Flow cytometry evidence about sperm competition in honey bee (Apis mellifera)

Tofilski, Adam; Chuda-Mickiewicz, B.; Czekońska, Krystyna; Chorbiński, Paweł

doi:10.1007/s13592-011-0089-6

Cited by 16 publications

(20 citation statements)

References 29 publications

(31 reference statements)

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“…Similar to another honeybee sperm FCM study , this investigation also identified additional populations of sperm in the data that appeared when freeze–thawing was used to kill cells. Initial experiments using an analog instrument (FACSCalibur, BD Biosciences) also used by Tofilski et al produced multiple populations, but without clear separation between live and dead cells. This study has found that use of digital flow cytometer allowed distinction of both peak area and peak height, important in the analysis of bee sperm because of their unusual shape.…”

Section: Discussionmentioning

confidence: 63%

“…We show that FCM, compared with fluorescence microscopy, is a faster and more accurate method to assess sperm viability in sperm samples of honeybees. Furthermore, we are able to resolve some of the technical issues reported in earlier published literature about the possible use of FCM for honeybee sperm . We show that killing sperm by freeze–thawing with liquid nitrogen (LN), which has previously been used to generate reference semen samples with known sperm viabilities, creates a distinct population with high PI fluorescence, likely due to membrane disruption and/or changes to chromatin structure which affect PI binding.…”

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confidence: 86%

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Flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee Apis mellifera

et al. 2014

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An important measure of male quality is sperm viability; i.e., the percentage of live sperm within an ejaculate, as this provides an accurate measure of the number of sperm potentially available for egg fertilization. Sperm viability is often determined by fluorescence microscopy using dyes that differentially stain viable and nonviable sperm, but the technique has a number of limitations. Here, a flow cytometry (FCM) method was developed, which allows the rapid determination of honeybee sperm viability, facilitating high throughput analyses. Using samples with known sperm viabilities, it was found that data obtained from FCM were more accurate and less variable compared with data obtained for the same samples using fluorescence microscopy. It was also found that a previously reported additional population of honeybee sperm found in datasets using FCM is caused by freeze-thawing samples. In conclusion, the method described here allows to quantify sperm viability of honeybees quickly and with high accuracy. This will be of great value for future scientific research and could also be of value to guide future bee breeding programs, given the agricultural importance of honeybees as pollinators. V C 2014 International Society for Advancement of Cytometry Key terms honeybee; sperm; flow cytometry; microscopy; viability; SYBR 14; propidium iodide; liquid nitrogen EJACULATE quality is a fundamentally important reproductive male trait as the number of live sperm available for egg fertilization after being transferred to the female's reproductive tract is a key determinant of male competitiveness, reproductive success, and fitness (1-4). A number of methods have been developed in the past to quantify semen quality, including parameters such as sperm viability, motility, morphology, and total number (5-7). Such inspections are often done microscopically or using machinery in combination with software, capable of simultaneously tracking large numbers of sperm cells within a sample (8). Sperm viability, in particular, has become a key measure for semen quality in recent years, and often refers to the proportion of live sperm in an ejaculate. It is generally quantified by measuring the plasma membrane integrity, as the plasma membrane becomes more permeable in dead sperm and coincides with the loss of motility (9,10) and therefore fertilization capacity. Consequently, assessing sperm viability within ejaculates has become of major interest for the study of reproductive biology as well as for medical research and practices, for example to understand and treat male infertility (11).To determine the proportion of live and dead sperm in a semen sample, a number of dyes have been developed, which allow relatively easy quantification of sperm viability. This approach has been used for a number of different species and for the study of a wide range of different questions (6,(12)(13)(14). SYBR 14 and propidium

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Section: Discussionmentioning

confidence: 63%

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confidence: 86%

Flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee Apis mellifera

et al. 2014

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“…Sperm motility reflects both energetic efficacy and the quality of the motility apparatus; it is also important for sperm transfer to a queen's spermatheca (Ruttner and Koeniger 1971;Baer 2005). Viability staining reflects functional quality of membrane permeability and was found to be especially useful for the evaluation of sperm competition in insects, including honeybees (Hunter and Birkhead 2002;Tofilski et al 2012). Measurements of mitochondrial membrane potential (MMP) is important for evaluating the function of mitochondria, which are particularly vital for the production of energy during sperm movement.…”

Section: Introductionmentioning

confidence: 99%

Sperm parameters of honeybee drones exposed to imidacloprid

et al. 2016

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-The objective of this study was to evaluate the effects of chronical exposure of honeybee drones to environmental (5 ppb) and non-environmental concentration (200 ppb) of imidacloprid (IMD) on sperm concentration, motility, viability, and mitochondrial membrane potential measured in semen obtained from 180 drones originating from 18 colonies. The results demonstrate that IMD exposure did not affect sperm concentration; however, there were significant differences in concentration within colonies. IMD exposure was associated with reductions in sperm motility, which also varied within colonies. Statistically significant interactions between IMD exposure and colony were found for active mitochondria and sperm viability. Our results strongly suggest that neonicotinoids can negatively affect honeybee drone sperm quality. It is important to emphasize that IMD actions can be strongly modulated according to the colony.Apis mellifera / imidacloprid / spermatozoa / motility / viability

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“…Coagulation of seminal fluids is related to sperm competition (Dixson & Anderson, 2002) and proteins contained in particular semen coagulate when in contact with the spermatozoa of another drone in order to immobilize them (Tofilski et al 2012). Although the drones in our study originated from one paternal colony, the chromosome complement transferred by the mother may have been different.…”

Section: Discussionmentioning

confidence: 91%

Proteolytic system of the sperm of Apis mellifera drones

et al. 2013

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During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.

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Flow cytometry evidence about sperm competition in honey bee (Apis mellifera)

Cited by 16 publications

References 29 publications

Flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee Apis mellifera

Flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee Apis mellifera

Sperm parameters of honeybee drones exposed to imidacloprid

Proteolytic system of the sperm of Apis mellifera drones

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