The authors have no acknowledgements to make.

All procedures were approved by the Tongji Medical College Council on the Animal Care Committee of Huazhong University of Science and Technology (Wuhan, China). The manuscript was prepared according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines6.
1. Husbandry
<ol>
	<li>Use Sprague-Dawley (SD) rats (n = 20, 10 males, 8 week-old, weighing 261 to 291 g, mean = 272.85 &#177;9.07 g) from the Experimental Animals Center of Huazhong University of Science and Technology and maintain rats in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No.85-23, revised 1996)</li>
	<li>House under standard conditions with free access to food and drinking water. Keep rats in 530 cm2 cages with wood-shaving bedding. House two rats in each cage. Keep all cages open to the room environment (no micro-isolation or ventilated caging)</li>
	<li>Ensure the staff involved in the experimental protocol regularly handle the rats.</li>
	<li>Maintain room temperature between 21 &#176;C and 23 &#176;C. Give rates a normal salt diet (0.3% NaCl) throughout the study.</li>
</ol>
2. Blood Collection Procedure
NOTE: The vacuum blood collection system consists of a double-pointed needle [22 G (0.7 mm x 25 mm)], a plastic holder, and a series of vacuum tubes (2 mL) with rubber stoppers (Figure 1).
<ol>
	<li>Place the rat in a plastic restraining holder and wash the tail with warm (20-30 &#176;C) water to remove any visible dirt or feces. While an assistant holds the tail in an extended position, wipe the tail with 70% ethanol, and clean with gauze to make the vein clear.</li>
	<li>Insert the 22 G butterfly needle tip into one of the lateral tail veins (around 5 mm) at a position approximately 2-3 cm away from the tip of the tail at angle of approximately 20&#176; (Figure 2). Collect blood into the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube (Figure 3). 
	NOTE: The maximal collection blood volume is 1.2 mL7.</li>
	<li>After blood collection, remove the needle and gently apply pressure to the puncture site with gauze for 15-30 s to stop the flow of blood. Then, release the rat from the plastic restraining holder and return the rat to its cage.</li>
	<li>Use tubes that contain EDTA as an anticoagulant to collect blood plasma. Gently invert the tube several times to mix anticoagulant in the blood and place the samples on ice vertically.</li>
	<li>Centrifuge the blood sample collection tubes in a refrigerated centrifuge at 2,000 x g for 10 min to separate plasma or serum.</li>
	<li>Extract the plasma/serum, taking care not to disturb the red and white blood cell layers. To collect blood serum, use tubes without anticoagulant. Both types of sample can be used immediately, or stored at -80 &#176;C for up to one year. 
	NOTE: Successful blood collection was defined as obtaining 1.2 mL blood each time. Maximal puncture time was set to three trials, if the total blood volume was lower than 1.2 mL after the third puncture, it was defined as a failed blood collection. The sampling duration starts from the tail vein puncture to the removal of the butterfly needle after blood collection.</li>
	<li>Collect blood twice within two weeks7.
	<ol>
		<li>To test and verify the feasibility of this method, train two medical students (beginners) for 2 h before performing this blood collection protocol. Use twenty rats and draw blood from 10 rats by 2 experienced fellows (experimental group) and from 10 rats by the 2 medical students (beginner group).</li>
	</ol>
	</li>
</ol>
3. Statistical Analysis
<ol>
	<li>Express data as a mean value &#177; standard deviation and analyze with SPSS Statistics 17. An &#945; value of 0.05 was chosen, therefore p &#60;0.05 was considered statistically significant.</li>
</ol>

