Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

Taylor, Mark A.; Guzmán-Novoa, Ernesto; Morfin, Nuria; Buhr, M.M.

doi:10.1016/j.theriogenology.2009.02.012

Cited by 46 publications

(58 citation statements)

References 37 publications

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“…However, the morphological changes observed in Figure B, as well as the greatly increased intensity of PI staining in LN‐killed cells (compared to dead cells in the sample, or heat killed cells) has not been reported in other species, and is not apparent in the images of freeze–thaw killed turkey and bull sperm reported previously . Researchers involved in developing cryopreservation techniques for honey bee sperm have struggled to maintain viability following the freeze–thaw process . Peng et al also found that honey bee sperm incur extensive cellular injuries from freeze–thawing.…”

Section: Discussionmentioning

confidence: 85%

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: 85%

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

et al. 2014

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“…A longer drying time results in a higher number of dead spermatozoa; some spermatozoa lose their structural integrity during drying and are thus stained by PI. Taylor et al (2009) used SYBR-14/PI to assess the viability of honeybee semen by scoring 100 cells twice per sample. Results were accepted only if the duplicate counts differed by less than 5%; otherwise, samples were recounted using the same method.…”

Section: Discussionmentioning

confidence: 99%

“…Following semen collection, the samples were analyzed for sperm motility (Taylor et al 2009), concentration (Taylor et al 2009), viability (Taylor et al 2009), and sperm plasma integrity using both the water test (Lomeo and Giambersio 1991) and the HOS test (Jeyendran et al 1984). …”

Section: Semen Collection and Dilutionmentioning

confidence: 99%

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The use of the hypo-osmotic swelling test, water test, and supravital staining in the evaluation of drone sperm

et al. 2011

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International audienceThe aim of the present study was to investigate the suitability of the water test and hypo-osmotic swelling (HOS) test for analyzing honeybee semen. In particular, the relationships between these tests of the integrity of the sperm plasma membrane and tests of sperm motility and sperm viability were measured using SYBR-14/PI. To this end, semen was obtained from mature drones (16 days or older) collected from four colonies. The means of the per-drone sperm concentration, sperm motility, and live spermatozoa were 1.47 × 106, 76.0%, and 87.2%, respectively. The percentage of spermatozoa with swollen tails was 92.2% as measured with the water test. With the HOS tests at 50, 100, and 150 mOsm/kg, the percentages of spermatozoa with swollen tails were 94.2%, 90.5%, and 85.6%, respectively, after a 30-min incubation and 92.2%, 90.5%, and 88.0%, respectively, after a 60-min incubation. It was observed that subjecting honeybee spermatozoa to a hypo-osmotic solution resulted in clearly identifiable swollen-tail spermatozoa. The percentages of swollen-tail spermatozoa obtained using the water and HOS tests were higher than the percentages of viable and motile spermatozoa. Similar results were obtained using the water test and the HOS test at 50 mOsm/kg. It was concluded that HOS and water tests are suitable and simple measures of the functional integrity of the plasma membranes of bee spermatozoa, and they may be useful additions to standard methods of semen evaluation in mammals

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“…The immotility is reversible as long as the spermatozoa are alive (Verma 1978). In nature, honey bee semen is diluted with spermathecal fluid but the spermatozoa survive also in many human made diluents (Moritz 1984;Taylor et al 2009). …”

mentioning

confidence: 99%

A scientific note on amoeboid movement of honey bee semen

Tofilski

2014

Apidologie

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Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

Cited by 46 publications

References 37 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

The use of the hypo-osmotic swelling test, water test, and supravital staining in the evaluation of drone sperm

A scientific note on amoeboid movement of honey bee semen

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