ABSTRACT
Cetaceans swim via vertical movements of the tail. The tendons located in the caudal peduncle are attached to the caudal vertebrae to generate propulsive oscillations. Arguments have centered on whether the upstrokes and downstrokes of the tail and propulsive flukes are symmetrical or asymmetrical in time. Previous research from kinematics of swimming animals, muscle architecture and histology has supported both conditions. However, the composition and structure of the tendons suggest a potential mechanism to evaluate this disparity. In this study, the tendons of the caudal peduncle of the harbor porpoise (Phocoena phocoena) – specifically, the extensor caudae medialis (ECM) and the extensor caudae lateralis (ECL) from the epaxial muscle, and the medial hypaxialis lumborum (MHL) from the hypaxial muscle – were mechanically tested. Ramp to failure was performed on isolated tendon fascicles. Stress relaxation tests to 3% strain were also performed on fascicles. Polarized light microscopy was used to visualize the fibril crimp as tensile forces were applied to fascicles. Uncrimping of isolated fascicles was visualized at mean strain values between 0.031% and 0.048%. The maximum elastic moduli of fascicles taken to failure were between 1039.5 and 1185.8 MPa. No differences were found in the mechanical performance of the fascicles of the epaxial and hypaxial tendons. The mechanical properties of peduncle fascicles suggest a symmetrical stroke cycle for swimming by the porpoise.
INTRODUCTION
Cetaceans oscillate their tails and flukes dorsoventrally to generate thrust for swimming (Fish and Hui, 1991). The spinal flexibility of the tail occurs mainly among the caudal vertebrae (Long et al., 1997) and predominately around the ball vertebrae at the junction of the caudal peduncle and the anterior insertion of the flukes (Tsai, 1998; Pabst, 2000; Fish et al., 2006). Movement of the caudal region of the cetaceans is actuated by contraction of anterior epaxial (dorsal) and hypaxial (ventral) muscles that insert via tendons on the caudal vertebrae in the peduncle (Pabst, 1990; Adams and Fish, 2019) (Fig. 1). In addition to controlling the flexion of the tail, the tendons aid in the bending of the flukes (Fish et al., 2006; Adams and Fish, 2019). Tendons experience large deflection (strain) owing to increased bending of the peduncle around the ball vertebrae (Fish et al., 2006).
Extensor caudae medialis (ECM; blue) and extensor caudae lateralis (ECL; yellow) tendons with their corresponding muscle attachments. The right and left images show the multiple dividing branches of ECM tendon that attach to the caudal vertebrae of the peduncle. The ECL tendon inserts exclusively on the four to five terminal vertebrae (orange dots) shown in the right image.
Extensor caudae medialis (ECM; blue) and extensor caudae lateralis (ECL; yellow) tendons with their corresponding muscle attachments. The right and left images show the multiple dividing branches of ECM tendon that attach to the caudal vertebrae of the peduncle. The ECL tendon inserts exclusively on the four to five terminal vertebrae (orange dots) shown in the right image.
Previously, researchers have argued on whether there is a difference in the timing and force production between the upstroke and downstroke of the flukes (Parry, 1949; Slijper, 1962; Purves, 1963; Strickler, 1980; Arkowitz and Rommel, 1985; Bello et al., 1985; Fish, 1993; Fish and Rohr, 1999; Fish et al., 2023). It was asserted that the upstroke and downstroke were asymmetrical in time and strength based on cine analysis of slow swimming dolphins and the difference in epaxial and hypaxial muscle mass (Parry, 1949; Slijper, 1962; Purves, 1963; Bello et al., 1985; Videler and Kamermans, 1985). However, more recent video recording of steadily swimming dolphins (Fish, 1993, 1998) and analysis of muscle architecture and histology indicate a symmetrical tail stroke (Arkowitz and Rommel, 1985; Mankovskaya, 1975; Ponganis and Pierce, 1978; Bello et al., 1983, 1985; Suzuki et al., 1983).
The tendons that connect muscle to bone are subject to high stresses from the contraction of the attached muscle, the biomechanics of the bony leverage system, and the loading to counter the resistive forces in order to effect movement. For cetaceans, oscillatory movements of the tail are produced by contraction of the large epaxial and hypaxial muscles with the transmission of muscular force through the tendons inserting on the caudal vertebrae (Long et al., 1997). The resistive forces on the tail are directly related to interaction of the mass of the flukes with the water varying with the speed, acceleration and drag on the body. Although the flukes are only 1% of the mass of a dolphin, the added mass of water affected by the oscillations of the flukes increased the total mass to 7% of body mass (Bennett et al., 1987).
