Fascicle length is a key anatomical variable used to estimate functional adaptations of muscles. Although a recent study concluded that the fascicles measured on different specimens using in situ measurement and chemical dissection yield results that are not significantly different, these two methods have not been systematically tested within individual specimens. To that end, we compare the two measurement techniques on contralateral sides of (1) carnivoran masticatory muscles and (2) rat leg muscles to evaluate their statistical comparability. Our results show that the two methods result in statistically significant fascicle length differences, with in situ measurements being significantly greater in architecturally simpler muscles across a weighted average of masticatory muscles and among individual muscles. This trend holds for the rat soleus muscles but is reversed for the architecturally more complex gastrocnemius, with chemical dissection resulting in greater fascicle lengths. This suggests that pennation may influence methodological compatibility, with more complex connective tissue potentially obscuring fascicle ends and architecturally simpler muscles being prone to stretching when measured in situ. Given the importance of this anatomical measure, these results have potentially wide implications. Although small muscles with complex connective tissue yield shorter fascicle lengths when measured in situ, in general, in situ measurements are significantly longer by a factor of 1.3. Comparing fascicles measured using these traditional methods and through imaging techniques like DiceCT and micro-MRI, which allow evaluation of intact specimens, may provide further insight into the true anatomical reliability of these methods that unambiguously produce significantly different measurements.

To test these hypotheses, we use each of the two methods (described in detail below) to measure fascicle length on contralateral sides of the same specimens in two different anatomical systems (masticatory and crural) and lineages (carnivorans and rodents) of mammals. 2.1 | Sample Each of the masticatory muscles was dissected bilaterally. These were taken from 23 wild, adult individuals (as determined by affirmation of dental maturity) of seven species of North American carnivorans (Table 1). Additionally, both hindlimbs of 40 adult (equal sex ratio) Wistar rats (Rattus norvegicus) were removed for dissection of the soleus and medial head of the gastrocnemius. Although Leonard et al. (2022) showed that formalin fixation does not have an effect on fascicle length, we confirmed this by structuring our rodent sample as follows: half were dissected bilaterally and processed immediately, whereas hindlimbs of the other 20 rats were skinned and fixed in formalin prior to bilateral sharp dissection (see details below). No animal was killed for this research; all carnivore specimens were byproducts of the fur trade, and all rats were discarded control specimens from research not pertaining to this study. North Carolina State University does not require IACUC approval for use of specimens as cadavers.

2.2 | Carnivoran masticatory myology (including abbreviations)

The masticatory muscles of carnivorans, like those of most mammals, are characterized by multi-layered architecture and variable fascicle orientation (Hartstone-Rose et al., 2012, 2019, 2022). The spatial complexity of the fascicle orientation has only recently been documented in carnivorans in three-dimensional space (Davis et al., 2026); however, these methods follow those that have revealed fascicle orientation spatial complexity in primates (Dickinson et al., 2020, 2024). Those studies have shown that the directionality and depth of fascicle layers can vary considerably not only intraspecifically and between individual muscles but also within individual muscles. Following Hartstone-Rose et al. (2012) we separated the masticatory muscles into the following divisions: superficial, deep, and zygomatic temporalis (ST, DT, and ZT, respectively); superficial, deep, and zygomatic masseter, with the zygomatic portion being subdivided (following Brassard et al., 2020) into anterior and posterior portions (SM, DM, ZMA, and ZMP, respectively); medial and lateral pterygoids (MP and LP, respectively) and digastric (Dig), which in carnivorans is one body made up of the inseparable anterior and posterior bellies found in most mammals. For each specimen, the fascicles of the muscles of one side were measured using the in situ method, wherein muscles were sectioned along their lengths, and 3–5 representative fascicles were measured using calipers. Fascicles of the contralateral side were measured following the chemical dissection protocol.

All carnivore specimens were frozen fresh for storage and then thawed before each masticatory muscle was removed following standard sharp dissection protocol; immediately after its removal, morphometric data were collected including mass to the nearest 0.01 g, density, and length following Boettcher et al. (2019). Because all muscles could not be processed simultaneously, excised muscles from all but five carnivore specimens were fixed in 10% buffered formaldehyde (formalin) after dissection, but prior to chemical processing. Although fixing muscles after removal from bony attachments has been shown to cause moderate volumetric fascicle length shrinkage in some studies (e.g., Cutts, 1988), other studies have shown that although ethanol storage substantially affects muscle architecture muscles are not substantially influenced by freezing or formalin fixation (Leonard et al., 2022). All gross morphometrics and in situ fascicle measurements of the carnivoran masticatory muscles were taken on muscles unaltered by formalin fixation.

2.3 | Fixation

In order to confirm the effects of fixation on fascicle length (e.g., to determine the compatibility of the fascicle lengths derived from the fixed and unfixed carnivoran masticatory muscles), half of the rodent specimens were fixed prior to fascicle measurement. Following the method described in Hinks and Power (2024) for the fixation of rat hindlimbs prior to dissection, rat hindlimbs were skinned and submerged in 10% buffered formaldehyde (formalin) with their ankles and knees pinned at 90-degrees for 2 weeks. After the fixation period, specimens were then dissected following the same procedures used for the fresh specimens.