<ol><li>Brown, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+collection+from+the+tail+of+a+rat%5BTitle%5D)+AND+%22Lab+Anim+%28NY%29%22%5BJournal%5D)">Blood collection from the tail of a rat.</a> Lab Anim (NY). 35 (8), 24-25 (2006).</li>
<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+sample+collection+in+small+laboratory+animals%5BTitle%5D)+AND+%22J+Pharmacol+Pharmacother%22%5BJournal%5D)">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</li>
<li>Staszyk, C., Bohnet, W., Gasse, H., Hackbarth, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+vessels+of+the+rat+tail%3A+a+histological+re-examination+with+respect+to+blood+vessel+puncture+methods%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Blood vessels of the rat tail: a histological re-examination with respect to blood vessel puncture methods.</a> Lab Anim. 37 (2), 121-125 (2003).</li>
<li>Lee, G., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sampling+blood+from+the+lateral+tail+vein+of+the+rat%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Sampling blood from the lateral tail vein of the rat.</a> J. Vis. Exp. (99), (2015).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22WHO+Guidelines+on+Drawing+Blood%3A+Best+Practices+in+Phlebotomy+WHO+Guidelines+Approved+by+the+Guidelines+Review+Committee%22">WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy WHO Guidelines Approved by the Guidelines Review Committee</a>. , (2010).</li>
<li>Kilkenny, C., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+bioscience+research+reporting%3A+ARRIVE-ing+at+a+solution%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Improving bioscience research reporting: ARRIVE-ing at a solution.</a> Lab Anim. 44 (4), 377-378 (2010).</li>
<li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood%2C+including+routes+and+volumes%5BTitle%5D)+AND+%22J+Appl+Toxicol%22%5BJournal%5D)">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J Appl Toxicol. 21 (1), 15-23 (2001).</li>
<li>Eder, J. M., Cutter, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+device+for+collecting+cord+blood%5BTitle%5D)+AND+%22Obstet+Gynecol%22%5BJournal%5D)">A new device for collecting cord blood.</a> Obstet Gynecol. 86 (5), 850-852 (1995).</li>
<li>Reynolds, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+a+new+device+for+blood+sampling+in+cats+with+a+vacuum+tube+collection+system+-+plasma+biochemistry%2C+haematology+and+practical+usage+assessment%5BTitle%5D)+AND+%22J+Feline+Med+Surg%22%5BJournal%5D)">Comparison of a new device for blood sampling in cats with a vacuum tube collection system - plasma biochemistry, haematology and practical usage assessment.</a> J Feline Med Surg. 9 (5), 382-386 (2007).</li>
<li>Wiwanitkit, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+blood+specimens+from+plain+and+gel+vacuum+blood+collection+tubes%5BTitle%5D)+AND+%22J+Med+Assoc+Thai%22%5BJournal%5D)">Comparison of blood specimens from plain and gel vacuum blood collection tubes.</a> J Med Assoc Thai. 84 (5), 723-726 (2001).</li>
<li>Wollowitz, A., Bijur, P. E., Esses, D., John Gallagher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+butterfly+needles+to+draw+blood+is+independently+associated+with+marked+reduction+in+hemolysis+compared+to+intravenous+catheter%5BTitle%5D)+AND+%22Acad+Emerg+Med%22%5BJournal%5D)">Use of butterfly needles to draw blood is independently associated with marked reduction in hemolysis compared to intravenous catheter.</a> Acad Emerg Med. 20 (11), 1151-1155 (2013).</li>
</ol>