One hypothesis states that the stresses on the tendons of cetaceans could alternate with an asymmetrical stroke, producing an acceleration and deceleration throughout the stroke cycle. A pattern was proposed such that a net hydrodynamic force would be negative during the upstroke, but positive during the downstroke (Bello et al., 1985; Videler and Kamermans, 1985). Videler and Kamermans (1985) reported that the magnitude of thrust production differed between the upstroke and downstroke. Thus, with an asymmetrical stroke, the stress on the tendons during both phases would differ, potentially affecting the structure of the tendons.
Tendons are composed of subunits known as fascicles. Fascicles are mainly comprised of collagen fibers, which allows the individual fascicle and whole tendons to withstand high tensile forces. The collagen fibers are aligned in an anisotropic fashion, primarily aligned with the load direction. Changes in the stiffness of the tendon (i.e. Young's modulus) from lower to higher stiffnesses occurs owing to realignment and sliding of the collagen fibrils (i.e. fascicles) composing the tendon (Biewener and Patek, 2018). The tendons and their component fascicles can be loaded and recoil back to their original shape without stretching (Vogel, 2013). In addition, the tendons can store elastic energy (Vogel, 2013; Thorpe et al., 2015; Wainwright, 2020). The number of fascicles in a tendon varies from tendon to tendon and is based on the source (Kannus, 2000). Indeed, the mechanical properties of tendons were found to vary among different species, different muscles within a body, functionally distinct tendons, as well as with quantity of elastin, injury, aging, exercise and loading (Bennett et al., 1986; LaCroix et al., 2013; Burgio et al., 2022; Eekhoff et al., 2023).
The purpose of this research study was to determine whether the fascicle units of the tendons of the harbor porpoise (Phocoena phocoena) show mechanical properties indicating differences between the upstroke and downstroke. An asymmetrical stroke would generate different loadings on the dorsal and ventral tendons and their components, which would be evident in their mechanical properties. Based on previous kinematic data on swimming dolphins with symmetrical strokes (Fish, 1993, 1998), it was hypothesized that mechanical properties of the fascicles would be the same for the tendons of epaxial and hypaxial propulsive muscle. The mechanical behavior of the tendon fascicles was examined to determine elastic modulus, relaxation properties and the percent strain at which uncrimping occurs.
Stress relaxation testing and ramp-to-failure testing of the component fascicles are standard means of assessing the material properties of biological structures (Rigby et al., 1959; Ker, 1981, 2007; Bennett et al., 1986, 1987; Danto and Woo, 1993; Pollock and Shadwick, 1994; Toedt et al., 1997; Hamilton et al., 1998; Atkinson et al., 1999; Pioletti and Rakotomanana, 2000; Alexander, 2002; Shadwick et al., 2002; Wang, 2006; Maganaris et al., 2008; Duenwald et al., 2009, 2010; Huang et al., 2011; Screen et al., 2013; LaCroix et al., 2013; Legerlotz et al., 2013). This study also utilized polarized light microscopy, a common methodology to visualize collagen fiber crimp in connective tissues (Spiesz et al., 2018; Zuskov et al., 2020; Bell et al., 2022; Ishizaki et al., 2024).
MATERIALS AND METHODS
Specimen collection and dissection procedure
Caudal flukes with approximately 0.18 m of peduncle were collected from 10 stranded, adult harbor porpoises [Phocoena phocoena (Linnaeus 1758)] by the Marine Mammal Center (Sausalito, CA, USA) and the New Jersey Marine Mammal Stranding Center (Brigantine, NJ, USA). The specimens were obtained from the stranding centers under a letter from the National Marine Fisheries Service to F.E.F. The specimens were stored in air-tight bags at −20°C until dissection. Following thawing in a room-temperature bath, the extensor caudae medialis (ECM), extensor caudae lateralis (ECL) and medial hypaxialis lumborum (MHL) tendons were retrieved, placed in mammalian Ringer’s solution, and stored at −20°C until testing. Tendons of approximately 50 mm in length were retrieved from each caudal peduncle: two ECM, two ECL and two MHL from the same caudal peduncle. Multiple fascicles could be isolated from a single tendon as each of the tendons (ECM, ECL and MHL) comprises approximately three to four fascicles. Mechanical tests were performed on individual fascicles owing to the structural nature of the tendons and the issue of slippage between the component fascicles during whole tendon testing (Shadwick et al., 2002). We collected 21 ECM fascicles, 18 ECL fascicles and 24 MHL fascicles obtained from 30 tendons.
Mechanical testing procedure
Mechanical tests were performed with an electromechanical testing system (ADMET eXpert 7603, ADMET, Norwood, MA, USA) equipped with a 4.5 kN capacity load cell; force and grip displacement data were collected at 200 Hz. The fascicles were kept hydrated with mammalian Ringer’s solution between tests. The baseline number of fascicles mechanically tested consisted of 10 ECM, 10 ECL and 10 MHL fascicles. In some cases, fascicles from the same tendon were retested if slipping occurred.
