Research Note Spatiotemporal mapping of the muscular activity of the gizzard of the chicken (Gallus domesticus) R. G. Lentle,*1 G. Reynolds,* C. de Loubens,† C. Hulls,* P. W. M. Janssen,* and V. Ravindran* *Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11222, Palmerston North, New Zealand; and †UMR 782 Génie et Microbiologie des Procédés Alimentaires, INRA, AgroParisTech, CBAI 78850 Thiverval Grignon, France ABSTRACT We report the results of spatiotemporal mapping of the spontaneous actions of component muscles of the gizzard and associated structures in ex vivo preparations with combined superfusion and vascular perfusion. Ongoing spontaneous contraction of cranial and caudal thin muscles occurred at a frequency of 2.2 ± 0.1 cycles per minute. Contractions of M. tenuis craniodorsalis with mean duration of 2.8 ± 0.2 s commenced ventrally adjacent to the distal limit of the proventriculus and progressed dorsally at 2.02 ± 0.03 mm·s−1 in a concerted front. Near simultaneous contraction of M. tenuis caudoventralis of mean duration of contraction of 4.7 ± 0.7 s commenced dorsally and progressed ventrally at a similar rate (2.1 ± 0.1 mm·s−1) and in a similar manner. Contraction of the
caudoventralis preceded that of craniodorsalis (mean 1.1 ± 0.15 s). Contraction of the 2 tenuis muscles was synchronous with the first component peak of the cyclic increase in lumen pressure and with distension of the crassus musculature. Contraction of the M. crassus caudodorsalis muscle coincided with the second component peak and was followed by distension of the tenuis musculature. The latter commenced before the relaxation of the tenuis muscles. Contractions of the crassus muscle propagated rapidly at right angles to the orientation of the muscle fibers at a faster velocity than that of the tenuis musculature. The durations of the component peaks in lumen pressure indicated that the duration of crassus contraction was similar to that of the tenuis musculature.
Key words: ex vivo gizzard, spatiotemporal mapping, muscular action 2013 Poultry Science 92:483–491 http://dx.doi.org/10.3382/ps.2012-02689
INTRODUCTION
feeds increasing growth performance and causing gizzard size to be reduced (Behnke, 1996). A thorough understanding of the mechanical action of the gizzard is essential for gaining insight into strategies that optimize foraging in avian species as well as into the formulation of feeds to optimize digestive efficiency. Although the principal outcomes of gizzard action (i.e., the selective retention and comminution of larger food particles) are well described (Moore, 1999), little is known of the mechanical means by which this action is achieved. In spite of the ability of the avian gizzard to retain deliberately ingested (Alonso, 1985; Soler and Soler, 1993) large, hard, nonnutrient particles (i.e., gizzard stones) for long periods (Walter and Aitken, 1961; Gionfriddo and Best, 1999), it is known that (as in the human stomach; Meyer et al., 1979; Burton et al., 1995; Pera et al., 2002) retention is not absolute (Amerah et al., 2007), and larger particles of grain and other nutrients do exit to the distal gastrointestinal tract at intervals (Trost, 1981; Amerah et al., 2007). Thus it appears that selective retention of larger food particles depends on a probabilistic sorting action generated during a specific contractile sequence rather
The proximal digestive system of birds is thought to differ from that of mammals as a result of evolutionary adaptations to improve flying efficiency, notably the adaptation of the pyloric stomach (Pernkopf, 1929; Smith et al., 2000) rather than the development of teeth and heavy jaws (Ziswiler and Farner, 1972; Duke, 1997). The pyloric stomach of most aves is adapted to triturate, macerate, and pump the food (Stevens and Hume, 1995), the latter function being modified in some species to selectively retain larger particles (Moore, 1999). The development of the gizzard musculature is greatest in granivorous and herbivorous species in which its action may govern nutrient intake. Hence, the efficiency of feed conversion by poultry is known to be significantly influenced by the action of the gizzard (Gionfriddo and Best, 1999; Svihus, 2011), with the fine grinding of ©2013 Poultry Science Association Inc. Received August 15, 2012. Accepted October 14, 2012. 1 Corresponding author:
[email protected]
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than the sieving action of a net-like morphological feature such as in the omasa of ruminants. The broad sequence in which the various muscular and associated components of the gizzard are activated after stimulation of the vagus nerves is well described (Duke et al., 1972; Dziuk and Duke, 1972; Chaplin and Duke, 1990; Chaplin et al., 1992). The qualitative descriptions of movements in exposed gizzards of anaesthetized birds elicited by intermittent electrical vagal stimulation suggest that contractions commence simultaneously in, and subsequently spread across the thin craniodorsal and thin caudoventral muscle in a concerted clockwise manner, toward the centrally situated thick cranioventral and caudodorsal muscle where they subsequently induce its contraction (Dziuk and Duke, 1972). Thus, contractions appear to cycle simultaneously from dorsal thin to ventral thick muscle at the distal end and from ventral thin to dorsal thick muscle at the proximal end of the gizzard. Later work showed that lesions in the myenteric plexus overlying the caudal and cranial regions of thin muscle disrupted the concerted sequence of thin muscle activation but did not influence the generation of contractions in the thick musculature, which were then out of phase with those of the thin muscles, and hence were thought to be nonneurogenically generated (Chaplin and Duke, 1990). It is important to note that the events in several the above experiments may be complicated by the stimulation of the vagus. For example, vagal activity appears to influence nonadrenergic, noncholinergic-mediated inhibitory junction potentials in the gizzard (Komori et al., 1997), suggesting that stimulation may influence both tonic and phasic activity (Kenney et al., 1990; Ogut et al., 2007). Detailed direct evaluation of gizzard movements in vivo is hindered by the 3-dimensional complexity of the muscular elements and lumen space within the gizzard (Gabella, 1985; Moore, 1998b, 1999) and by practical difficulties in monitoring. Further, the establishment of ex vivo preparations of the gizzard is complicated by the bulk of muscular tissue and the susceptibility of spontaneous muscular contraction to hypoxia (Bennett, 1969). Again the spontaneous action of the gizzard is said to be inhibited following administration of anesthetic agents or cervical dislocation (Mangold, 1906; Rogers, 1916). These problems necessitate the use of Langendorf preparations that are surgically demanding owing to the relative inaccessibility and structural complexity of the celiac artery (Bennett, 1969). Thus, 40 yr after the descriptions of gizzard action by Dziuk and Duke (1972), it remains to be verified whether the contraction sequence that follows vagal stimulation is identical to that which occurs spontaneously. Further, a more quantitative description of the timing, magnitude, and radiation of contraction is needed to gain a greater understanding of the mechanical processing of particulate matter within the gizzard. Although the embryonic anlage of the gizzard has been shown to be the same as that of the pylorus in the uniloculate stomach (Smith
et al., 2000), it is not currently known whether retention of coarse particles results from a “reversal of the normal orad to aborad progression of digesta” (Chaplin and Duke, 1990) by the action of M. tenuis caudodorsalis driving particulate material into the lumen between the crassus musculature or solely from the restrictive action of the crassus musculature similar to that of the pyloric sphincter in the uniloculate stomach (Indireshkumar et al., 2000). The purpose of the current work was to quantify the muscular action of gizzard preparations with combined superfusion and arterial perfusion, notably the relative magnitude and direction of contractile progression and the level of coordination between the component muscles of the gizzard, so as to achieve a greater understanding of its mechanical action.