2.4 | Rodent crural myology

To observe whether the two fascicle measurement methods vary in comparability relative to muscle architecture complexity, the medial head of gastrocnemius and the soleus (Figure 1a, left and right, respectively) were bilaterally dissected from our rodent sample. These muscles are both relatively simple compared to most of the masticatory muscles and functionally distinct hindlimb muscles in rats and were selected due to their well-defined fascicle orientation and consistent anatomical organization. This contrasts markedly with the layered and variable structure of masticatory muscles. While both muscles predominantly extend the ankle, the medial gastrocnemius is characterized by shorter fascicles arranged in a more pennate architecture at an angle to the tendon, and the soleus is comprised of longer fascicles running a longer span of the length of the muscle with a much lower degree of pennation (Figure 1b, right). These differences in architecture and function between the two muscles provide an additional opportunity to assess how dissection method affects fascicle length measurement across two simple fundamental muscle types.

2.5 | Rat leg dissection

The soleus and medial head of gastrocnemius were removed from the left and right sides of each rat following sharp dissection procedures and then morphometric data were collected including mass to the nearest 0.01 g and muscle length in mm. of each specimen. The fascicles of one randomly assigned side were measured using the in situ method, wherein the muscle was sectioned and three fascicles of various depths were sampled. Conversely, muscles of the other side were measured following chemical dissection.

2.6 | Chemical dissection

Following sharp dissection and morphometric measurement, both the rat leg and carnivoran masticatory muscles assigned to the chemical dissection protocol were placed in petri or larger glass dishes and submerged in 20%–30% nitric acid, as described by Martens et al. (2024), to dissolve connective tissue and allow for manual separation of fascicles. Although sulfuric acid has been used successfully in the past, it generally requires heat (e.g., to 70C) to be effective and while nitric acid works well at room temperature. Using an acid that does not require an oven or incubator (which are at risk of corrosion) allows for more economical fume hood space use. Having extensive experience with both acids, it appears that they produce equivalently disarticulated fascicles. Time spent processing in acid varies for muscles based on acid choice and concentration, size and fixation with heated sulfuric being generally faster than room temperature nitric at the concentrations used. Smaller, unfixed muscles using 20%–30% nitric acid took between 2 and 5 days, whereas larger, formalin fixed muscles in the current study took 1–2 weeks to fully process. With regular monitoring and topping off of fluids when necessary, there does not seem to be any danger of drying. Once enough connective tissue had been dissolved so that fascicle bundles could be teased apart with minimal force, the nitric acid was removed and replaced with 50% glycerol. The fascicles of each individual muscle were then gently teased apart, and a subsample of ~40 intact fascicles (fewer in smaller muscles that contain fewer fascicles overall) from throughout the muscle were distributed evenly across the dish (Figure 1c). Each dish was then photographed with identifying information and a scale bar. Fascicles were then measured using ImageJ (Version 1.54). In this software, a straight line is stretched across the scale bar to “set the scale” and then the ratio of this known measurement to the pixel size is automatically used for all subsequent measurements of fascicles drawn along their curve (Figure 1, green lines). This scale is reset for each photograph.

Measurements were only taken of fascicles that could be fully visualized and deemed entirely intact. Broken fascicles often exhibit frayed ends and feathering of muscle fibers, so fascicles with these characteristics are easily excluded from measurement or removed from the dishes prior to photographing (Figure 2). Additionally, fascicles were only measured if the entire length was unobstructed. Overlapping or poorly visualized fascicles were not measured.

Prior to data collection, observers are trained on collections of photographs of previously established muscle fascicle lengths to ensure observer reliability. As in many studies using this method (e.g., Martens et al., 2024) measurements were taken on approximately 20–40 representative fascicles per rat leg muscle and 30–60 fascicles per carnivoran masticatory muscle, depending on the overall size of the muscle, to produce a single average fascicle length. Smaller muscles such as LP had fewer fascicles compared to others such as divisions of masseter and temporalis. Masticatory muscles were all measured by a single author, ESB. This was repeated by 1–2 additional experienced individuals on the same set of fascicles from the same dish photographs to ensure repeatability. Interrater reliability tests were conducted, and all achieved greater than 95% concordance. Rat leg fascicles were all measured by authors RAB and KMC. The average fascicle lengths for each muscle measured by each observer were averaged to produce a single value for each respective muscle.

2.7 | In situ sectioning

In situ sectioning measurements were taken following the procedures described by Taylor et al. (2015). While the majority of us have extensively and nearly exclusively used the chemical dissection approach (Martens et al., 2023, 2024), coauthor Herrel has used both procedures in many studies (e.g., in Ginot et al., 2018; Brassard et al., 2021, 2022, 2025; Herrel et al., 2008, 2024, 2025). Therefore, he collected all the in situ measurements for the more complex masticatory muscles. One of us (RAB) collected the in situ measurements for the simpler leg muscles. To do this, first, muscles were removed from their osteological origins and insertions. Next, an incision was made along the belly of the muscle following the orientation of the superficial-most fibers. Measurements were then taken of the exposed fibers using digital calipers, perpendicular to this cut, from the superficial muscle surface to their deep attachment (Figure 1b).

2.8 | Statistical analysis

All statistical analyses were conducted using a pooled t-test for each muscle to determine the comparability of methodological approach and impacts of fixation. For the carnivoran masticatory muscle dataset, weighted average fascicle length was calculated by taking the sum of each muscle's average fascicle length value multiplied by its mass obtained from both in situ measurements and chemical dissections and then dividing each sum by the total mass of each muscle set, allowing comparisons across a single representative masticatory muscle value (Figure 3). All analyses were performed using JMP Student Edition 19, with an a priori significance level of 0.05.

Please see the paper for references.