For each sample, a series of stress relaxation and ramp-to-failure tests were performed. Approximately 15 mm of each end of the fascicle was placed into serrated/pyramid grips (GP-1T pneumatic grips, ADMET; Fig. 2). A 1 N preload ensured there was no slack in the fascicle prior to sample loading. Before each test, a 1 N preload was (re)established and gauge length and major/minor diameters were measured with digital calipers (0.01 mm resolution). Engineering stress and strain were both calculated for each test, which allowed for calculation of the elastic modulus, and total relaxation of fascicles.
Mechanical testing setup for the stress relaxation and ramp-to-failure procedures.
Mechanical testing setup for the stress relaxation and ramp-to-failure procedures.
Mechanical testing procedure
Stress relaxation testing
Ramp-to-failure testing
The linear region of each successful test was manually identified by the first author as the area between the non-linear toe region (∼0–2% strain) and the non-linear region immediately preceding sample failure.
Fibril crimp visualization
Polarized light microscopy was used to visualize changes in fascicle collagen fiber crimp in relation to the measured fascicle stress and strain during elongation in a custom test frame (Fig. 3). The ends of the fascicle were tied with surgical silk (Ethicon, size 1) with one end anchored and the other end threaded through a pulley to a strain gauge, which was attached to a load motor. The fascicles were submerged in a well filled with mammalian Ringer's solution to maintain the natural mechanical properties of the fascicles and avoid dehydration (Fig. 3). Each fascicle was marked with two dots using black dye in the view of the camera to track the stretching of the fascicle. Python software (3.8.16) was used to control the load motor, which increased strain in a stepwise fashion. The software was dependent on strain control as it drives the operation of a component of the software Raspberry Pi and the operation of the load motor. The software code was written specifically to determine the stress and strain on the fascicle.
Fibril crimp visualization test stand with fascicle tied on both ends and an enlarged view of the fascicle through a microscope.
Fibril crimp visualization test stand with fascicle tied on both ends and an enlarged view of the fascicle through a microscope.
Fascicles were stretched at a strain rate of 0.005% s−1 until the visible banding patterns (i.e. crimp) disappeared under polarized light microscopy. Six trials were run on each fascicle to ensure the procedure was run smoothly. Data were only considered acceptable if the fascicle was not twisted and the mark on it could be tracked. Strain was measured visually from video recordings by tracking the change in length between two points on the fascicles. The video recordings were made with a Koolertron 1080p Digital Microscope situated above the testing well. The cross-sectional area of the fascicle was assumed to be circular, and the thickness of the fascicle was measured from scaled video recordings. Videos were played back frame-by-frame until the banding patterns were no longer visible through the microscope to determine the strain at which the collagen fibers were uncrimped. The change in length of the fascicle was measured using Tracker software (6.0.10). R software (4.1.2) was used to calculate the corresponding strain as the collagen fibers uncrimped.
Statistical analysis
Statistics were run using Astatsa (one-way ANOVAs), VassarStats (Tukey post hoc t-tests) and GraphPad Prism (normality and equal variance). All data groups were tested for normality and equal variance using Shapiro–Wilk tests and Brown–Forsythe tests, respectively. All data groups passed both tests except the total relaxation of the ECM samples (Shapiro–Wilk test indicated the data were not normally distributed); the data did not pass the Shapiro–Wilk test even following log-transformation, so the extent of relaxation was only compared between the MHL and ECL using an unpaired t-test. An unpaired t-test was also run to compare the dorsal tendons (ECM and ECL) and ventral tendons (MHL). Statistical significance for the t-tests was defined as P≤0.05. One-way ANOVAs were used to determine any statistical differences among the tendon types in terms of elastic moduli and strain at uncrimping. Peak stress values obtained during stress relaxation testing and ramp-to-fail testing of each tendon type were also statistically compared using one-way ANOVAs. A Tukey post hoc test was used for all pairwise comparisons following an ANOVA P-value of less than 0.05. To examine differences between dorsal and ventral tendons, results from the ECM and ECL were pooled and compared with the MHL results using an unpaired t-test. A P-value of less than 0.05 was used to indicate significance for the t-tests. Variation about means was expressed as ±1 s.d.
