Request for Information (RFI) on Flexibilities to Reduce Administrative Burden While Continuing to Apply the PHS Policy to Zebrafish Immediately After Hatching
Notice Number:

Key Dates

Release Date:

May 7, 2021

Response Date:
August 09, 2021

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Issued by

Office of The Director, National Institutes of Health (OD)


Through this Request for Information (RFI), the NIH Office of Laboratory Animal Welfare (OLAW) is seeking input on flexibilities to reduce administrative burden while continuing to apply the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (Policy) to zebrafish (Danio rerio) immediately after hatching.


The 21st Century Cures Act, section 2034(d), directed the NIH to conduct a review of applicable regulations and policies for the care and use of laboratory animals and to make revisions, as appropriate, to reduce the administrative burden on investigators. It mandated that any revisions must maintain the integrity and credibility of research findings and protection of research animals.

One action identified by the research community and included in the report Reducing Administrative Burden for Researchers: Animal Care and Use in Research in response to the 21st Century Cures Act is to consider changing the applicability of the PHS Policy to zebrafish larvae from immediately after hatching (typically 3 days post fertilization [dpf] under optimal conditions) to when larvae begin free feeding (at approximately 5 – 7 dpf). In this RFI, OLAW provides justification for maintaining oversight authority for zebrafish larvae immediately after hatching and seeks input on the flexibilities that would reduce the administrative burden on institutions while continuing to protect zebrafish immediately after hatching.

Information Requested

The NIH is seeking input on the flexibilities available to reduce administrative burden with research involving zebrafish larvae while continuing to apply the PHS Policy to zebrafish immediately after hatching.


OLAW offers the following justification for maintaining oversight of zebrafish larvae immediately after hatching.

The PHS Policy defines an animal as “any live, vertebrate animal used or intended for use in research, research training, experimentation, or biological testing or for related purposes.” Although zebrafish embryos develop anatomical structures characteristic of vertebrates such as a notochord, pharyngeal arches, a neural tube, and somites prior to hatching, OLAW interprets the PHS Policy’s definition of a live vertebrate animal to apply to zebrafish (and other embryonated eggs) immediately after hatching. This reduces the burden that would have been imposed by OLAW if researchers were required to identify and account for zebrafish embryos at earlier developmental stages.

Insufficient evidence currently exists to indicate beyond a reasonable doubt that zebrafish larvae are incapable of pain and suffering at hatching because the earliest age that many physiological and behavioral studies are conducted with zebrafish larvae is 5 dpf. According to US Government Principle IV, “Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals.” Because contrary evidence is not currently compelling, the precautionary principle applies, which emphasizes that scientific uncertainty or a lack of knowledge should not be used as a reason to postpone protecting zebrafish welfare.

At 5 dpf, zebrafish larvae have already developed some of the anatomical, physiological, and behavioral characteristics for sensing pain, distress, or discomfort, and demonstrate an aversion to noxious stimuli. Because scientific evidence regarding the ability to feel pain, distress, discomfort is currently lacking for zebrafish larvae immediately upon hatching, continued oversight of zebrafish research prior to 5 dpf is warranted. Revising the OLAW guidance based on other developmental stages, such as free feeding or swimming, independent of consideration for the potential of zebrafish to feel pain or distress, may negatively impact animal welfare. Based on the above justifications, OLAW cannot justify postponing the oversight of zebrafish larvae to 5 – 7 dpf.


To reduce the administrative burden in research involving zebrafish while maintaining oversight immediately after hatching, institutions may find it beneficial to:

  • Only generate approximate numbers of animals to be used, as described in PHS Policy IV.D.1.A. How to determine what constitutes approximate numbers, within reason, is established by the institution. Some institutions cover all live stages of zebrafish in their research protocols reviewed by the Institutional Animal Care and Use Committee (IACUC). Others have a core protocol that covers larvae and embryos until they are transferred to the research protocol at the required stage needed for research (i.e., post-hatching). Yet other institutions have IACUC protocols that cover all zebrafish immediately after hatching.
  • Use designated member review to review research projects with zebrafish unless full committee review is requested.
  • Include a zebrafish researcher or technician as an ad hoc consultant or a subject matter expert to assist in developing tracking, animal care, and euthanasia methods.


OLAW’s mission is to ensure the humane care and use of laboratory animals, including zebrafish, and the 21st Century Cures Act mandated that data integrity and animal welfare not be negatively impacted to comply with its goals. Based on the above justifications, postponing the applicability of the PHS Policy to zebrafish larvae until later developmental stages may negatively impact animal welfare. It would also be inconsistent with OLAW’s mission, and noncompliant with the PHS Policy, the U.S. Government Principles, and the Guide for the Care and Use of Laboratory Animals. Therefore, OLAW will continue requiring full implementation of the PHS Policy to research studies involving zebrafish immediately after hatching. This includes requiring Assured institutions to have policies and procedures in place that address the care or euthanasia of animals that hatch unexpectedly.

Please see OLAW's zebrafish webpage for more information.

How to Submit a Response

All responses to this RFI must be submitted electronically on the RFI webpage by August 9, 2021, at 11:59 PM ET. The comments may be made available on the OLAW website.

Responses to this RFI are voluntary and may be submitted anonymously. Please do not include any personally identifiable or other information that you do not wish to make public. Proprietary, classified, confidential, or sensitive information should not be included in responses. The Government will use the information submitted in response to this RFI at its discretion. The Government reserves the right to use any submitted information on public websites, in reports, in summaries of the state of the science, in any possible resultant solicitation(s), grant(s), or cooperative agreement(s), or in the development of future funding opportunity announcements. This RFI is for informational and planning purposes only and is not a solicitation for applications or an obligation on the part of the Government to provide support for any ideas identified in response to it. Please note that the Government will not pay for the preparation of any information submitted or for use of that information.


Please direct all inquiries to:

Office of Laboratory Animal Welfare
Telephone: 301-496-7163

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