The authors have nothing to disclose

The present report describes an easy-to-learn blood collection method in rats. Our results showed that this method is feasible for repeatedly collecting a large volume of blood from rats. Moreover, this method could be easily mastered with a short-term learning curve. This procedure can be performed in non-anesthetized rats and only causes minimal stress to the animals.
Blood can be collected from rats in various locations. The tail vein is a superior location for blood sampling4. However, it is not always easy to obtain enough blood volume from the thin and low blood flow tail vein of the rats. The vacuum blood collection system is usually used for collecting blood samples from patients in daily clinical practice. This vacuum blood collection system has been used in patients and research animals for a long time8,9,10. This system consists of a vacuum blood collection tube with silicone coated inner tube wall and butterfly needle. It was based on the vacuum negative pressure principle and allows the continuous drawing of blood without the risk of blood collecting, which also reduces the chance of contamination and hemolysis11. The volume of blood sample in the catheter is about 0.3 mL. After withdrawing the needle from the tail vein, the blood blocked in the catheter will flow into the collecting vacuum tube due to the vacuum. This method is also applicable for collecting blood from rats weighing less than 100 g with the use of a smaller puncture needle.
This blood collection method has several advantages. This procedure only causes minimal injury to rats and blood collection can be performed without the use of anesthetics, thus the influence of the stress response and anesthetics on the blood sample can be avoided. Our observations showed that rats were quiet in the plastic restraining holder during the procedure and there were no deaths during these experiments. Secondly, repeated blood collection every one or two days is possible, which enables frequent sampling of blood with an adequate volume for various research purposes. Finally, this method is easy to learn and was easily mastered by beginners after a short (2 h) learning period, as indicated by the similar success rates and puncture times between the experimental and beginner groups in this report. In a previous report, Lee et al. demonstrated a similar method of sampling blood from the lateral tail vein of the rat4. Our method is simpler than that reported by Lee et al.4 in that we directly insert the puncture needle into the tail vein and the other end of the tube is directly connected to the vacuum tube for collecting the blood, without the use of an extra syringe and manual aspiration of the blood and without the need of &#39;milking&#39; the vein to facilitate blood flow. Collecting blood with this closed system reduces the chance of blood contamination. Use of the plastic restraining holder also reduces the stress to the rat and facilitates the fixation of the tail.
The following two steps are critical for the successful application of this method. 1) Extend the tail and avoid the movement of the tail; 2) Gently puncture the vein at an angle of 20&#176; to avoid puncture through the vein and keep the puncture needle in the vein.
In conclusion, the adopted vacuum blood collection method in rats is safe, feasible, and easy to practice. This method enables frequent sampling of blood with an adequate blood volume obtained from non-anesthetized rats.

Blood sampling from rats is required for a wide variety of experimental studies. Techniques for blood collection from the rat include puncture of the heart, retro-orbital plexus, jugular vein, saphenous vein, tail blood vessels, carotid artery, abdominal aorta, and vena cava. Most techniques (except saphenous vein and tail blood vessel puncture) require anesthesia1,2. Veins of the rat tail are routinely punctured for blood sampling and vascular injection. After treating the tail with warm water, the blood vessels are well dilated and suitable for manipulation, even when rats are anesthetized3. 
Despite the development of new and improved techniques to obtain blood samples in small laboratory animals2, it is not always easy to obtain enough blood when sampling from small animals. The tail vein of rats is a superior location for blood sampling4. However, the tail vein is thin, thus it is sometimes hard to puncture and would take a long time to get enough blood due to its low blood flow. Blood collection via a vacuum extraction tube system is a typical method in daily clinical practice. This closed system for blood collecting reduces the risk of direct exposure to blood and has made it easier to take multiple samples from a single venipuncture5. The present report describes a simple tail vein blood sampling method in non-anesthetized rats using the vacuum extraction tube system. This method is easy to master and can be used to obtain a large volume of blood repeatedly in rats.

<table><tbody><tr><td>a double-pointed needle</td><td>Shanghai Kang Nong medical instrument co., LTD, China</td><td>22G&amp;nbsp;</td><td>(0.7&amp;times;25mm)</td></tr><tr><td>vacuum tubes</td><td>Wuhan Zhi Yuan, medical science and technology co., LTD, China</td><td>2 ml</td><td></td></tr><tr><td>rat restraining holder&amp;nbsp;</td><td>Shanghai Kang Nong medical instrument co., LTD, China</td><td>250g rat hoder model</td><td></td></tr><tr><td>normal salt diet for rats</td><td></td><td></td><td>Rats received a normal salt diet (0.3% NaCl) throughout the study</td></tr><tr><td>SPSS software for statistical analysis</td><td>SPSS Inc,&amp;nbsp; Chicago; USA&amp;nbsp;</td><td>Version 17.0.&amp;nbsp;</td><td></td></tr></tbody></table>

repeated blood collection from tail vein of non-anesthetized rats with a vacuum blood collection system

