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

Abstract

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

Why the viability of spermatozoa diminishes in the honeybee (Apis mellifera) within short time during natural mating and preparation for instrumental insemination

“…Up to now, several authors have reported data on spermatozoa viability in honeybee drones representing great variation, measured by fluorescent staining technique combined with microscopy (Collins and Donoghue 1999;Collins and Pettis 2001;Collins 2003Collins , 2004Lodesani et al 2004;Burley et al 2008;Gençer and Kahya 2011a;Bieńkowska et al 2011;Czekoňska et al 2013a, b;Stürup et al 2013) or with flow cytometry (Rzymski et al 2012;Tofilski et al 2012;Paynter et al 2014). Our finding that the spermatozoa viability in seminal vesicles is higher than that in ejaculate collected into the syringe tip is in agreement with the finding of Collins (2004).…”

Assessing Sperm Quality in Stingless Bees

“…Although less accurate than flow cytometry methods, fluorescence-based methods have been widely used and they are well accepted as standard methods (Thomas & Simmons., 2007;den Boer et al, 2008den Boer et al, , 2010Cobey et al, 2013). Moreover, sperm viability obtained with fluorescence microscopy and flow cytometry has been found indistinguishable in some cases (Paynter et al, 2014).…”

“…It is also commonly used in other organisms to study various aspects of their reproductive biology (Garner et al, 1994;Ball et al, 2001;Paulenz et al, 2002). Recent advances in the use of flow cytometry to assess sperm traits in social insects allowed significant improvements, decreasing processing times and increasing accuracy (Cornault & Aron, 2008;Paynter et al, 2014). However, flow cytometry equipment is not common across the tropics (where stingless bees occur), and the costs involved in their use and maintenance are substantially higher than those involving fluorescence-based methods.…”

Sperm parameters of honeybee drones exposed to imidacloprid

“…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. The use of flow cytometry, rather than fluorescence microscopy, facilitates high-throughput analysis and enables the quantification of thousands of sperm cells per sample and between the implementation of blind counting and randomization (Holman 2009;Rzymski et al 2012;Paynter et al 2014).…”