MATERIALS AND METHODS All the experimental procedures were approved by the Massey University Animal Ethics Committee (MUAEC approval no. 1104), and complied with the New Zealand Code of Practice for the Care and Use of Animals for Scientific Purposes. Twenty-five White Leghorn chickens of age between 8 and 20 wk were maintained on Tegel broiler diet (New Plymouth, New Zealand) grower pelleted feed in caged groups with water available ad libitum until the moment of slaughter. The birds were euthanized by cervical dislocation, and the abdomen was immediately opened by a vertical incision. The esophagus and terminal colon were clamped with hemostats and the gut divided at these points. The celiac artery was clamped and divided at its junction with the abdominal aorta. The entire gastrointestinal tract was then freed by severing the mesenteric attachments to the posterior wall of the abdomen and immediately immersed in ice-cold, oxygenated Earle’s Hepes solution (HBS) before arterial cannulation. Ligatures were placed around the duodenojejunal and pancreaticoduodenal arteries, which were then divided distal to the points of ligation. The portion of the celiac axis that contained the origin of the principal arteries supplying the proventriculus gizzard and proximal duodenum was subsequently isolated and cannulated with a polyethylene cannula (0.97-mm internal and 1.27-mm outside diameter, Dural Plastics, Sydney, Australia). The blood supply of the gut distal to the first 2 cm of duodenum was then isolated from that of the gizzard by ligation and freed from the gizzard. The gizzard was then superfused in a bath of continuously recirculated and oxygenated HBS maintained at 39.0°C; the attached celiac cannula was mounted in a Minipuls 3 (Gilson, Middleton, WI) peristaltic perfusion pump, and the preparation was perfused with oxygenated HBS. A pressure transducer was attached to the perfusion line to measure perfusion pressure, which was maintained above 70 mmHg (range 70 to 100 mmHg) by adjusting the perfusion rate between 10 and 15 mL/min. An air-filled 10-mL syringe was con-
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syringe pump that enabled intralumenal pressure to be increased. The duodenum was clamped and the clamp used as a basis for suspending the organ in the bath (Figure 1A). The gizzard was positioned in the tank with the clamp holding the duodenum uppermost and with the surface of the M. crassus caudodorsalis and plane of the overlying aponeurosis facing the camera in a position next to the window of the tank. Hence, the proventriculus lay to the left and the underlying gizzard lumen ran from left to right with its enlarged superficial and deep dorsoventral surfaces parallel to the surface and its narrower superior and inferior dorsoventral surfaces at right angles to the camera. The point at which M. crassus caudodorsalis merged with M. tenuis craniodorsalis was superficial to the junction of the cavities of the gizzard and proventriculus and to the left, whereas the point of merging of M. crassus caudodorsalis with M. tenuis caudoventralis lay to the right. In 3 preparations, the vagus nerve was isolated and dissected clear from the surface of the proventriculus (Bennett, 1969) with its distal attachment to the gizzard preserved. The cut end of the nerve was subsequently stimulated (see below). The nerve was kept immersed in HBS when not being electrically stimulated.
Pressure Recordings
Figure 1. A. Layout of the apparatus used for the combined superfusion and arterial perfusion of the chicken gizzard ex vivo. Arterial cannula (AC); celiac artery (CA); gizzard (CG); reservoir of Earle’s Hepes buffer solution (HBS); peristaltic pump (PP); pressure dampening device (PD); organ bath (OB); clamp (CL); proventriculus cannula (PV); pressure transducer (PT); bioamplifier (BA); computer (PC); syringe pump (SP); and camera (CM). B. Detail of the gizzard in relation to its position in the apparatus. The tendinous continuation of the crassus musculature is seen above the central portion of the lumen with M. crassus caudodorsalis to the front.
nected via the side arm of a 3-way tap to the perfusion line to reduce pulse pressure due to the pulsatile action of the peristaltic pump to below 10 mmHg (Figure 1A). The contents of the gizzard and associated structures were flushed clear with HBS. In one set of experiments (10 birds), 6.4 g of 5-mm spherical glass beads were then instilled directly into the cavity of the gizzard (Figure 1B). In another (5 birds), a balloon cannula was placed in the distal gizzard lumen. A fluid-filled 5-mm silicone cannula was inserted into the proximal end of the gizzard cavity via the proventriculus and connected to a T piece, on one arm of which was the Statham P23XL (Spectramed, Oxnard, CA) physiological pressure transducer connected to a DC Bioamplifier (Neomedix, Sydney, Australia) and Powerlab 8SP (AD Instruments, Sydney, Australia), and on the other, a
The durations of each of the 2 component peaks in the biphasic pulse in lumen pressure that occurred during each contraction cycle were calculated by projecting the slopes of the initial and final peaks to the baseline values immediately before the commencement and the termination of the cycle.