RESULTS
Stress relaxation testing
The data displayed in Fig. 4 indicate the typical stress relaxation behavior of the fascicles to a 3% strain, whereas Fig. 5 shows the extent of relaxation for all samples. The ECL fascicles (33.6±7.2%) showed greater total relaxation as compared with the ECM (32.7±15.4%) and MHL (28.3±6.9%) (Fig. 5A). In addition to total relaxation, peak stress values were compared (Fig. 4). The MHL (16.3±7.9 MPa) had a higher mean peak stress compared with the ECM (14.4±7.7 MPa) and ECL (14.3±6.3 MPa) (Fig. 5B), although these differences also failed to reach statistical significance as one-way ANOVA found no significant differences in the peak stress between the three tendons (P=0.65). Comparing the peak stress between the dorsal and ventral tendons found no significant difference (P=0.35). An unpaired t-test was performed on the ECL and MHL tendons, indicated a significant difference in their total relaxation (P=0.03).
Example stress relaxation curves for ECM, ECL and MHL fascicles loaded to 3% strain at a 0.05–0.055 s−1 strain rate, and then allowed to relax for 100 s.
Example stress relaxation curves for ECM, ECL and MHL fascicles loaded to 3% strain at a 0.05–0.055 s−1 strain rate, and then allowed to relax for 100 s.
The extent of relaxation and peak stress for all the ECM (n=18), ECL (n=15) and MHL (n=20) samples tested. (A) Stress relaxation; (B) peak stress. The means for each tendon type are indicated by the black horizontal line.
The extent of relaxation and peak stress for all the ECM (n=18), ECL (n=15) and MHL (n=20) samples tested. (A) Stress relaxation; (B) peak stress. The means for each tendon type are indicated by the black horizontal line.
Ramp-to-failure testing
Fig. 6 shows the typical stress–strain behavior of fascicles when pulled at a strain rate of 0.05 s−1 until failure or slippage from the grips. This gives a characteristic J-shaped curve. For all three tendon types, the curves follow a fairly similar path in the toe and linear regions. Following the linear region, microscopic fracture or tearing (i.e. narrowing of the fascicle mid-section as collagen fibers begin to fail) began to occur. The peak stress represents the ultimate tensile strength of the fascicles before failure or slippage. On average, the ECL fascicles (37.5±14 MPa) required more stress to fail or slip compared with the ECM (35.5±17.5 MPa) or MHL (35.9±12.2 MPa) fascicles (Fig. 7B). However, a one-way ANOVA determined that there was no statistical difference (P=0.85) in peak stresses among the fascicles. Toe region extended approximately from 0% to 3% strain and the linear region ranged approximately from 3% to 8% strain for each of the ECM, ECL and MHL fascicles. Fig. 7 displays the elastic moduli for all the fascicles tested. The maximum E measured for each of the fascicle types ranged from 1039.5 MPa (MHL) to 1185.8 MPa (ECM), whereas the mean values of E for the fascicles ranged from 574.3±262.8 MPa (ECM) to 650.9±229.1 MPa (ECL) (Fig. 7A). A one-way ANOVA determined that there was no statistical difference (P=0.40) among fascicle types in terms of elastic modulus. One-way ANOVAs were run between the dorsal and ventral tendons but also failed to detect significant differences in elastic modulus (P=0.45) and peak stress (P=0.89).
Example ramp-to-failure curves for ECM, ECL and MHL fascicles. Fascicle samples were pulled at a ramp strain rate of 0.05 s−1 until failure or slippage occurred.
Example ramp-to-failure curves for ECM, ECL and MHL fascicles. Fascicle samples were pulled at a ramp strain rate of 0.05 s−1 until failure or slippage occurred.
Elastic modulus and peak stress of ramp-to-failure for all the ECM (n=34), ECL (n=29) and MHL (n=30) samples tested. (A) Elastic modulus; (B) peak stress. The means for each tendon type are indicated by the black horizontal line.
Elastic modulus and peak stress of ramp-to-failure for all the ECM (n=34), ECL (n=29) and MHL (n=30) samples tested. (A) Elastic modulus; (B) peak stress. The means for each tendon type are indicated by the black horizontal line.
Fibril crimp visualization
The images displayed in Fig. 8 are representative of collagen fibers responding to applied loads. The isolated collagen fiber responded to applied forces causing the fiber to straighten out (1) compared with the collagen fiber resting in a neutral state with no forces applied (2) (Fig. 8A). When there are no forces applied to the fascicle, striations are visible under polarized light microscopy, indicating crimping of the collagen fibers (Fig. 8B). With an applied load, the striations on the fascicle disappear, indicating uncrimping of the fibers (Fig. 8C). Fig. 9 shows the strain at which uncrimping occurred for acceptable trails for each tendon type tested (ECM, n=3; ECL, n=3; MHL, n=4). Uncrimping of the ECM, ECL and MHL fascicles determined via polarized light microscopy occurred at higher strain values (4.8%, 3.2%, and 3.1%, respectively) than the ∼2% end of the toe region noted during ramp-to-failure testing. A one-way ANOVA for the strain at uncrimping found no significant difference (P=0.35) among the tendon types. One-way ANOVAs were run between the dorsal and ventral tendons but also failed to detect significant differences in the strain at uncrimping (P=0.40).