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Sampling duration, body mass, and blood collection volume of the two groups 
Blood was collected from 10 rats (5 males) twice within two weeks in each group. The mean body mass in both groups was similar (272.00 ±9.66 g and 273.70 ±8.87 g). Sampling duration was significantly shorter in the experimental group compared to beginner group (2.77 ±0.53 min vs. 3.28 ±0.83 min, p &lt;0.05) (Table 1).
Success rate and puncture times of two groups 
The success rate was 95% (19/20) in the experimental group and 90% (18/20) in the beginner group (p &gt;0.05). Puncture times were not significantly different between the two groups (1.50 ±0.61 min vs. 1.85 ±0.75 min, p &gt;0.05) (Table 2). These results indicate this method is easy to learn, and the technique could be mastered after training for 2 h.
<img alt="Rodent stereotaxic surgery setup, featuring anesthesia chamber and syringe for drug administration." class="xfigimg" src="/files/ftp_upload/55852/55852fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: 2 mL vacuum blood collection tube (left); 22 G butterfly needle (center); the plastic restraining holder (right). <a href="https://cloudflare.jove.com/files/ftp_upload/55852/55852fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Catheter placement in hospital setting; gloved hands inserting tube; medical procedure setup." class="xfigimg" src="/files/ftp_upload/55852/55852fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: The needle tip was inserted into the lateral tail vein (around 5 mm) at the position approximately 2-3 cm away from the tip of the tail at angle of approximate 20°. <a href="https://cloudflare.jove.com/files/ftp_upload/55852/55852fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Blood sampling setup with syringe and vial; laboratory experiment for biochemical analysis." class="xfigimg" src="/files/ftp_upload/55852/55852fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Successful venipuncture. <a href="https://cloudflare.jove.com/files/ftp_upload/55852/55852fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="5">                        Experimental group</td>
			<td></td>
			<td colspan="5">                                  Beginner group</td>
		</tr>
		<tr>
			<td></td>
			<td>Gender</td>
			<td>Body weight (g)</td>
			<td>Collected blood  volume (mL)</td>
			<td>sampling duration (min)</td>
			<td></td>
			<td></td>
			<td>Gender</td>
			<td>Body weight (g)</td>
			<td>Collected blood  volume (mL)</td>
			<td>sampling duration (min)</td>
		</tr>
		<tr>
			<td>E1-1</td>
			<td>male</td>
			<td>291</td>
			<td>1.2</td>
			<td>3.12</td>
			<td></td>
			<td>B1-1</td>
			<td>male</td>
			<td>282</td>
			<td>1.2</td>
			<td>3.18</td>
		</tr>
		<tr>
			<td>E1-2</td>
			<td>male</td>
			<td>291</td>
			<td>1.2</td>
			<td>2.73</td>
			<td></td>
			<td>B1-2</td>
			<td>male</td>
			<td>282</td>
			<td>1.2</td>
			<td>2.18</td>
		</tr>
		<tr>
			<td>E2-1</td>
			<td>male</td>
			<td>276</td>
			<td>1.2</td>
			<td>2.3</td>
			<td></td>
			<td>B2-1</td>
			<td>male</td>
			<td>264</td>
			<td>1.2</td>
			<td>3.73</td>
		</tr>
		<tr>
			<td>E2-2</td>
			<td>male</td>
			<td>276</td>
			<td>1.2</td>
			<td>2.37</td>
			<td></td>
			<td>B2-2</td>
			<td>male</td>
			<td>264</td>
			<td>1.2</td>
			<td>2.47</td>
		</tr>
		<tr>
			<td>E3-1</td>
			<td>male</td>
			<td>267</td>
			<td>1.2</td>
			<td>2.73</td>
			<td></td>
			<td>B3-1</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>3.52</td>
		</tr>
		<tr>
			<td>E3-2</td>
			<td>male</td>
			<td>267</td>
			<td>1.0</td>
			<td>3.88</td>
			<td></td>
			<td>B3-2</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>2.53</td>
		</tr>
		<tr>
			<td>E4-1</td>
			<td>male</td>
			<td>263</td>
			<td>1.2</td>
			<td>2.41</td>
			<td></td>
			<td>B4-1</td>
			<td>male</td>
			<td>289</td>
			<td>1.2</td>
			<td>2.32</td>
		</tr>
		<tr>
			<td>E4-2</td>
			<td>male</td>
			<td>263</td>
			<td>1.2</td>
			<td>2.52</td>
			<td></td>
			<td>B4-2</td>
			<td>male</td>
			<td>289</td>
			<td>1.2</td>
			<td>3.62</td>
		</tr>
		<tr>
			<td>E5-1</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>3.61</td>
			<td></td>
			<td>B5-1</td>
			<td>male</td>
			<td>277</td>
			<td>1.2</td>
