Flight toward Sustainability in Poultry Nutrition with Black Soldier Fly Larvae.

Black soldier fly larvae (BSFL), Hermetia illucens (L.) (Diptera: Stratiomyidae), have emerged as a promising feed ingredient in broiler chicken diets, known for their high protein content, nutritional richness, and environmental sustainability. This review examines the effects of integrating BSFL into broiler feeds, focusing on aspects such as growth performance, nutrient digestibility, physiological responses, and immune health. The ability of BSFL to transform waste into valuable biomass rich in proteins and lipids underscores their efficiency and ecological benefits. Protein levels in BSFL can range from 32% to 53%, varying with growth stage and diet, offering a robust source of amino acids essential for muscle development and growth in broilers. While the chitin in BSFL poses questions regarding digestibility, the overall impact on nutrient utilization is generally favorable. The inclusion of BSFL in diets has been shown to enhance growth rates, feed efficiency, and carcass quality in broilers, with the larvae's balanced amino acid profile being particularly advantageous for muscle development. BSFL may also support gut health and immunity in broilers due to its bioactive components, potentially influencing the gut's microbial composition and enhancing nutrient absorption and overall health. Moreover, the capacity of BSFL to efficiently convert organic waste into protein highlights their role as an environmentally sustainable protein source for broiler nutrition. Nonetheless, further research is necessary to fully understand the long-term effects of BSFL, ideal inclusion rates, and the impact of varying larval diets and rearing conditions. It is crucial for poultry producers to consult nutritionists and comply with local regulations when incorporating new feed ingredients like BSFL into poultry diets.

Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.

Characteristics of Enteroendocrine Cells of White Leghorn Chickens, Gallus gallus, Before and After Hatching.

The aim of this study was to identify the histological features of chicken enteroendocrine cells before and after hatching. Tissue samples from the duodenum, proximal and distal parts of the jejunum and ileum, cecum and colorectum were collected from the embryos at days 18, 19, 20, and 21 of incubation, and from 3-day-old chicks. The expression of glucagon-like peptide (GLP)-1, somatostatin (SST), and neurotensin (NT) in the enteroendocrine cells was detected using the streptavidin-biotin method, and the colocalization of these peptides was revealed using the double immunofluorescence method. All of assessed peptides were expressed in the enteroendocrine cells at day 18 of incubation. GLP-1-immunoreactive cells were only observed in the jejunum and ileum. The cell numbers gradually increased as incubation progressed. NT-immunoreactive cells were detected in all intestinal parts at all incubation days, and the highest expression was observed in the colorectum of 3-day-old chicks. SST-immunoreactive cells were observed from the duodenum to the ileum, excluding the colorectum. The double immunofluorescence method revealed that GLP-1 and NT colocalized in the same endocrine cells of the jejunum and ileum. The colocalization ratio of GLP-1 with NT was the highest in the distal ileum of 3-day-old chicks. However, neither GLP-1 nor NT colocalized with SST. These results indicate that chicken enteroendocrine cells markedly change their density and colocalization ratios before and after hatching.

Dietary carbohydrate modifies the density of L cells in the chicken ileum.

Glucagon-like peptides (GLPs) are secreted from intestinal L cells and stimulate various physiological functions in the gastrointestinal tract. The secretion of GLPs is influenced by macronutrient ingestion. This study aims to clarify the effects of dietary carbohydrate (CHO) on L cells in the chicken ileum. Six-week-old, male White Leghorn chickens were divided into three groups: control, low-CHO and CHO-free, with five chickens in each group. Paraffin sections were made from the proximal and distal ileum of each animal and subjected to immunohistochemistry for GLP-1 and GLP-2 peptides and in situ hybridization for proglucagon (PG) mRNA. A significant reduction of GLP-1- and GLP-2-immunoreactive cells was observed in the two experimental groups compared with that in the control. A reduction of cells expressing PG mRNA was observed in the proximal and distal ileum of the CHO-free group compared with that in the control. The ratio of GLP-1-immunoreactive cells showing Ki-67 immunoreactivity was significantly lower in the distal ileum of the CHO-free group than that in the control group. These data suggest that dietary CHO is an effective stimulator for modifying L cell density in the chicken ileum.

The unique physiological features of the broiler pectoralis major muscle as suggested by the three-dimensional ultrastructural study of mitochondria in type IIb muscle fibers.

Typical skeletal muscles are composed of mixed muscle fiber types, which are classified as slow-twitch (type I) and fast-twitch (type II) fibers, whereas pectoralis major muscles (PMs) in broiler chickens are 100% composed of type IIb fast-twitch fibers. Since metabolic properties differ among muscle fiber types, the combination of muscle fiber types is involved in physiological functions and pathological conditions in skeletal muscles. In this study, using serial block-face scanning electron microscopy, we compared three-dimensional (3D) mitochondrial properties in type IIb fibers in broiler PMs and those in type I fibers of broiler gastrocnemius muscles (GMs) heterogeneously composed of slow- and fast-twitch muscle fibers. In type I fibers in the GMs, elongated mitochondria with numerous interconnections to form a substantial network among myofibrils were observed. Along with lipid droplets sandwiched by mitochondria, these features are an adaptation to effective oxidative respiration and constant oxidative damage in slow-twitch muscle fibers. In contrast, type IIb fibers in the PMs showed small and ellipsoid-shaped mitochondria with few interconnections and no lipid droplets, forming a sparse network. The mitochondrial spatial network comprises of active mitochondrial dynamics to reduce mitochondrial damage; therefore, type IIb fibers possess physiologically low capacity to maintain mitochondrial wellness due to static mitochondrial dynamics. Based on 3D mitochondrial properties, we discussed the contrasting physiological functions between type I and IIb fibers and proposed a high contractile power and low stress resistance as unique physiological properties of broiler PMs.