Microscopic images of fascicles. (A) The crimp in the collagen fibers of a tendon from the peduncle. The degree of crimp of an unloaded fiber (2) is shown relative to the fiber when fully loaded and straightened (1). (B,C) Images of fascicles with no force applied (B) and force applied (C). The striations indicate that there is crimping of the collagen fibers, whereas no striations indicate stretched and straightened collagen fibers. The lowest strain level where striations were no longer visible was recorded as the strain at uncrimping.
Microscopic images of fascicles. (A) The crimp in the collagen fibers of a tendon from the peduncle. The degree of crimp of an unloaded fiber (2) is shown relative to the fiber when fully loaded and straightened (1). (B,C) Images of fascicles with no force applied (B) and force applied (C). The striations indicate that there is crimping of the collagen fibers, whereas no striations indicate stretched and straightened collagen fibers. The lowest strain level where striations were no longer visible was recorded as the strain at uncrimping.
Strain at uncrimping for fibers that were not twisted and could be tracked for the ECM (n=3), ECL (n=3) and MHL (n=4) samples tested. The means for each tendon type are indicated by the black horizontal line.
Strain at uncrimping for fibers that were not twisted and could be tracked for the ECM (n=3), ECL (n=3) and MHL (n=4) samples tested. The means for each tendon type are indicated by the black horizontal line.
DISCUSSION
Upstroke versus downstroke
The controversy around the assertion that there was an asymmetry between the upstroke and downstroke of the flukes of a swimming dolphin was initiated by Parry (1949). The idea was predicated by differences in the epaxial and hypaxial muscle masses (Parry, 1949; Purves, 1963; Fish and Hui, 1991). This idea was initially confirmed by differences in stroke duration from a cine film of the posterior view of a swimming dolphin (Parry, 1949). A count of the frames of the film for one stroke showed that the upstroke was longer than the downstroke. However, the film was taken of the dolphin during parturition. The flukes of the dolphin neonate could be observed extending from the mother. Such a condition would have obviously affected the normal swimming pattern as an abnormal swimming condition. Unfortunately, subsequent reproductions of the swimming sequence excluded the tail flukes of the neonate, perpetuating the assertion (Slijper, 1962; Hertel, 1966).
It was considered that the upstroke generated the thrust whereas the downstroke was passive and functioned as a recovery stroke (Purves, 1963, 1969; Pilleri et al., 1976; Fish and Hui, 1991). In contrast, an alternate view was proposed that thrust can be produced mainly in the downstroke in steady swimming or when bursting (Bello et al., 1985; Videler and Kamermans, 1985; Tanaka et al., 2019; Han et al., 2020; Guo et al., 2023; Ten et al., 2025). Indeed, the symmetrical sinusoidal pathway of the flukes, phase times and measurable angles of attack during each phase of the stroke cycle indicate thrust production during both phases (Lang and Daybell, 1963; Goforth, 1990; Fish, 1993, 1998).
Despite the disparity between the epaxial and hypaxial muscle masses, each muscle mass could generate equivalent propulsive forces and dorsoventral bending movements (Arkowitz and Rommel, 1985). Other aspects of the musculoskeletal design also affect the magnitude of the propulsive forces. There may be a significant mechanical advantage from tendons inserted on the long lever arms of the spinous and transverse processes and chevron bones of the tail vertebrae (Slijper, 1962; Smith et al., 1976; Pabst, 1990). These lever arms amplify the forces compared with those resulting from tendon insertions closer to the vertebral bodies. Pabst (1987) has suggested that insertion of the epaxial muscles on the connective tissue sheath under the blubber allows for a portion of the force to be developed at the caudal vertebrae. The results of the mechanical tests on the fascicles of the dorsal and ventral tendons in the present study show no significant differences, which may be indicative of a symmetrical stroke.
Slippage and potential error
Accurate measurement of the mechanical properties of soft biological tissues is inherently challenging. The mechanical properties of isolated tendons are particularly difficult to measure owing to their non-homogeneous structure (Shadwick et al., 2002; Ker, 2007). To avoid many of the issues related to mechanical testing of whole tendons, this study performed mechanical tests on isolated fascicles. Despite addressing the issue of relative movement of component fascicles and tendon sheath, slippage at the grips was still an issue. During the testing period, some of the fascicles would slip out of the grips. This problem occurred for both testing protocols (stress relaxation and ramp-to-failure), but was a particular challenge at the higher stresses of the ramp-to-failure tests. When slippage occurred at the grips during any of the tests, the fascicles were repositioned and retested until no slippage was evident only if the main body of the tendon remained intact.