			<td>3.48</td>
		</tr>
		<tr>
			<td>E5-2</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>2.42</td>
			<td></td>
			<td>B5-2</td>
			<td>male</td>
			<td>277</td>
			<td>1.2</td>
			<td>3.12</td>
		</tr>
		<tr>
			<td>E6-1</td>
			<td>female</td>
			<td>285</td>
			<td>1.2</td>
			<td>3.33</td>
			<td></td>
			<td>B6-1</td>
			<td>female</td>
			<td>274</td>
			<td>0.9</td>
			<td>4.87</td>
		</tr>
		<tr>
			<td>E6-2</td>
			<td>female</td>
			<td>285</td>
			<td>1.2</td>
			<td>3.03</td>
			<td></td>
			<td>B6-2</td>
			<td>female</td>
			<td>274</td>
			<td>1.2</td>
			<td>3.37</td>
		</tr>
		<tr>
			<td>E7-1</td>
			<td>female</td>
			<td>271</td>
			<td>1.2</td>
			<td>2.82</td>
			<td></td>
			<td>B7-1</td>
			<td>female</td>
			<td>268</td>
			<td>1.0</td>
			<td>3.93</td>
		</tr>
		<tr>
			<td>E7-2</td>
			<td>female</td>
			<td>271</td>
			<td>1.2</td>
			<td>2.42</td>
			<td></td>
			<td>B7-2</td>
			<td>female</td>
			<td>268</td>
			<td>1.2</td>
			<td>3.82</td>
		</tr>
		<tr>
			<td>E8-1</td>
			<td>female</td>
			<td>268</td>
			<td>1.2</td>
			<td>1.93</td>
			<td></td>
			<td>B8-1</td>
			<td>female</td>
			<td>262</td>
			<td>1.2</td>
			<td>2.07</td>
		</tr>
		<tr>
			<td>E8-2</td>
			<td>female</td>
			<td>268</td>
			<td>1.2</td>
			<td>3.05</td>
			<td></td>
			<td>B8-2</td>
			<td>female</td>
			<td>262</td>
			<td>1.2</td>
			<td>3.67</td>
		</tr>
		<tr>
			<td>E9-1</td>
			<td>female</td>
			<td>273</td>
			<td>1.2</td>
			<td>2.52</td>
			<td></td>
			<td>B9-1</td>
			<td>female</td>
			<td>281</td>
			<td>1.2</td>
			<td>4.38</td>
		</tr>
		<tr>
			<td>E9-2</td>
			<td>female</td>
			<td>273</td>
			<td>1.2</td>
			<td>3.17</td>
			<td></td>
			<td>B9-2</td>
			<td>female</td>
			<td>281</td>
			<td>1.2</td>
			<td>4.57</td>
		</tr>
		<tr>
			<td>E10-1</td>
			<td>female</td>
			<td>261</td>
			<td>1.2</td>
			<td>1.82</td>
			<td></td>
			<td>B10-1</td>
			<td>female</td>
			<td>275</td>
			<td>1.2</td>
			<td>2.53</td>
		</tr>
		<tr>
			<td>E10-2</td>
			<td>female</td>
			<td>261</td>
			<td>1.2</td>
			<td>3.25</td>
			<td></td>
			<td>B10-2</td>
			<td>female</td>
			<td>275</td>
			<td>1.2</td>
			<td>2.28</td>
		</tr>
		<tr>
			<td>Average</td>
			<td></td>
			<td></td>
			<td></td>
			<td>2.77</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>3.28</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="11">En-1: serial number of rats in the experimental group for the first blood collection.</td>
		</tr>
		<tr>
			<td colspan="11">En-2: serial number of rats in the experimental group for the second blood collection.</td>
		</tr>
		<tr>
			<td colspan="11">Bn-1: serial number of rats in the beginner group for the first blood collection.</td>
		</tr>
		<tr>
			<td colspan="11">Bn-2: serial number of rats in the beginner group for the second blood collection.</td>
		</tr>
	</tbody>
</table>
Table 1: Results of blood collected in the experimental group and the beginner group.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Blood collection times</td>
			<td>Average puncture times</td>
			<td>One time  puncture</td>
			<td>Two times puncture </td>
			<td>Three times puncture </td>
			<td>Failed</td>
			<td>Success rate</td>
		</tr>
		<tr>
			<td>Experimental group</td>
			<td>20</td>
			<td>1.50 ±0.61</td>
			<td>11</td>
			<td>8</td>
			<td>0</td>
			<td>1</td>
			<td>95%</td>
		</tr>
		<tr>
			<td>Beginner group</td>
			<td>20</td>
			<td>1.85 ±0.75</td>
			<td>7</td>
			<td>9</td>
			<td>2</td>
			<td>2</td>
			<td>90%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="6">One time puncture: Number of rats with successful blood collection after the first  puncture</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="6">Two times puncture:Number of rats with successful blood collection after the second puncture </td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="6">Three times puncture:Number of rats with successful blood collection after the third puncture </td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">Failed:Number of rats with failed blood collection after the third puncture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of the puncture times and success rates of the experimental group and the beginner group.