Spatiotemporal Analysis Sequences of images of the gizzard were acquired and strain rate (SR) maps were derived using a technique based on that described in earlier work (Lentle et al., 2007; Janssen et al., 2009; Hulls et al., 2012). Cross-correlation between successive images was used to find the displacements of evenly spaced reference points within a rectangular region of interest (ROI). The displacement data were differentiated with respect to horizontal and vertical distance to obtain the horizontal and vertical strain rates for all reference points. These strain rates could be superimposed on the original movie to visualize the strength, extent, and timing of contractions. The SR maps were derived along lines of interest (LOI) positioned either horizontally or vertically on the surface of the gizzard. The strain rates could be specified to be either in the direction of the LOI, perpendicular to the LOI, or a combination of the two. This allowed us to differentiate the principal axis in which the muscle contracted in different locations within the different compartments of the gizzard. The duration of muscular contraction (i.e., the period of time that a contraction was maintained at a given
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point in the gizzard muscle) was derived where possible from SR maps for strain rates that were oriented parallel to the LOI for the various muscular sites in the gizzard. Synchrony in the contraction of the component muscles was assessed by examining the phasic relationship of the cyclic contractile activity using fast Fourier transforms of vertical transects of SR maps from each muscle.
Electrical Stimulation of the Vagus Stimulation of the exposed vagus nerve for 90 s was carried out after initial spatiotemporal mapping in 3 preparations. The cut end of the nerve was lifted clear of the HBS solution and stimulated using handheld platinum wire electrodes connected to a Powerlab 8SP (AD Instruments). Stimulus strength was 10 V for a duration of 2 ms and at a frequency of 10 or 30 Hz.
RESULTS Pressure Recordings Regular concerted contractions of the gizzard musculature commenced shortly after the organ had been transferred from the ice bath in which dissection and cannulation were performed, and installed in the organ bath. These contractions increased in amplitude as warming progressed, eventually becoming distinctly biphasic (Figure 2). The peaks in intraluminal pressure varied within and between preparations (Figure 3). The mean rise in intraluminal pressure was similar during each of the 2 peaks (31.8 mmHg in the first and 29.77 in the second). The overall mean durations of the 2 extrapolated peaks were similar (first 19.0 ± 2.3 s and second 17.8 ± 1.7 s). Hence, the second peak appeared to commence around 10 s before the termination of the first (mean overlap between extrapolated peaks 9.12 ± 1.4 s). However, it was noteworthy that in some traces the interval
Figure 2. Pressure tracing showing cyclic contraction during the warming of a gizzard. At the commencement of the trace, there is a gradual increase in pressure, presumably from increasing tone. After 3.5 min, the typical biphasic form of coordinated contraction is seen, which results from alternate contraction of the tenuis and crassus musculature. Note the spontaneous variation in the relative amplitude and interval between of the component peaks of each complex.
Figure 3. Lumen pressure and temporal profiles of contraction of the tenuis and crassus muscles determined at the midpoint of each muscle. M. tenuis craniodorsalis (proximal thin muscle) activity is shown in light gray; trace from M. tenuis caudoventralis (distal thin muscle) shown in black; trace from M. crassus cranioventralis shown in gray. The pressure trace is shown as a dashed line. Note the slight distension of the crassus musculature coincident with concerted contraction of craniodorsal and caudodoventral tenuis muscles and the more marked distension of craniodorsal and caudodoventral tenuis muscles at the conclusion of the contraction of the crassus musculature.
between the first and second peaks varied cyclically, whereas in others it was more constant (Figure 2). The frequencies of the biphasic peaks in pressure were generally identical to the synchronous contractions of the thin and thick muscles of the gizzard seen on the SR maps (Figures 3 and 4). The maximum pressure values recorded during contraction in the ex vivo gizzard preparation were 87.9 mmHg.