Slippage between the connective tissue sheath and fascicles in the grips of the mechanical test apparatus, sliding of the component fascicles within the excised tendon (i.e. cut ends), and crimping of the tendon within the grips (Alexander, 2002; Shadwick et al., 2002; Ker, 2007) all contribute to testing challenges. As slippage at the grips can markedly reduce the maximum stress measured for an entire tendon, it is the primary concern (Ker, 2007). Shadwick et al. (2002) noted that slippage of tuna tendons from the grips occurred while performing mechanical testing, which required the removal of those tests from further analysis. Similarly, when clamping mammalian tendons, Ker et al. (2000) ran into issues associated with the independent fibrous units and hydration of the tendons. He noted that the fibrous units of the tendons needed to be held together and kept hydrated to avoid premature failure during mechanical testing. Alexander (2002) also noted issues with tendon testing owing to the concentrated stress at the grip and tendon interface, which led to premature failure.
Stress relaxation testing
Stress relaxation testing of fascicles was used to examine the viscoelastic behavior of porpoise tendons. In general, a stress relaxation test of soft tissues begins with a rapid increase in stress during the ramping phase followed by a quick relaxation, then a slow relaxation as the test reaches the end (Pioletti and Rakotomanana, 2000) (Fig. 4). The behavior of the ECM, ECL and MHL fascicles followed similar relaxation patterns (rapid ramp-up followed by quick to moderate to slow relaxation), but at overall different stress levels (Pioletti and Rakotomanana, 2000). The ECL fascicles demonstrated an overall relaxation of 33.6±7.2% (Eqn 1). However, the ECM and the MHL fascicles demonstrated a lower average overall relaxation of 32.7±15.4% and 28.3±6.9%, respectively (Fig. 5A). Peak stress values were also examined among the tendon types, but no statistical difference was found (P=0.65; Fig. 5B). There was no significant difference in peak stress values between the dorsal and ventral tendons (P=0.35), whereas significance was determined between the ECL and MHL tendons when comparing their total relaxation using an unpaired t-test (P=0.03).
Relaxation properties of harbor porpoise tendons provide an understanding of the response to different loading conditions. The data show that each tendon type will relax approximately one-third of its peak stress. Given the statistical analysis result of the peak stress values and percent of relaxation, the ECM, ECL and MHL fascicles exhibit similar relaxation and viscoelastic properties.
Stress relaxation testing determined the behavior of fascicles when subjected to a constant strain to understand the viscoelastic properties, which was seen as an exponential decay in stress (Pioletti and Rakotomanana, 2000) (Fig. 4). Various factors can influence the results of stress relaxation tests, such as the sample geometry, sample hydration, sample storage and gripping conditions (Screen et al., 2013). The relaxation behavior of soft tissues, such as tendons, relate to the structural makeup and the stiffness/mechanical integrity of the collagen fibers (Pioletti and Rakotomanana, 2000). Stress relaxation tests are used to characterize collagen fiber recruitment and determine the response of the tendons under constant strain (Legerlotz et al., 2013).
Screen et al. (2013) performed stress relaxation tests on porcine fascicle subunits of positional tendons and energy-storing tendons at low strains. Their study concluded that both positional tendons and energy-storing tendons result in collagen fibers sliding past each other, but the degree of reorganization varied. Positional tendons and energy-storing tendons adapt to their location and stress environment, where their response to similar loads differs. Positional tendons rely more on fibril relaxation, which can lead to earlier tendon failure as they are unable to withstand large loads, whereas energy-storing tendons rely more on fibril reorganization (Screen et al., 2013). Stress relaxation results of soft tissues follow a similar trend and behavior, but the viscoelasticity of the soft tissue differs (Pioletti and Rakotomanana, 2000).
Ramp-to-failure testing
Ramp-to-failure testing was performed to determine the tensile properties, including elastic modulus (E) for fascicles from the ECM, ECL and MHL. Statistical comparisons of E were based on the maximum and the mean values within a tendon type. Each analyzed test could have concluded in one of three ways: slippage from the grips, semi-failure (collagen fibers/component fascicles sliding past each other) or complete failure. Among the ECM, ECL and MHL tendons, the mean E-values of the fascicles were 574.3±262.8, 647.9±271.4 and 650.9±229.1 MPa, respectively (Fig. 7A). The maximum E-values were more aligned with previous work. The ECM fascicle yielded a maximum E of 1185.8 MPa (1.19 GPa) whereas the ECL and MHL fascicles exhibited lower E-values of 1090.3 MPa (1.09 GPa) and 1039.5 MPa (1.04 GPa), respectively. However, these values are still lower than the mean values reported by Bennett et al. (1987). Examining the peak stress of each tendon type tested demonstrates the ultimate tensile strength of the fascicles immediately before failure. A one-way ANOVA determined no statistical difference among tendon types for the mean E-values (P=0.40). The ultimate tensile strength or peak stress was statistically compared using a one-way ANOVA, which resulted in no statistical difference being found among the tendon types (P=0.85; Fig. 7B). When comparing the dorsal and ventral tendons, no significant difference was detected for elastic modulus (P=0.45) and peak stress (P=0.89).
