Location: Insect Genetics and Biochemistry Research
2016 Annual Report
Objectives
Develop means for long-term storage of bee germplasm, including cryopreservation of embryos. [NP 305, Component 2, Problem Statements 2A, 2B, 2C]
Approach
Pollinating insects are important to the U.S. agricultural economy, contributing an estimated $16 billion to annual crop production. This critical component is currently at risk due to a dramatic decrease in managed and native pollinators, and will face additional challenges due to climate change in the future. Despite this importance, there is no organized germplasm biorepository (genebank) for bees. This project will develop protocols that support the establishment of a National Bee Germplasm Repository. Specifically, the project will focus on: 1) the development of an improved protocol for the cryopreservation of bee spermatozoa, 2) the development of a novel protocol for the cryopreservation of bee embryos, and 3) the development of a protocol for the in vitro rearing of embryos after storage into reproductively viable adults. This research will ultimately result in the preservation of elite and genetically diverse pollinator strains, the development of a cryogenically-based system for the safe importation of bee germplasm, and the systematic delivery of high-quality germplasm and insects to end users.
Progress Report
With reference to Milestone 1A.1, we have developed two seminal plasma substitutes namely, ASP1 and ASP2. Both the solutions contain minimal salts and organic compounds that mimic seminal plasma characteristics. These seminal plasma substitutes will be used as extender/dilution media for bee spermatozoa cryo-storage. In all of our testing, the newly designed extender media have exhibited many of the characteristics of seminal plasma.
Under sub-objective 3A.1, we have analyzed the effect of the media developed under sub-objective 1A.1 on both the motility and long term viability of spermatozoa. Bee semen, when cryopreserved using the above said medium plus 2- 5% dimethyl sulphoxide, retained its viability (>94%) over months of storage on thawing. The semen remained viable on thawing and storing at either room temperature or at 7C for over 12 months. The controls tested with Harbo’s medium did not survive over 24 hours after thawing. The assessments were done by estimating motility characteristics (straight, coiled, in circle, and vibratory) as well as live-dead cell assay.
Accomplishments
1. Development of seminal plasma replacement. A vast majority of the maintained honeybee populations in the USA and Europe use queens that are artificially inseminated. Artificially inseminated queens typically under-perform after a season of egg laying due to deteriorating semen quality. This is primarily due to the diluents that are mixed into the semen prior to insemination. Scientists at the USDA-ARS in Fargo, North Dakota developed two newer diluents, taking into consideration some of the most recent developments in the field of sperm biology. Testing of the diluents on the artificially collected bee semen indicated that the semen quality remains intact for more than 14 months (our longest test period). This technology permits a significantly longer period of storage of semen under what are generally considered suboptimal conditions (room temperatures, field conditions, etc.) for both sperm banking as well as transportation.
2. Cryopreservation of bee semen in a newly developed medium. Increasingly, cryopreserved semen (semen stored in liquid nitrogen for indefinite storage) is used to inseminate queen bees. However, this semen does not last the life-time of the queen bee, contrary to observations if insemination had occurred in the wild. The dilution medium for bee semen developed at the USDA-ARS in Fargo, North Dakota was used as a cryoprotective medium with the addition of an antifreeze (dimethyl sulphoxide). With the newly designed cryoprotective medium, USDA-ARS scientists were able to obtain viable sperm cells with an average viability of >94%. This is a significant advancement in the field of semen cryobiology and will form the foundation for the national honey bee germplasm repository ensuring preservation of this vital agricultural resource.