Spatiotemporal Mapping Cyclic contractions of the gizzard were sustained when the lumen was distended with glass beads but were not sustained when the gizzard was distended with a simple balloon catheter. For the purposes of clarity in comparison, anatomical relationships will be described with respect to the left lateral view that has been used in prior work (Dziuk and Duke, 1972) rather than the orientation of the gizzard in the organ bath. Gizzard contractions commenced almost simultaneously in the ventral portion of M. tenuis craniodorsalis at its junction with the isthmus of the proventriculus and at the cranial limit of the junction of M. tenuis caudoventralis with M. crassus caudodorsalis, the commencement of the former occurring 1.25 s ahead of the latter on phase comparisons of fast Fourier transforms of vertical transects of spatiotemporal maps. Analyses of strain rate either parallel to or at right angles to an LOI drawn from the aforementioned inferior junction to the upper caudal limit of M. tenuis craniodorsalis at its junction with M. crassus craniodorsalis as it passed from the field of view (AB in Figure 4) showed that the contraction propagated dorsally (ventrally across the anatomically inverted image in Figure 4) across the surface of M. tenuis craniodorsalis at a mean overall rate of 2.0 ± 0.03 mm∙s−1 in the broad direction of the LOI. The mean overall duration of contraction of M. tenuis craniodorsalis was 2.8 ± 0.2
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Figure 4. Spatiotemporal maps of the spontaneous cycle of contraction in the proximal, middle, and distal segments of the gizzard. The photo shows the gizzard with the orientation of the lines of interest (LOI) used to generate the strain rate (SR) maps. The hatched areas show the area of contraction in 2 successive regions (x and y) of M. tenuis craniodorsalis. The graph to the left shows variation in lumen pressure with time. The SR maps to the right show contraction (positive strain rate) as white and dilation (negative strain rate) in black. It should be borne in mind that the maps are of strain rate, and hence, the maximum strain and probably the peak in the resultant pressure will occur at the end of the contraction on the map. The SR map of M. tenuis craniodorsalis is on the left (LOI corresponds to AB on gizzard photo), M. crassus caudodorsalis is in the center (LOI corresponds to CD), and M. tenuis caudoventralis is on the right (LOI corresponds to EF). The SR map along LOI AB shows the maximum of either the parallel or orthogonal strain rates in the direction of the LOI. The SR maps for the CD and EF LOI show only the strain rates orthogonal to (i.e., perpendicular to) the LOI. The contraction labeled z appears to be occurring on the partially obscured side of the organ and indicates an asymmetry between the contractions of the 2 sides. The video sequence with superimposed strain rates for the period corresponding to the SR maps has been included as supplemental material (available online at http://ps.fass.org/).
s, and the frequency of contraction was 2.3 ± 0.1 cycles per minute (cpm). However, spatial resolution of these contractions in the 2 dimensions showed that propagation occurred within 2 successive regions of this muscle. The strain rate in the first region (x in Figure 4), which was close to the site of origin, was oriented principally parallel to the LOI, whereas the strain rate in the second (y), which occupied a larger area of the muscle, was greater at right angles to the LOI (see video in supplemental material S1; available online at http:// ps.fass.org/). At the same time as the contraction of M. tenuis craniodorsalis, a contraction with maximal strain rate at right angles to the direction of propagation propagated inferiorly (dorsally in the inverted image in Figure 4)
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across M. tenuis caudoventralis. The mean overall rate of propagation of contraction determined from strain rate parallel to a line drawn from the aforementioned superior junction to its lower limit at the junction with M. crassus caudodorsalis (EF in Figure 4) was 2.1 ± 0.1 mm∙s−1. The mean overall duration of the muscle contraction was 4.7 ± 0.7 s. The SR maps of this muscle with strain rate at right angles to the LOI (Figure 4) were clearer than those obtained with strain rate parallel to the LOI in showing a similar organization to that in M. tenuis craniodorsalis in which regions within this muscle contracted sequentially, each with slightly differing speed and directions of propagation. The near synchronous contraction of the 2 tenuis muscles caused lumen pressure to rise and then to fall to a point below its initial value as contraction in these muscles abated (Figure 3). The concerted contraction of the tenuis musculature was followed by slight distension of the crassus musculature (CD in Figure 4). Following this and coincident with the upslope of the second component rise in the intralumenal pressure, low amplitude contraction of M. crassus caudodorsalis occurred with a maximal strain rate at right angles to an LOI oriented across the breadth of this muscle (note that the simultaneous contraction of the other thick muscle M. cranioventralis could not be verified because it was obscured from camera view). Hence, the contraction of M. crassus caudodorsalis was largely out of phase with the contractions of the tenuis muscles and was more variable in timing and extent. Low overall strain rates, evidenced by the lesser contrast of contracting areas on the spatiotemporal map (CD in Figure 4), were accompanied by large increments in intralumenal pressure. The generally low amplitude of the strain rate (Figure 3) made it difficult to determine the point at which contraction of the crassus musculature ceased. Judging by the duration of the first and second component peaks in pressure (Figure 3) and the overall width of the (lighter gray) contraction band of the spatiotemporal map (Figure 4), the propagation of contraction of the thick (crassus) musculature across the middle portion of the gizzard lumen occupied a similar period of time in the contraction cycle to that taken for contraction of the thin (tenuis) muscles to propagate across the proximal and distal portions of the gizzard lumen. Toward the end of the period of propagation of a contraction through the crassus musculature, the strain rate rapidly declined in the tenuis musculature, indicating distension of the proximal and distal components of the gizzard lumen. It is noteworthy that the near horizontal band of lighter gray indicating a rapidly propagating increase in strain rate at right angles to the LOI oriented across the lower part of the crassus musculature (Figure 4) was not M shaped as would occur with activation from the proximal and distal junctions with the tenuis musculature. Further, this band was parallel to a dark line that was coincident with contraction of the tenuis musculature and indicated similarly rapid distension of the middle section of the gizzard.
