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Research Compliance and Tech Transfer

Retro-orbital Bleeding and Injections in Mice and Rats

Number: IACUC-POL-033 

Responsible Office: Office of Research and Creative Scholarship (ORCS) 

Applies to: Principal Investigators Conducting Animal Research or All animal research and teaching protocols involving live vertebrate animals 

1. Purpose 

To comply with government regulations (PHS and USDA), AAALAC, The Guide for the Care and Use of Laboratory Animals, and standard veterinary care techniques, The 91次元 IACUC has implemented a policy concerning retro-orbital eye bleeding and injections in mice and rats. Retro-orbital injections and blood collection can be acceptable alternatives to other routes (e.g., tail vein, saphenous vein, or submandibular vein) when performed correctly by trained personnel. Retro-orbital bleeding represents more than “minimal or transient pain and distress” and, according to the USDA, is an example of a “Category D” procedure.  

2. Training  

  1. In the hands of an unskilled phlebotomist, retro-orbital sampling has a greater potential than other blood collection routes to result in complications. When personnel are undergoing training in retro-orbital blood collection, initial training must first be performed on a carcass prior to practicing on an anesthetized live animal. During live animal training, general anesthesia is required, and the animals must be euthanized immediately following the procedure. 

  2. Investigators must demonstrate proficiency in the procedure before initiation of the study. Procedural competency will be signed off by the Attending Veterinarian (AV). 

  3. Protocol participants transferring from another institution may provide proof of training from that institution to the AV. The protocol participant will be required to display competency in the method to the AV. 

3. Requirements  

Investigators should maintain familiarity with the IACUC guidelines regarding retro-orbital sinus bleeding or injections in mice. In rats, the presence of a venous plexus rather than a sinus can lead to greater orbital tissue damage than in the mouse.  

  1. Scientific rationale must be provided in the proposed animal use protocol with justification of why other routes are not desired.  
  2. All animals must be appropriately anesthetized prior to performing procedure. Since this is a short procedure, Isoflurane is recommended unless this procedure is to be followed by another, more lengthy procedure requiring the administration of injectable anesthetic agents.  
  3. In addition, a topical ophthalmic anesthetic, e.g., proparacaine or tetracaine drops, is recommended immediately prior to the procedure, and may be considered as an analgesic. 
  4. Each eye may only be used once within a minimum 2–3 week period, and repeated sampling from the same eye is discouraged unless scientifically necessary and specifically approved. The maximum number of bleeds or injections must be clearly defined in the protocol and justified by scientific need, with IACUC approval required. 
  5. For bleeding, sterile micro-hematocrit tubes should be used to help avoid periorbital infection and potential long-term damage to the eye. The edges of the tubes should be checked for smoothness to also decrease the likelihood of eye damage. The use of sterile hypodermic needles can only be used following IACUC approval and if sound scientific justification is provided and proficiency is demonstrated.  
  6. For blood collection, refer to Table 1 for the maximum allowable circulating blood volume per draw. For injections, a volume of up to 1% of the animal’s body weight may be administered per injection. The maximum injectable volume in adults is <200 μL, using a 27–29 gauge, 0.5-inch insulin needle and syringe (recommended). 
  7. An individual can receive no more than one injection per day or undergo one RO blood collection. When more than one injection is required, alternate between eyes, and allow at least 24 hours between injections. Do not exceed two injections per eye in any individual.  
  8. No bleeding may be performed from a damaged eye. If both eyes are damaged, eye bleeding must cease.  

Based on animal welfare indices the NIH veterinary recommended blood volume to use is 55 to 70 ml/kg when calculating quantity. Volumes greater than recommended should be justified in the AUP and appropriate fluid replacement provided. Calculated blood sample ranges, based on recommended body weight are provided in Table 1. 

Calculated Blood Sample Volumes for Species and Range of Body Weights

Table 1: Calculated Blood Sample Volumes for Species and Range of Body Weights 

Species 

Body weight (g) 

*CBV(ml) 

~1% CBV every 24 hrs† 

~7.5% CBV every 7 days† 

~10% CBV every 2 - 4wks† 

Mouse 

 

 

 

 

20 

1.10 - 1.40 

11 - 14 µl 

90 - 105 µl 

110 - 140 µl 

25 

1.37 - 1.75 

14 - 18 µl 

102 - 131 µl 

140 - 180 µl 

30 

 

1.65 - 2.10 

17 - 21 µl 

124 - 158 µl 

170 - 210 µl 

35 

1.93 - 2.45 

19 - 25 µl 

145 - 184 µl 

190 - 250 µl 

40 

2.20 - 2.80 

22 - 28 µl 

165 - 210 µl 

220 - 280 µl 

Rat 

  

  

  

  

  

  

125 

6.88 - 8.75 

69 - 88 µl 

516 - 656µl 

690 - 880 µl 

150 

8.25 - 10.50 

82 - 105 µl 

619 - 788 µl 

820 - 1000 µl 

200 

11.00 - 14.00 

110 - 140 µl 

825 – 1050 µl 

1.1 - 1.4 ml 

250 

13.75 - 17.50 

138 - 175 µl 

1.0 – 1.3 ml 

1.4 - 1.8 ml 

300 

16.50 - 21.00 

165 - 210 µl 

1.2 – 1.6 ml 

1.7 - 2.1 ml 

350 

19.25 - 24.50 

193 - 245 µl 

1.4 – 1.8 ml 

1.9 - 2.5 ml 

*Circulating blood volume (1ml = 1000µl) 

†Maximum sample volume for that sampling frequency 

4. Monitoring and Follow-up 

Post Procedure Monitoring and Follow-up: Animals must be monitored until fully recovered from anesthesia and again within 24hrs for signs of ocular injury, infection and distress.  

Potential Adverse Effects of Retroorbital Bleeding in Mice 

Retroorbital bleeding may result in a number of adverse outcomes. Ocular complications can include corneal abrasions or ulceration, conjunctival trauma, periorbital swelling or inflammation, hemorrhage into ocular tissues, proptosis of the eye, periocular atrophy resulting in a sunken globe, and in rare cases, rupture of the globe. Neurologic and sensory effects may involve pain, distress, or temporary to permanent vision impairment in the affected eye due to tissue or nerve damage. 

Systemic complications can occur if excessive blood is withdrawn, leading to anemia, hypovolemia, or shock. Tissue trauma may also predispose animals to secondary infections and increase susceptibility to stress, thereby reducing overall welfare. 

Long-term sequelae may include chronic ocular irritation, scarring, persistent or progressive vision impairment, corneal or lens opacification, and changes in ocular structure such as sunken globe or asymmetry of the orbits. These complications can contribute to ongoing discomfort, alter behavior, and reduced quality of life. 

Injury Management 

Any ocular injury or adverse event must be reported to the AV immediately; animals with severe injury may require euthanasia for humane reasons.  

5. References 

  1. Guide for the Care and Use of Laboratory Animals (NRC, 2010).
  2. NIH Guidelines for Survival Blood Collection in Mice and Rats, updated 12/14/2022.
  3. Type, Duration, and Incidence of Pathological Findings After Retroorbital Bleeding of Mice by Experience and Novice Personnel. J Am Assoc Lab Anim Sci. 2015 May;54(3):317-27 
  4. Comparison of Serial Blood Collection by Facial Vein and Retrobulbar Methods in C47Bl/6 Mice. J Am Assoc Lab Anim Sci. 2018 Jul;57(4): 382-391  

6. Review, Approval and Version History 

Review and approval version history for this policy

Version 

Date 

Description of Changes 

Approved By 

1.0 

 September 9, 2025 

Initial policy creation 

IACUC