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Title: VIABILITY ASSESSMENT OF HONEY BEE, APIS MELLIFERA, SPERMATOZOA USING DUAL FLUORESCENT STAINING.

Author
item Collins, Anita
item Donoghue, Ann - Annie

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Previous work with the storage of honey bee semen depended on instrumental insemination of queens and subsequent production of new bees, to determine the quality of the semen after treatment. This is a time-consuming process. Cellular stains have been developed for many other species that allow the relative proportions of live and dead sperm present in a sample to be directly counted microscopically, based on color. This study showed that two such pairs of stains correctly discriminated honey bee semen, coloring live cells green and dead cells red. Research to improve the storage of honey bee semen, including cryopreservation, is especially needed at this time because two parasitic mites have caused severe losses of colonies in the U.S. We need to breed parasitic-mite resistant stains of bees for control of mite-induced colony deaths, and to maintain necessary levels of genetic variation in our honey bee populations. Thousands of healthy colonies are needed to perform pollination services for a variety of our agricultural crops, home gardens and wild plants.

Technical Abstract: 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 used in combination have been effective in assessing sperm viability in mammalian and avian species. Our objectives were to test two combinations of live:dead fluorescent stains, SYBR-14 with propidium iodide (PI), or Calcein-AM with PI, and validate the used of these probes with honey bee semen. SYBR-14 is a nuclear stain producing green fluorscent 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 live stains fluoresced bee sperm, but the SYBR-14: PI produced a clearer distinction between the live and dead sperm. A graduated series of known live: dead sperm proportions was used to validate the accuracy of the stains for determing sperm viability in honey bees.