Watch this Scientific Journal Video about Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System at JoVE.com

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

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35.7K Views.  Jianghan University. Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture. 

Video: Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

Behavior

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Biology

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Neuroscience

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Comparative Study of Blood Collection Techniques in Rats

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Author Spotlight: Context Dependent Blood Collection Methods in Rats 

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood ...

Subclavian Vein Puncture Method for Repeated Blood Collection in Conscious Rats

Subclavian Vein Blood Sampling in Conscious Rats

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein sampling without anesthesia and jugular vein sampling with anesthesia. However, most of these methods require assistance and anesthetic equipment and sometimes pose difficulties in terms of blood collection or the poor quality of blood samples. In addition, these methods of blood collection consume significant time and human resources when repeated blood sampling is required for a large number of rats. This study presents a technique for repetitive blood sampling in non-anesthetized rats by a single proficient individual. Highly satisfactory blood samples can be obtained by puncturing the subclavian vein. The method demonstrated an impressive overall success rate of 95%, with a median time of merely 2 min from rat restraint to the completion of blood collection. Furthermore, performing consecutive blood collections within the designated range does not inflict any harm on the rats. This method is worth promoting for blood collection, especially in large-scale pharmacokinetic studies.

Author Spotlight: Developing an Efficient Blood Collection Method in Non-Anesthetized Rats 

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein ...

Blood Withdrawal I

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Blood collection is a common requirement for research studies that involve mice and rats. The method of blood withdrawal in mice and rats is dependent upon the volume of blood needed, the frequency of the sampling, the health status of the animal to be bled, and the skill level of the technician.1 All methods discussed-retro-orbital sinus bleeds, initial tail snip bleeds, and intracardiac bleeds-require the use of a general anesthesia.

Retro Orbital, Tail and Intra-cardiac Blood Collection in Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Blood collection is a common requirement for ...

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Blood Withdrawal II

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for analysis can be done through a variety of methods. Each method of collection has variations in the type of restraint required, the invasiveness of the procedure, and the necessity of a general anesthetic.1Historically, the use of the retro-orbital sinus cavity has been used, but not without debate. The controversy related to the potential tissue damage,or even blindness,caused by retro-orbital bleeds has led to the development of facial and submandibular vein bleeding methods in mice.Blood collection from the saphenous vein in both mice and rats is another technique that has been developed. These procedures do not require anesthesia and therefore are suitable when the use of anesthetics may confound blood results or other data.

Collecting Blood from Facial, Submandibular, Saphenous &amp; Femoral Veins in Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for ...