Mechanical testing by Bennett et al. (1987) compared with the present study differs based on the types of samples, the locations of samples in the porpoise, and gripping techniques. Each of these differences poses potential variability in the E-values obtained in both studies. Statistical comparison determined there was no significant difference among the ECM, ECL and MHL fascicles, which indicates that they respond similarly to tensile loading before failure.
Tendons and their component fascicles exhibit significant stiffness under tensile loading, which can be quantified by the elastic modulus (E). E can be calculated by analysis of the slope of the linear region of a stress–strain curve (LaCroix et al., 2013). LaCroix et al. (2013) noted that studying E is also beneficial to understanding how tendon damage and repair occurs in vivo and the effects of aging. For tendons from various adult vertebrates, E-values can range widely from 800 up to 2000 MPa, but the average falls around 1500 MPa (Pollock and Shadwick, 1994; Ker, 2007). Testing rates used in previous studies include 0.05 s−1 (Bennett et al., 1986), 0.05 to 0.1 min−1 (Shadwick et al., 2002) and 0.10 min−1 (Ker, 1981). The strain rate, 0.05 s−1, was chosen based on the similarity of the species used for the tendon experiments. Ker (2007) noted that the wide range of E-values was due to the high variability when mechanically testing tendons of different sizes and species.
Bennett et al. (1987) highlighted the tensile properties for tendons of the sacrocaudlis dorsalis and ventralis in the tail of the harbor porpoise (P. phocoena) and the white-sided dolphin (Lagenorhynchus acutus), and E was reported as 1.43 and 1.53 GPa, respectively. In a previous study, Bennett et al. (1986) noted that the E of dolphin tail tendons ranged from 1.22 to 2 GPa, with the large range attributed to difficulty clamping the tendons during mechanical testing. Shadwick et al. (2002) also performed mechanical tensile testing on tendons from the caudal peduncle of tuna with E-values ranging from 1.19 to 1.43 GPa.
Terrestrial animal tendons function similarly to the tendons of aquatic animals as suggested by Pollock and Shadwick (1994) as they performed mechanical testing to determine E for a range of vertebrates (Ker, 2007). Therefore, researchers have grouped terrestrial and aquatic animals together when comparing E-values. Similarly, Alexander (2002) performed mechanical testing on various vertebrate tendons and determined E was approximately 1.24 GPa. Ker (1981) determined that tendons in the legs of sheep have an average elastic modulus of 1.65 GPa with great variation.
Fibril crimp visualization
When the tendons are not subjected to any force, the collagen fibers display a wavy banding pattern (i.e. they are crimped). As axial tensile force increases, the collagen fibers straighten out, becoming uncrimped. Once uncrimped, the application of greater force is resisted by a linear increase in the tensile strength of the tendon. With the relaxation of the tensile force, the fibers rebound, and nearly all the energy is recycled with minimal loss (Vogel, 2013; Thorpe et al., 2015; Thorpe and Screen, 2016; Wainwright, 2020).
Under a polarized light, the transition point defining the extent of the toe region between crimped and uncrimped collagen fibers within a fascicle was observed. Percent strain values were recorded to determine the differences between the tendon types under increasing tension. The ECL and MHL fascicles had a mean strain value of 3.2% and 3.1% where the tendon experienced uncrimping, respectively. Conversely, the ECM fascicles experienced fibril uncrimping at mean values of 4.8%. The differences in strain values that define the transition from the toe region to the linear region could be due to varying waviness structures of the collagen fibers among the tendon types. Wang (2006) noted that the angle of the collagen fiber crimp determined the tendon's ability to resist deformation or failure (i.e. tendons with smaller crimp angles will fail before those with larger crimp angles). When statistically comparing the dorsal and ventral tendons, no significance was detected (P=0.40).
The transition between the toe region and the linear region seen in the fibril crimp visualization occurred at higher strain values compared with the ramp-to-fail testing and prior studies. The end of the toe region in ramp-to-fail testing occurred at about 2–2.5% strain whereas the fibril crimp visualization test showed strain values at fiber uncrimping of 3.11–4.8% (Fig. 9). The ramp-to-failure test was set to examine resistance to deformation and the full mechanical integrity by pulling the fascicle to the plastic region to see failure. The fibril crimp visualization test pulled the fascicles enough to visualize the transition from the toe region to the linear region to see the crimping and uncrimping of the collagen fibers.
