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Generation of zebrafish embryos by In Vitro Fertilization: Effectiveness of Using Different Media

15TH ANNUAL NMS RESEARCH SYMPOSIUM

Lydia N. Loos and Daniel P. Szeto

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Specific Aims and Significance

    • Project focus on:
      • Establish protocol for in vitro fertilization for zebrafish embryos
      • Test protocols with different conditions

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    • Significance
      • More consistent genetic background of zebrafish embryos

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Experimental Design and Procedure

  1. Extraction of gametes from male and female zebrafish

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Weinstein, B. (2019). [digital]. National Institute of Health. https://www.nichd.nih.gov/research/atNICHD/Investigators/weinstein/zebrafish

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Male

Female

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Extraction of Female Gametes

  • After anaesthezing with tricaine solution for a few minutes
  • Before extraction
    • No water
    • Media added
      • Fish Ovarian Fluid (classic)
      • Hank’s Balanced Buffer
      • Hank’s Balanced Buffer with Bovine Serum Albumin (BSA)

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Extraction of Female Gametes

Female gamete extraction

    • Laid flat
    • Fingers are used
    • Washed with media
    • Recovery

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    • Gametes put aside

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Extraction of Male Gametes

  • Male zebrafish
    • Anesthetized
    • Belly up
    • Forceps
    • Capillary tube
    • Recovery

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Experimental Design and Procedure

2) In vitro combining the male and female gametes in selected medium

After mixing, incubated at room temperature for 5 minutes

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Experimental Design and Procedure

  • 3) Completing in vitro fertilization by adding fish system water

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  • 4) Embryos are analyzed after 24 to 48 hours after fertilization

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Experimental Design and Procedure

  • 5) Analysis was done using Keyence microscope in Mission Hall
    • Looking at normal physical appearance
    • Heartbeat
    • Blood circulation
  • Gives indication that zebrafish embryos developed normally

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Observations

Selected Media

Observed Fertilization Rate

Fish Ovarian Fluid

+++ (90%) 45 embryos

Hank’s Balanced Buffer

++ (75%) 65 embryos

Hank’s Balanced Buffer with BSA

+ (?) 4 embryos

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Observations

Heartbeat

Selected Media

Embryonic Heart Beating

Fish Ovarian Fluid

Normal >90%

Hank’s Balanced Buffer

Normal >90%

Hank’s Balanced Buffer with BSA

Normal >90%?

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Observations

Blood Circulation

Selected Media

Vasculature Circulation

Ovarian Fluid

Normal >90%

Hank’s Balanced Buffer

Normal >90%

Hank’s Balanced Buffer with BSA

Normal >90%?

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Summary

Selected Media

Observed Fertilization Rate

Embryonic Heart Beating

Vasculature Circulation

Fish Ovarian Fluid

+++ (90%) 45 embryos

Normal >90%

Normal >90%

Hank’s Balanced Buffer

++ (75%) 65 embryos

Normal >90%

Normal >90%

Hank’s Balanced Buffer with BSA

+ (?) 4 embryos

Normal >90%?

Normal >90%?

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Conclusions

  • Established standardized protocol
  • Improved protocol by using Hank’s Balanced Buffer

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Future Experimental Plans

  • Hank’s Balanced Buffer with Bovine Serum Albumin
  • Optimal time of in vitro fertilization for oocytes
  • Effects of cryopreservation and different chemicals on spermatozoa
  • Efficiency of other media on fertilization rate

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Thank You