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Figure 5. Variation in lumen pressure and the contraction strain rates in the proximal middle and distal segments of the gizzard following vagal stimulation. The strain rates were all derived from vertical transects of strain rate maps of parallel strain rate. Following commencement of vagal stimulation (indicated by solid bar above time axis), the peaks associated with tenuis contraction (light gray and black lines) are reduced, whereas those of crassus contraction (gray line) are at first maintained. Subsequent loss of coordinated tenuis and crassus activity is evidenced by loss of all peaks associated with tenuis contraction and distension and is coincident with loss of the second phase of rise in intraluminal pressure.
Stimulation of the vagus caused the normal contractile sequence to be interrupted (Figure 5). The contraction of the proximal tenuis musculature briefly persisted, though with decreased amplitude, while cyclic contraction of the crassus musculature (and the subsequent distension of the compartment enclosed by the tenuis muscles) was maintained during this period but was abolished in the subsequent period when coherent cyclic contraction of the tenuis musculature ceased. Hence, vagal stimulation appeared to have an immediate effect on the contraction of tenuis musculature and a more delayed effect on the contraction of the crassus musculature.
DISCUSSION Physiological Findings It is noteworthy that our procedure for removal of the gizzard from the chicken shortly after cervical dislocation, under hypothermic conditions, with subsequent installation in a superfused bath and with concurrent perfusion of the celiac artery, produced several preparations that demonstrated active spontaneous movements once they had warmed to 39°C. This result runs contrary to the observations of early researchers (Mangold, 1906; Stübel, 1911; Rogers, 1916) who stated that spontaneous movements of the stomach were absent both in chickens and hens that had been anaesthetized with chloroform or ether, and in freshly killed hens. Further, it demonstrates unequivocally that, like the mammalian uniloculate stomach (Lentle et al., 2010), the gizzard has an intrinsic rhythm that is modulated by extrinsic innervation. The frequency of spontaneous gizzard contractions determined in this study was 2.2 ± 0.2 cpm. The value
is similar to that of spontaneous spike bursts (2.5 to 4.5 cpm) recorded from the thick gizzard muscle of roosters with implanted electrodes in the fed state (Clench and Mathias, 1996). These workers reported that each spike burst was associated with a slow wave complex, although previous workers had failed to demonstrate spontaneous slow wave activity in the gizzard of the chicken (Roche, 1974) or other avian species (Duke et al., 1972, 1975, 1976). The frequency is also close to that reported in fistulated ambulant turkeys (3.3 cpm) with implanted cannulas or electrodes (Duke et al., 1972) and with implanted strain gauges (3.1 cpm) (Chaplin et al., 1992), but higher than that of spike bursts (0.7 to 1.9 cpm) recorded in chickens with implanted gizzard electrodes (Roche and Ruckebusch, 1978). The occurrence of regular contraction sequences only when the gizzard contained glass beads suggests that local distension by solid particles as well as general lumen distension (such as would be present in the fed state) are required before spontaneous contraction is instituted, presumably mediated by slow waves. Hence, the variation with fed state in the frequency of spike potentials associated with muscular contraction that has been reported previously (Chaplin and Duke, 1990) may to some extent reflect local distension and receptor stimulation. Comparison of the lumen pressure recordings with the spatiotemporal traces (Figures 3 and 4) confirms that the initial rise in intraluminal pressure is coincident with the contraction of the tenuis musculature, whereas contraction of the crassus musculature is coincident with the subsequent peak. The same temporal association of component pressure peaks with contraction of the tenuis and crassus musculature has been reported in anesthetized turkeys following vagal stimulation (Duke et al., 1972). It is noteworthy that