The expected data and outcomes between the two tests varied, which could potentially allude to the difference of strain values. As collagen fibers are stretched under an increasing load, the waviness of the unloaded crimped fibers straightens to an uncrimped arrangement that typically occurs between 1 and 2% strain (Danto and Woo, 1993; Pollock and Shadwick, 1994; Wang, 2006; Duenwald et al., 2009). This gradual straightening of the collagen fibers is represented as the toe region in a stress–strain curve, which is followed by subsequent loading of the fiber itself in the linear region of the stress–strain curve, where tendon stiffness is determined by the effect increasing force has on the molecular bonds of a material (Pollock and Shadwick, 1994; Wang, 2006; Biewener and Patek, 2018). As the tendons are stretched in the linear region, the interactions between the collagen and the proteoglycans begin to fracture, but these interactions can store elastic energy (Duenwald et al., 2009). With increasing loads, the molecular bonds will begin to break once the ultimate tensile strength is met, indicating the material cannot withstand any more loading. Tendons experience viscoelastic behaviors based on the abundance of collagen, water, and interactions between the collagenous and non-collagenous proteins (proteoglycans) found in tendons. This composition allows tendons to be more deformable at lower strain rates (Wang, 2006). Thus, they are able to absorb more energy rather than transfer loads (Wang, 2006).
The toe region corresponds to applied forces that do not inflict damage to the tendon as the collagen fibers are stretched (Maganaris et al., 2008). Maganaris et al., 2008 noted that when tendons are stretched, they do not behave perfectly elastically even before leaving the toe region. The toe region is known as the pre-damage stage where the tendon will not undergo physical changes as increasing forces are applied. The stress–strain curve leaves the toe region and enters the linear region once the collagen fibers are straightened out (Rigby et al., 1959). Once the collagen fibers are straightened out, they begin to behave like a stiff spring until maximum stress is reached, causing the fascicles to break and/or begin to slide past each other (Rigby et al., 1959). As forces are increasingly applied to tendons, thus stretching them, they begin to alter their mechanical properties, composition and structure to withstand the forces (Wang, 2006).
Conclusions
The mechanical properties of tendons are critical to the transmission of muscular forces to the skeleton, enabling efficient swimming of cetaceans such as the harbor porpoise. The tendons of the caudal peduncle actuate the oscillatory motions of the tail and flukes to generate thrust (Adams and Fish, 2019). The present study highlights the biological and biomechanical properties of tendon fascicles when subjected to a series of tensile tests. The fascicles from each tendon type can experience large amounts of force before failure. All tests performed resulted in no statistical differences found among the dorsal and ventral tendon types, which indicate that the fascicles share comparable mechanical properties. The controversy into whether the propulsive strokes of cetaceans are symmetrical or asymmetrical may stem from the fact that cetaceans are capable of controlling their swimming kinematics as they choose. Examination of the biological and biomechanical properties of the caudal peduncle tendons of the harbor porpoise provides a greater understanding of the mechanism of force transmission.
Acknowledgements
We are grateful to Kavish V. Saini for assistance during mechanical testing, Caitlyn Swiston for preparation of specimens, Miruna Vasilescu for assistance with dissections and mechanical testing, and Anthony Nicastro and Matthew Waite for recommendations to the manuscript. We are also appreciative of the Marine Mammal Center (Sausalito, CA, USA) and New Jersey Marine Mammal Stranding Center (Brigantine, NJ, USA) for collection of specimens.
Footnotes
Author contributions
Conceptualization: F.E.F., A.R.C., D.S.A.; Data curation: A.R.C.; Formal analysis: A.R.C., J.K.P., N.L.R., M.M., D.S.A.; Funding acquisition: F.E.F.; Investigation: A.R.C., N.L.R., J.K.P., M.V.R.; Methodology: N.L.R., M.V.R., D.S.A.; Project administration: F.E.F.; Resources: N.L.R., F.E.F., M.V.R.; Writing – original draft: F.E.F., A.R.C.; Writing – review & editing: J.K.P., N.L.R., M.V.R., D.S.A.
Funding
This work was supported by grants from the Multidisciplinary University Research Initiative of the Office of Naval Research (N00014110533 and N00014-21-1-2210). Open Access funding provided by West Chester University. Deposited in PMC for immediate release.
Data availability
Data are available from the West Chester University institutional data repository at http://digitalcommons.wcupa.edu/bio_data/10.
References
Competing interests
The authors declare no competing or financial interests.