Insect Embryo Cryopreservation Through Waxy Layer Permeabilization
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Solution Overview
Problem
Current methods for cryopreservation of insect embryos, particularly those of cold-sensitive species like the Black soldier fly, are inefficient, result in low survival rates, and are not scalable, leading to challenges in maintaining genetic lines and mass production.
Innovation Solution
A method involving the removal of chorion layers and permeabilization of the waxy layer with a composition of limonene and 2-methyl tetrahydrofuran (2-MeTHF), followed by loading with cryoprotective agents and uniform freezing, allows for reproducible cryopreservation and revivification of embryos, suitable for large-scale applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional cryopreservation methods are used on insect embryos with intact chorion and waxy layers, then the process is simple, but the survival rate of cryopreserved embryos is low
Solution Approach 1:
The patent applies preliminary action by removing the chorion layers and permeabilizing the waxy layer before cryopreservation. This pre-treatment allows cryoprotective agents to penetrate the embryo effectively, significantly improving survival rates. The method involves incubating embryos in sodium hypochlorite solution to remove chorion, followed by treatment with limonene and 2-MeTHF to permeabilize the waxy layer, and then loading with cryoprotective agents before freezing.
2Productivity
If small-scale cryopreservation experiments are conducted, then specialized equipment can be used, but the yield of viable embryos is low and results are highly variable
Solution Approach 1:
The patent applies parameter changes by optimizing multiple parameters including embryo developmental stage (selecting specific stages), chemical concentrations (sodium hypochlorite at 1-6%, limonene at 50-95%, 2-MeTHF at 5-50%), treatment times, and cryopreservation temperatures. These optimized parameters consistently produce high yields of viable embryos with reliable development to adult flies, overcoming the variability of previous small-scale experiments.
3Reliability
If insect colonies are maintained live for genetic line preservation, then genetic material can be preserved, but resources are significantly consumed and genetic drift or contamination can occur
Solution Approach 1:
The patent applies the extraction principle by removing embryos from the living colony system and preserving them in a dormant cryopreserved state. This extracts the genetic material from the resource-intensive live colony environment, eliminating ongoing resource consumption while preventing genetic drift and contamination. The cryopreserved embryos can be stored indefinitely and revived when needed, providing a static but reliable genetic archive.
4Productivity
If egg production is increased to meet demand, then production capacity increases, but yield varies dramatically week to week and colonies may collapse
Solution Approach 1:
The patent applies preliminary action by producing and cryopreserving embryos in advance during optimal production periods. This allows decoupling of egg production from the resource-intensive colony maintenance and immediate hatching processes. Cryopreserved embryos can be stored and hatched on demand, smoothing out production variations and preventing colony collapse from over-exploitation while maintaining steady supply.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method achieves high survival rates of cryopreserved embryos, with a significant percentage developing into healthy, fertile adults, suitable for maintaining genetic lines and mass production.
Implementation Method 1
permeabilizing the exposed waxy layer by contacting said layer with a permeabilization composition comprising a permeabilizing compound selected from the group consisting of a cyclic terpene and/or an essential oil comprising same
Implementation Method 2
loading the embryo obtained at step (b) with at least one cryoprotective agent (CPA), thereby obtaining an embryo loaded with the at least one CPA; and exposing the embryo obtained at step (c) to a cryogenic temperature
Implementation Method 3
exposing the embryo obtained at step (c) to a cryogenic temperature; thereby obtaining cryopreserved viable embryos
Data Source
AI summary
The present invention provides methods for cryopreservation of insect embryos comprising chorion layers and waxy layer, particularly fly embryos including embryos of Black Soldier Fly (Hermetia illucens, BSF) useful in mass rearing of beneficial fly adults. The present invention further provides cryopreserved embryos having an industrially suitable adult recovery and survival rate.