Dietary carbohydrate effects on histological features of ileal mucosa in White Leghorn chicken.

White Leghorn chickens were divided into the control, low-carbohydrate (CHO), and CHO-free groups to investigate dietary CHO's significance on histological features of chicken ileal mucosa. Paraffin sections of distal ileum from each chicken were stained by periodic acid-Schiff reaction and subjected to morphometrical analysis. Most villi in the control group had a fingerlike shape but those of the experimental groups showed irregular shapes. Villus height, crypt depth and the number of mitotic cells per crypt were significantly lower in the CHO-free group than in the control group. The density of goblet cells also showed a significant decreasing trend with a reduction in dietary CHO level. In conclusion, dietary CHO positively affects the proliferation of epithelial cells in the chicken ileum.

Dietary Protein Level Influences on Neurotensin-immunoreactive Cells in the Chicken Ileum.

Neurotensin is secreted from intestinal N cells in response to the food ingestion. Influences of different dietary protein levels on neurotensin-immunoreactive cells in the chicken ileum were examined by using immunohistochemical and morphometrical techniques. The results showed that dietary protein had an obvious influence on neurotensin-immunoreactive cells in the chicken ileum. Four experimental groups were used, with dietary crude protein (CP) levels of 18% (control), 9%, 4.5% and 0%. Enteroendocrine cells showing neurotensin-immunoreactivity were located in crypts and villous epithelium in all groups. Most of the neurotensin-immunoreactive cells in the villous epithelium showed pyramidal or spindle-like shape with a long cytoplasmic process reaching the intestinal lumen, but cells with round or oval shape were found in the CP4.5% and 0% groups. Frequencies of occurrence of neurotensin-immunoreactive cells in the CP18%, 9%, 4.5% and 0% groups were 42.4±3.3, 36.6±2.2, 30.8±2.6 and 25.4±3.8 (cell count per mucosal area: cells/mm2, mean±SD), respectively. There were significant differences in neurotensin-immunoreactive cell frequency between the control and lower CP level, 4.5% and 0%, groups. A significant correlation was found between frequency of occurrence of neurotensin-immunoreactive cells and daily protein intake. These results indicate that ingested protein is likely to be a potential signal for neurotensin production and secretion of N cells in the chicken ileum.

Three-Dimensional Analysis of the Nasolacrimal Duct and Nasal Cavity and Arrangement of Mucosal Tissue in Chickens.

The nasal mucosa plays an important role in the immune system, with nasal mucous cells secreting mucin that, along with pili, exclude foreign substances from intervening. Nasal mucosal-associated lymphoid tissue (NALT), present in the nasal lamina propria, acts as a local immune system. In birds, the Harderian gland in the orbit also plays an important role in the local immune system. In this study, we analyzed the pathway from the nasolacrimal duct to the nasal cavity in chickens and the distribution of the nasal mucous cells responsible for defense mechanisms against pathogens. To determine the three-dimensional structure of the pathway from the nasolacrimal duct to the nasal cavity, we made casts of the anatomy by injecting an acrylic resin into the area. We then prepared paraffin sections to determine the distribution of the NALT and mucous cells. The mucous gland was clearly seen in the mucosal epithelium of the nasal cavity, suggesting that the pathway along the nasal cavity develops a nonspecific immune system to deal with large foreign substances, such as bacteria, using mucins that are secreted from the mucous glands. Hence, there is not only a physical barrier but also an antibacterial activity. Unlike in other animals, morphologically, the nasolacrimal duct in chicken becomes the ventral nasal meatus and opens into the choanae in the caudal portion of the nasal cavity. NALT was prominently present in the lamina propria of the ventral nasal meatus, suggesting the presence of a specific immune system protecting against avian viruses. Thus, responses to vaccine stimulation could be developed from tissues along the pathway of the ventral nasal meatus via the nasolacrimal duct running from the punctum. These morphological studies suggest that the instillation of eye drops could be used as an efficient vaccination method for avoiding respiratory diseases.

Chicken Intestinal L Cells and Glucagon-like Peptide-1 Secretion.

