In Vitro Fertilization



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Ronna Hertzano

IVF - protocol

In Vitro Fertilization




1.1 Introduction

The following protocol has been adopted from the Institute of Experimental Genetics, in the GSF – Germany1. The protocol provides a full set of instructions for both ‘simple’ in vitro fertilization and for cryopreservation and the usage of thawed spermatozoa for in vitro fertilization.



In vitro fertilization can be used to produce large colonies of mice in a relatively short period of time, saving also on mouse caging space. Moreover, fewer males are needed to fertilize the same number of females and the procedure naturally overcomes problems such as periods of ‘no breeding’ or some fertility difficulties encountered especially in mutated strains.

The usage of cryopreserved spermatozoa enables long-term preservation of strains but has its limitations (mice on a C57Bl/6 pure background can not be used in this procedure, also maintenance of certain strains such as congenics and consomics requires matching oocytes).



Concept: sperm from one is taken to fertilize oocytes from 15-20 superovulated . On average, C3H females produce 15 oocytes per female. If cleavage rate is 60%, ~200 two-stage cells are attained, and 20 are retrieved per mouse – using 8-10 pseudopregnant ICR . As a mouse usually conceives up to 8 mice, usually at least 50 mice are generated in one reaction (80 being the upper limit). .

At first it seems as if it is much too much work for generating backcross mice for mapping. But considering mouse room limitations, and the F1 males limitation, the thought that in one week using 8 F1 mutants 400 progeny can be attained is quite encouraging. (ref 9 & 10 original protocol).



Snapshot protocol for IVF

Prepare




hCG



PMSG



CPA









HFT + mineral oil equilibrated



KSOM + mineral oil equilibrated



Glass capillary pipettes









Fertilization plate: 4 X35mm plates, 200μl HTF + 3ml HTF equilibrated oil.



4 well plate – 3 drops of NaCl + 170μl CPA for each mouse.



Prepare sperm evaluation plate: 100μl HTF + equilibrated oil.



60 mm dish with regular mineral oil.



60 mm dish with a drop of 400μl HTF covered with regular mineral oil.









Wash plates(1) – 4 drops of KSOM



Overnight culture dish – 100μl KSOM + KSOM equilibrated oil.









Wash plates(2) – three drops of M2 medium (50μl each) + 10μl hyaluronidase in the first drop.



100μl M2 drop covered with mineral oil.



Anesthesia for ICR females.


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