Viability assessment of honey bee, Apis mellifera, sperm using dual fluorescent staining

Since the development of instrumental insemination of honey bee (Apis mellifera) queens in the 1930s, there has been interest in the evaluation and in vitro storage of semen. Several fluorescent stains, when used in combination, have been effectively used to assess sperm viability in mammalian and avian species. Our objectives were to test two combinations of living:dead fluorescent stains, SYBR-14 with propidium iodide (PI), or Calcein-AM with PI, and validate the use of these probes with honey bee sperm. SYBR-14 is a nuclear stain producing green fluorescence of the DNA in living sperm, Calcein-AM is a membrane-permeant esterase substrate staining entire sperm green, and PI is a traditional dead cell stain giving a contrasting red color. Both living stains fluoresced bee sperm, but the SYBR-14:PI produced a clearer distinction between the living and dead sperm. A graduated series of known living:dead sperm proportions was used to validate the accuracy of the stains for determining sperm viability in honey bees.     

A supravital staining technique for honey bee spermatozoa

ABSTRACT A new supravital staining technique is described for honey bee, Apis mellifera L., spermatozoa using the fluorochromes, propidium iodide and Hoechst 33342 (H342), a bis-benzimidazole derivative. Propidium iodide binds to the DNA of sperm which lack membrane integrity and H342 binds to the DNA of all sperm. This assay is a simple and rapid method for determining the percentage nonviabiiity of a male honey bee's sperm. The recommended staining procedure is to incubate sperm in a solution of 5 μ.g/ml H342 and 10 μ.g/ml propidium iodide in modified Kiev solution for 15–20 min. After incubation, wet mounts of the sperm-stain suspension are examined using fluorescence microscopy. Percentage nonviabiiity is determined by the ratio of propidium iodide stained sperm to H342 stained sperm.     

In vivo validation of in vitro quality tests for cryopreserved honey bee semen

Development of cryopreservation protocols for honey bee semen is hampered by the lack of validated laboratory tests that allow the prediction of in vivo performance of frozen-thawed semen. Here we analyzed correlations between seven in vitro tests and indicators of semen performance after insemination. These tests included measures of motility, cell conformation, and membrane permeability before and after exposure to physiochemical stress. We show that the proposed protocol for motility measurement yields results that correlate well with the number of sperm reaching the storage organ of queens (correlation coefficient ρ=0.67) and the proportion of viable eggs in inseminated queens (ρ=0.48). The conventional live/dead assay of membrane permeability by dual fluorescent staining and a new test based on the leakage of the glycolytic enzyme glucose-phosphate-isomerase (GPI) from damaged cells were also correlated to the number of sperm reaching the spermatheca (ρ=0.54 and -0.61, respectively). We conclude that motility, live/dead-staining and the assay for GPI-leakage are valuable tools for the improvement of cryopreservation of honey bee semen.     

Effect of rapid freezing and thawing on cellular integrity of honey bee sperm

Abstract. Direct observations on the effect of rapid freezing and thawing on honey bee ( Apis mellifera L.) sperm were made by light, scanning and transmission electron microscopy. Rapid freezing of honey bee ejaculated sperm, suspended in freezing diluent, in liquid nitrogen followed by rapid thawing can cause cellular injuries which lead to the death of the sperm. The frozen-thawed sperm, supravitally stained, showed a significant decrease in cell viability compared with that of the control fresh sperm ( P <0.001). Significant uptake of the stain in the dead sperm resulted from damage in the cell membrane. The scanning electron micrographs of frozen-thawed sperm further demonstrated that the injury of cell membrane can lead to the splitting of mitochondrial derivatives from the flagellar axoneme. More cellular injuries including the release of acrosomal content and membrane damage at the acrosome, nucleus and the tail regions were further revealed by transmission electron microscopy. The impact of cellular injuries on the quality of honey bee sperm cryopreserved for artificial insemination of honey bee queens is discussed.     

Survival of honey bee (Hymenoptera: Apidae) spermatozoa stored at above-freezing temperatures

The development of practical techniques for the storage of honey bee, Apis mellifera L., semen would significantly improve our ability to breed for desirable genotypes and maintain genetic diversity in populations. Artificial insemination of queens has been possible for some time, but the semen used is usually freshly collected, or held for < 1 wk at room temperature. I examined the limitations of spermatozoal survival at nonfrozen temperatures. Pooled, diluted semen was stored in sealed capillary tubes at room temperature (25 degrees C) or in a refrigerator set to 12 degrees C, for periods up to 1 yr. Survival of spermatozoa was assayed by a dual fluorescent staining technique using SYBR-14 and propidium iodide stains, which readily distinguishes live and dead cells. No significant loss of viable spermatozoa occurred within the first 6 wk. Between weeks 6 and 9, the percent live spermatozoa fell from 80 to 58%, and remained at that level until after 39 wk. By week 52, samples at room temperature, but not at 12 degrees C, fell to 18.9% live spermatozoa. Nonfrozen storage of honey bee semen has potential for short-term preservation of germplasm, however several factors need to be studied further to optimize survival rates.     

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.     

Flow cytometry as an estimation tool for honey bee sperm viability

Flow cytometry is a method to conduct a multiparameter analysis of cells suspended in liquid and passing through a laser beam. Analyses of human and other mammal sperm using this method have already been performed but its application for insect semen is still the subject of investigation. Semen isolated from honey bee Apis mellifera seminal vesicles was dyed using SYBR-14 and propidium iodide (PI). The fluorescence of the SYBR-14 stained cells was analyzed in a green fluorescence channel (FL-1), while the PI fluorescence was analyzed in a red fluorescence channel (FL-3). Living and dead cell populations were separated using a density dot plot and the percentage of each in the sample was calculated. Flow cytometry seems to be an effective tool for assessing the viability of honey bee semen, solving the problems of distinguishing and counting the double-stained cells.     

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.     

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.     

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.     

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.     

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 degrees C is recommended for stallion semen.     

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.     

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.     

Zinc-Specific N-(6-Methoxy-8-Quinolyl)-Para-Toluenesulfonamide as a Selective Nontoxic Fluorescence Stain for Pancreatic Islets

Separation of the endocrine from the exocrine pancreatic tissue by fluorescence activated sorting has been limited by the lack of an ideal fluorescent label for islet tissue. Our studies indicates the zinc-specific stain N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), has characteristics ideal for use as a fluorescent label for islet tissue. Dispersed rat pancreas cells stained with TSQ produced bright blue fluorescence when excited by UV light [peak emission wavelength at 480 nm. maximal excitation at 365 nm). The fluorescence was specific for islet tissue as confirmed by counterstaining with the islet-specific stain dithizone and there was minimal background staining of exocrine tissue. Stained tissue remained brightly fluorescent for 2 hr. with some fading by 4 hr. Injection of TSQ into rats at a concentration sufficient to produce staining of islets produced no toxicity discernible at 4 months. The viability of isolated rat islets stained with TSQ was maintained as shown by supravital staining, in vitro secretion of insulin, and reversal of diabetes after transplantation of stained islets into diabetic syngeneic recipients.     

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.     

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32^°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.     

Evaluation of acrosomal status of bovine spermatozoa using concanavalin a lectin

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