspect ratio) were also determined. Both aspect ratio and oval factor were significantly affected by stallion (P < 0.001); however, oval factor was not affected by ejaculate and therefore may represent a less variable determination of sperm head shape across stallions. Overall, length and width of stallion sperm heads were larger (P < 0.01) for live, unfixed spermatozoa than for Feulgen-stained spermatozoa (length: 6.3 +/- 0.4 vs 5.08 +/- 0.44; width: 3.08 +/- 0.34 vs 2.71 +/- 0.28 mum, respectively). Computerized image analysis may be useful as a means to objectively measure sperm head dimensions in the stallion and could be useful in future studies to determine associations with stallion fertility.     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.     

Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 degrees C and 37 degrees C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 degrees C for 30 sec or at 75 degrees C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 degrees C and 75 degrees C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 degrees C and 75 degrees C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 degrees C compared with that at 75 degrees C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 degrees C instead of 75 degrees C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.     

伊红、台盼蓝检测河蟹精子存活率的比较

对台盼蓝和伊红染色法检测河蟹(Eriocheir sinensis)精子存活率的方法进行了评价研究。结果表明,两种染色法死、活精子分别呈现出明显不同的染色特征:活精子无色透明,顶体中央凸起呈圆锥状,光镜下辐射臂及细胞边界清晰;死亡精子顶体着色,且中央有一染色较深的圆斑,核杯染色不明显,细胞体积变大,边界模糊。通过不同染色时间和不同染料浓度的比较发现,两种染色法最适染液浓度分别是0·25%的伊红和0·5%的台盼蓝,染色时间均以15min为佳。在此基础上,将新鲜精子和60℃水浴处理致死精子以不同的体积比混合,配成含致死精子比例为10%～90%的9个梯度样品,用伊红和台盼蓝分别测定各样品精子死亡率,并进行相关性分析。结果发现,各样品实测精子死亡率均略高于样品的理论死亡率,同时两种染色法实测值与样品理论值呈显著正相关(P<0·05),两种染色法之间亦呈显著正相关(P<0·05)。上述结果表明,伊红和台盼蓝可用于河蟹精子的活体染色,且两种染色法在对河蟹精子染色中具有一定的稳定性和可比性。     

The influence of age and breed on stallion semen

This study reports on the variation in semen quality and in spermatozoal and behavioral characteristics of 168 stallions representing 9 breeds and ranging in age from 2 to 26 yr. Semen samples were collected into an artificial vagina and the number of mounts and urethral pulsations per semen sample were recorded. Semen characteristics were examined for total volume, gel-free volume, gel volume, color score, mass activity, nonmotile spermatozoa, dead spermatozoa, semen density, spermatozoa concentration, total number of spermatozoa and semen pH. Morphological characteristics of the spermatozoa included abnormal heads, abnormal mid-pieces, abaxial mid-pieces, protoplasmic droplets and abnormal tails. Sources of variation were evaluated and the overall means calculated by least-squares analyses of variance for nonorthogonal data. The significance of breed effects and between stallion variability were estimated using mixed-model procedures. All semen characteristics with the exception of color and urethral pulsations had significant variation due to age. Semen quality (gel-free volume, sperm concentration, total sperm numbers and sperm abnormalities) was poorest in stallions under 3 yr of age and over 11 yr. Significant breed variation was apparent in most characteristics except for pH, semen color, abnormal midpieces and urethral pulsations. It is recommended that both the age and breed of stallion be taken into consideration when evaluating stallion semen.     

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.     

The Differentiation of Live From Dead Sperms in Fowl Semen

A combined stain solution is made by dissolving 0.1 gm bromphenol blue and 0.2 gm nigrosin in 100 ml of a M/15 buffer solution of KH₂PO₄ and Na₂HPO₄ adjusted to pH 7.5. This staining solution was used to prepare stained fowl semen smears. Such smears give stable differentiation of live from dead sperms. The dead sperms are stained with a dark violet color while the live ones are not stained.     

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.     

Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry

An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (membrane damaged) cells to viable spermatozoa. There was a high correlation (r(2) = 0.996) between increased PI positivestained (dead) cells and the number of membrane-damaged spermatozoa added (% dead: 29 +/- 0.4, 44 +/- 1.4, 58 +/- 0.9, 75 +/- 0.7 and 91+/- 0.25 vs 0, 25, 50, 75 and 100% damaged cells, respectively). Optimal mitochondrial activity (OMA), as assessed by R123 uptake, was also reduced proportionally (r(2) = 0.976) by the percentage of membrane-damaged cells added (% OMA: 48 +/- 0.6, 37 +/- 1.7, 29 +/- 0.5, 16 +/- 1, 3.8 +/- 1.3 vs 0, 25, 50, 75 and 100% damaged cells, respectively). The mitochondrial inhibitors rotenone and monensin significantly depressed optimal mitochondrial activity (P < 0.001), and there was a significant positive correlation (r(2) = 0.959) between the dose of inhibitors added and the population of sperm cells exhibiting minimal R123 staining (4 -/+ 0.9, 12 -/+ 1.6, 14 -/+ 0.1 and 28 -/+ 2% for treatments with 0, 0.5, 1 and 2 x 10(-5) M rotenone and 0, 0.5, 1, and 2 x 10(-4) M monensin, respectively). Finally, it was shown that treatments containing identical proportions of membrane-damaged cells yielded similar results in terms of viability and mitochondrial activity, irrespective of whether the staining procedure was single or double (P > 0.05). The results of the double-staining method revealed that the percentage of spermatozoa with optimally functioning mitochondria was significantly correlated with the percentage of viable (PI negative) sperm cells (r(2) = 0.998). Flow cytometric analyses using this staining procedure provides reliable and rapid (10,000 cells/min) qualitative assessment of stallion semen.     

Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development

The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.     

Sources of variation in the morphological characteristics of sperm subpopulations assessed objectively by a novel automated sperm morphology analysis system

There is evidence that the mammalian ejaculate contains distinct subpopulations of spermatozoa and that the variability among these subpopulations may have adaptive and functional significance. This study investigated the precision, reproducibility and operating characteristics of a novel automated sperm morphology analysis system, the Hobson Morphology package, establishing protocols to investigate boar sperm characteristics. Five ejaculates were collected from each of three boars from different genetic lines: Landrace-Meishan introgression, Sireline Large White and Damline Large White. Five semen smears per ejaculate were stained with haematoxylin and eosin. Two hundred spermatozoa per slide were analysed. No significant differences among slides within an ejaculate were detected for sperm tail length (P = 0.770), head width (P = 0.736) and head length (P = 0.615), indicating that both staining and morphology analysis were precise and reproducible. Among the boars, variability in tail length was detected (P = 0.001), but head width (P = 0.114) and length (P = 0.069) did not differ significantly. Multivariate pattern analysis (PATN computer package) highlighted three sub-populations of spermatozoa objectively on the basis of tail length (10.0-22.0 microns, 22.1-73.0 microns and 73.1-130.0 microns). The Landrace-Meishan introgression boar possessed more spermatozoa (P < 0.0001) with tails 73.1-130 microns long. Subsequent analysis of morphology parameters in a pure-bred Meishan boar showed similar measurements for tail length (mean +/- SD; 66.36 +/- 24.70 microns) to the Landrace-Meishan introgression boar (mean +/- SD; 67.09 +/- 21.80 microns). Sperm subpopulations originate during spermatogenesis, when heterogeneous genotypic effects determine the structural features of spermatozoa. The findings of this study confirm that tail length differs between boars and that subpopulations of spermatozoa can be detected within a single ejaculate.     

Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry

A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.     

Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology

Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa.     

Effect of butylated hydroxytoluene on bull spermatozoa frozen in egg yolk-citrate extender

Effect of butylated hydroxytoluene (BHT) on the quality of frozen-thawed Holstein bull sperm in egg yolk-citrate extender was evaluated. High quality semen samples were diluted in egg yolk-citrate extenders containing 0, 0.5, 1, 2 and 4 mM BHT and subsequently frozen in liquid nitrogen. Pre-freeze and post-thaw progressive motility, and live/dead ratio and acrosomal integrity of 200 sperm per slide, stained with Eosin-Nigrosin and Giemsa, were evaluated at 0, 2 and 4 h after thawing. There was a significant decrease in forward motility, livability and acrosomal integrity up to 4 h after thawing the frozen sperm. Upon thawing, sperm progressive motility at 1 mM BHT was significantly (P<0.001) higher (11%) than other groups, but percentages of live sperm and live sperm with intact acrosomes were higher at 0.5 mM BHT. BHT at 4 mM BHT caused a significant decrease in motility, livability and acrosomal integrity during preparatory stages of freezing sperm. It is concluded that 0.5-1.0 mM BHT can be beneficial for freezing Holstein bull spermatozoa in egg yolk-citrate diluent, when inseminated immediately after thawing.