**3. Results**

### *3.1. Description of Studies*

Of the 31 full texts reviewed (Figure 1), 27 articles were eligible for inclusion. The date of publication of these articles ranged from 1995 to 2020, and no relevant studies were identified that were published prior to 1995. Five studies were retrieved through manual searching of the reference list of included studies. The characteristics of the included studies are summarised in Table 2. Observational studies were eligible for inclusion. However, the majority of the studies (25) adopted a randomized controlled study (RCT) design.

Out of the studies included, 78% evaluated more than one blood sampling method and tended to compare outcomes between blood sample methods. Methods evaluated included: 14 (52%) on facial vein sampling, 14 (52%) on the retrobulbar route, nine (33%) on tail incision, seven (26%) on tail tip amputation, five (19%) on the tail vein method, five (19%) using saphenous sampling, five (19%) on the sublingual, and two (7%) on a non-surgical jugular vein route. A further three studies used miscellaneous methods and uncommon routes of phlebotomy, including the use of blood-sucking bugs, a submental route, and puncture of the tail tip. For reporting, we have used consistent terminology in describing the methods. The definitions we propose are reported below.

Effects of serial blood sampling were examined in 16 (59%) of studies. Blood sampling interval varied widely across these studies, ranging from a few minutes to 8 week intervals. This finding creates challenges in comparing these studies to examine serial sampling e ffects. Only four studies (15%) used both male and female mice. Of these studies, 50% reported statistical analysis of sex di fference and incorporated findings in data presentation.

In a number of studies there were associated conditions, which ordinarily might be considered as confounders in study interpretation. These included the use of anaesthesia for sampling, and warming methods for obtaining tail vein samples. Only four out of the 14 studies on the retrobulbar route did not use anaesthesia, whilst 3/5 on the sublingual route were performed conscious. Given that these conditions are regularly used for these methods, and may be mandated by ethics committees, they were considered a part of the method itself and were incorporated in data synthesis. However, where these conditions varied across studies, rendering comparison inappropriate, this has been reported.

Sample quality measures were reported in six (22%) of the studies. Sample quality was not a primary focus of this review and consequently it should be noted that we utilised a restricted definition of quality, mainly focusing on sample haemolysis and clotting. Furthermore, our search was restricted to studies which looked at quality in conjunction with animal welfare outcomes. We may therefore have not identified all published studies evaluating quality of samples via the di fferent routes.


**Table 2.** Summary of included studies.

### *Animals* **2020**, *10*, 989


**Table 2.** *Cont.*


*Animals* **2020**, *10*, 989

**Table 2.** *Cont.*



### *Animals* **2020**, *10*, 989

needed for all sample routes.

### *3.2. Note on Terminology*

A variety of sampling routes from the tail were described with little consistency in naming. For the purposes of comparison, we have defined as follows: (1) tail amputation involves the removal of the tip of the tail with a blade, (2) tail incision uses a blade to cut the tail laterally, and (3) tail vein is the targeted collection of blood by insertion of a needle directly into the lateral tail vein.

The submandibular vein, as described in [5], targets the vascular bundle in the caudal part of the jaw. This terminology is commonly used, but perhaps erroneously [39]. The preferred term based on an examination of mouse anatomy is the facial vein [39]. This is the term used in this review, in spite of some usage of 'submandibular' in reviewed studies.

During retrobulbar bleeding, also called retroorbital bleeding, a capillary tube is used to disrupt the retrobulbar venous sinus located behind the eye [8]. Some authors refer to this route as the retroorbital plexus, inferring that a plexus is present in the mouse. This anatomical nomenclature may also be incorrect, although there exists controversy in the claim [39]. We have applied the term 'retrobulbar' in summarising these studies.

The terms saphenous vein, referring to a site near the ankle [8], sublingual for beneath the tongue [4], and jugular, for accessing the vein in the craniocervical region [8] were universally referred to in the studies evaluating them, and are reported as such.

Finally, the submental vein referred to in one study [33] has also been described as a misnomer by an eminent veterinary anatomist, with a suggestion that the site actually targeted was the inferior labial vein [40]. In spite of this, given that only one study reported on this technique, we have continued to refer to this as the submental route.

### *3.3. Animal Welfare Outcomes*

Animal welfare is an umbrella term, defined by the summation of the individual summed experiences of an individual [41]. The nature of a ffective states experienced by the animal, and their relative weighting over time, typically defines whether an animal has, on balance, good or poor welfare [42,43]. A ffective states comprise emotions such as pain, fear and joy [44]. For laboratory animals, the term "cumulative experience" has been coined [45]. Cumulative experience has been defined as: 'the sum of all the events and e ffects, including their quantity, intensity, recovery between and memory thereof, that impact adversely, positively, and by way of amelioration on the welfare of an animal over its lifetime' [45]. Whilst phlebotomy techniques are generally considered to produce a short-lived response in an animal, given that they are conducted frequently, they may have a significant impact on cumulative experience that can be minimised through appropriate evidence-based selection of sampling routes.

The included studies utilised a range of measures for assessing animal a ffect to provide an indication as to potential welfare impact. Utilising a combination of physiological and behavioural methods is generally regarded as superior in the holistic measurement of welfare state [46,47]. A short description of the main outcomes considered in this review follows. These measures have been categorised into (1) measures of physiology, (2) clinical and pathological parameters and (3) behavioural measures and (4) blood sample quality measures.