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the considerable distension of the distal portion of the lumen during contraction of the crassus musculature is rapidly reduced on contraction of M. tenuis caudoventralis. Given that in our experiment the duodenal outlet was completely occluded by a clamp, this action is testament to the ability of this muscle to retropulse material through the central portion of the lumen an action that may contribute to regurgitation of food residues in owls, hawks, kingfishers, and shrikes (Stevens and Hume, 1995). The maximal lumen pressure values were in the range of those reported previously. The intraluminal pressure is reported to vary between 4 and 300 mmHg (although pressures greater than 125 mmHg were rarely observed) in the turkey (Duke et al., 1972), and similar values have been reported in chickens (Mangold, 1906). The peak pressures reported during contraction of the thin and thick gizzard musculature in turkeys (122 and 180 mmHg, respectively) reported by Duke et al. (1972) are somewhat higher than those recorded in our ex vivo preparations. Given that the lumen pressure generated by the contraction of the thin and thick muscles was similar in our preparations, the markedly lower strain rate generated during contraction of the thick compared with the thin musculature indicates that the thick muscles operate more isometrically, whereas thin muscles must undergo considerable shortening to achieve the same effect. Beat-to-beat variation in the biphasic pattern of peaks in lumen pressure has been reported previously (Mangold, 1906; Ashcraft, 1930; Henry et al., 1933; Nolf, 1938). One group (Duke et al., 1972) attributed this to variation in the intraluminal position of the cannula. Our spatiotemporal results, which show there is spontaneous variation in the interval between successive peaks in lumen pressure when the cannula was ligated in a fixed position, suggest it is more likely to result from variation in the timing of commencement of the contraction in the thick musculature in relation to that in the thin musculature. Our work, showing that contraction of the tenuis and crassus musculature ceased after a period of vagal stimulation, fits in with previous work indicating that regular gizzard contractions commenced in anaesthetized turkeys only after vagal stimulation had ceased (Dziuk and Duke, 1972) and with work with arterially perfused chick gizzards showing that vagal stimulation induced (inhibitory) hyperpolarization of muscle with rebound firing occurring after cessation of the stimulus (Bennett, 1969). Hence, it appears that vagal stimulation inhibits the action of tenuis and crassus musculature possibly via nonadrenergic noncholinergic inhibitory junction potentials (Komori et al., 1997), perhaps by inducing tonal relaxation. The finding of immediate reduction of the amplitude of contraction of tenuis musculature while contraction of crassus musculature and subsequent tenuis distension continued fits in with the hypothesis of Chaplin et al. (1992) that the action of the crassus musculature is mediated by a mechanism
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that differs from that of the tenuis musculature. Hence, the persistence of the action of crassus musculature for a short period after the commencement of vagal stimulation likely reflects the persistence of stretch-induced inherent rhythmicity (Chaplin and Duke, 1990; Chaplin et al., 1992) mediated by elements of the enteric nervous system (Clench and Mathias, 1996) and interstitial cells of Cajal, which are absent in the crassus musculature (Reynhout and Duke, 1999), that normally operate independent of the CNS (Chaplin and Duke, 1990). Given the effects of vagal stimulation on rhythmicity, it may be expected that the sequence in which the musculature contracts after vagal stimulation would differ from that which occurs spontaneously. However, our quantitative description of the spontaneous sequences of contraction in various compartments of the gizzard is broadly similar to the qualitative descriptions of sequences induced by electrical stimulation of the vagus in anesthetized turkeys (Dziuk and Duke, 1972) as has been predicted by Nolf (1938). The variation of the direction of propagation in successive regions of tenuis musculature is broadly in line with the orientation of muscle fibers therein to form a sac (Moore, 1998a). Hence, the longitudinal axes of regions of contraction appear to follow the lines of longitudinally oriented muscle fibers that curve round from attachments at superior and inferior aspects of the crassus musculature. Thus, the broad directions of propagation of contraction across the thin musculature were similar to those previous reports based solely on direct observation (described as “clockwise from the ventral portion of the thin craniodorsal muscle and from the dorsal part of the thin caudoventral muscle”; Dziuk and Duke, 1972). The slight lag of 1.25 s in activation of M. tenuis caudoventralis following that of M. tenuis craniodorsalis has not been reported previously but fits in with previous work on site-specific lesioning of the myenteric plexus of the gizzard of turkeys, which concluded that activation of the caudoventralis muscle proceeds from the caudodorsalis muscle via the medial commissure (Chaplin and Duke, 1990). The direction of propagation of the thick muscular contraction in M. crassus caudodorsalis is at right angles to that of the longitudinal axis of orientation of the muscle fibers (Gabella, 1985). Hence, there is rapid activation of the whole array. It is noteworthy that such general rapid activation would be unlikely to induce directional flow and thus would cause any contained fluid to be ejected from either the cranial or the caudal limits of the lumen.
Mechanical Action The gizzard musculature may be considered to be organized into 2 functional components. First, the thin and curved (Gabella, 1985) bundles of tenuis musculature, which surround the lumen spaces at the proximal and distal limits of the structure, contract to engender significant shortening of the walls and their sub-
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sequent relaxation with little residual tone leaves the walls highly compliant. Second, the thicker and more linearly disposed crassus muscles (Gabella, 1985), which surround the central lumen space, are less compliant and contract more isometrically. The piecemeal propagation of the high amplitude contraction across the proximally situated M. tenuis craniodorsalis may induce progressive shortening, tending to lift particles from the proventriculus, away from the ventral opening of the duodenum (pylorus) and drive them dorsally and distally into spaces between the 2 pads of thick crassus muscle. Similarly, the piecemeal propagation of the near simultaneous contraction across the distally situated M. tenuis caudoventralis will tend to drive particles proximally into the space between the crassus muscles as well as inferiorly toward the pyloric opening. If the pylorus is autonomous (Keinke et al., 1984; Indireshkumar et al., 2000) and can open when the applied shear is low, then liquid phase along with fine particles may subsequently escape from the caudoventral lumen into the duodenum at this phase of the cycle. To translate the hoop stress generated in the crassus muscles into a compressive force between the opposing koilin plates, the plates must be rigid enough to withstand an end-on compressive load, and hence, the change in muscle length is small. This general low amplitude contraction will generate compression and shear in the narrow lumen space (Moore, 1998b, 1999) between the extensive koilin-coated (Stevens and Hume, 1995) right and left sides (Gabella, 1985) and transmit translational movement to any contained stones and larger interspersed food particles. At the same time, volumetric reduction generated by this action will cause the fluid component and any contained finer particles to be expelled through interparticle voids, and along transverse grooves in the koilin plates, into the more compliant proximal and distal components of the lumen. It is noteworthy that persistent low compliance of the crassus musculature and short-lived period of lumen distension (Figure 4) following the cessation of phasic contraction will act to resist distension of the central lumen and to retain larger particles and gizzard stones. Hence, the ingestion and retention of gizzard stones may increase the capacity of the central lumen to accommodate fluids and food particles displaced by contraction of tenuis muscles, in intervening voids, but that these same voids may allow premature escape of larger particulates into the duodenum. Moreover, the relative magnitude of these effects will vary with the size and load of gizzard stones. In conclusion, this work has confirmed that the mode of action of the gizzard musculature during spontaneous contraction is similar to that described following vagal stimulation (Dziuk and Duke, 1972). Moreover, the quantitative data that have been obtained are sufficient to gain greater insight into the manner in which trituration is achieved in chickens. We plan to further explore the effects of the physical properties of dietary
items and the effects of gizzard stones on triturative efficiency using in silico models based on these data.
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