Many types of endocrine cells have been identified in the gastroenteropancreatic system of vertebrates, which have subsequently been named with alphabet (s). L cells which secrete the glucagon-like peptide (GLP)-1 are scattered in the intestinal epithelium. This review discusses the morphological features of chicken L cells and GLP-1 secretion from intestinal L cells. L cells, identified using GLP-1 immunohistochemistry, are open-type endocrine cells that are distributed in the jejunum and ileum of chickens. GLP-1 co-localizes with GLP-2 and neurotensin in the same cells of the chicken ileum. Intestinal L cells secrete GLP-1 in response to food ingestion. Proteins and amino acids, such as lysine and methionine, in the diet trigger GLP-1 secretion from the chicken intestinal L cells. The receptor that specifically binds chicken GLP-1 is expressed in pancreatic D cells, implying that the physiological functions of chicken GLP-1 differ from its functions as an incretin in mammals.

Effect of posttransportation grazing on the physiological condition and meat quality traits of Black Bengal goats.

This study was designed to reveal the role of posttransportation grazing on the physiological condition and meat quality traits of Black Bengal goats. Twenty-four castrated male Black Bengal goats were divided into a control (untransported) group and three treated groups: Walking and Transportation with Human Interference group (WTHI) (30 min walking before 6 hr transport and then 30 min walking with human interference), posttransportation grazing for 48 hr (PTG1), and posttransportation grazing for 72 hr (PTG2). The WTHI and PTG1 groups had a significant reduction in their blood concentrations of tri-iodothyronine (T3 ) and thyroxine (T4 ), and a significantly higher neutrophil and lymphocyte (N:L) ratio compared with those of the control group. Blood concentrations of T3 and T4 and the N:L ratio of PTG2 returned to the control level after 72 hr of grazing. The final pH and water-holding capacity values of meat were significantly higher in the WTHI group than in the control group, but those in the PTG2 group returned to the control level after 72 hr of grazing. These results demonstrate that posttransportation grazing for 72 hr is effective for recovering from damage induced by transportation stress.

Glucagon-like Peptide-1 Receptor Expression in the Pancreatic D Cells of Three Avian Species; White Leghorn Chickens, Northern Bobwhites, and Common Ostriches.

Glucagon-like peptide (GLP)-1 is released from the intestinal L cells in response to food ingestion and stimulates insulin secretion from the pancreatic B cells, by binding to its specific receptor (GLP-1R), which is expressed on the pancreatic B cells in the mammalian pancreas. Previously, we demonstrated that chicken GLP-1R was expressed on the pancreatic D cells by using a specific antibody against chicken GLP-1R. In the present study, we compared the localization of GLP-1R in the pancreases of three avian species; white leghorn chicken, northern bobwhite, and common ostrich, using the double immunofluorescence technique. We found that the types of pancreatic islets in the northern bobwhite pancreas were similar to those found in the chicken pancreas. The ostrich pancreas contained several types of pancreatic islets. GLP-1R-immunoreactive cells were found in all types of pancreatic islets in both northern bobwhite and ostrich and expressed somatostatin immunoreactivity. The present results indicate that the pancreatic D cells are the target cells of GLP-1, and GLP-1 might play a physiological role via somatostatin in the avian species.

Glucagon-like peptide-1 is co-localized with neurotensin in the chicken ileum.

Glucagon-like peptide (GLP)-1 and neurotensin (NT) are distributed throughout the chicken ileum. Here, we attempt to determine if GLP-1 and NT co-localize in the chicken ileum by using immunofluorescence, immunocytochemistry and in situ hybridization techniques. Three types of enteroendocrine cells, GLP-1+/NT+, GLP-1+/NT- and GLP-1-/NT+ cells, were detected in the mucosal epithelium by the double immunofluorescence method. The ratio of GLP-1+/NT+ cells at the crypts in the distal ileum was significantly higher than that in the proximal ileum. The ratios of the three cell types were similar along the crypt-villous axis in the proximal ileum but the percentage of GLP-1+/NT+ cells significantly decreased at the middle part of villi relative to crypts and the bottom part of villi in the distal ileum. Enteroendocrine cells that were immunoreactive to both GLP-1 and NT peptides and showed both proglucagon and NT precursor mRNA signals were found in the crypts of the distal ileum but not in the villous epithelium. The results from performing an immunocytochemical method with colloidal gold indicated that the GLP-1 content within GLP-1+/NT+ cell secretory granules decreased stepwise from the crypt to the middle part of the villus but the NT content in these granules increased in this direction. These findings reveal that the cells producing both GLP-1 and NT are mainly localized in the crypts of the chicken ileum but these endocrine cells specialize in NT-producing cells at the villous epithelium of the distal ileum.

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy.

Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.

Oral ingestion of collagen peptide causes change in width of the perimysium of the chicken iliotibialis lateralis muscle.

Skeletal muscle is mainly composed of myofibers and intramuscular connective tissue. Bundles composed of many myofibers, with each myofiber sheathed in connective tissue called the endomysium, are packed in the perimysium, which occupies the vast bulk of the intramuscular connective tissue. The perimysium is a major determination factor for muscle texture. Some studies have reported that collagen peptide (Col-Pep) ingestion improves the connective tissue architecture, such as the tendon and dermis. The present study evaluated the effects of Col-Pep ingestion on the chicken iliotibialis lateralis (ITL) muscle. Chicks were allocated to three groups: the 0.15% or 0.3% Col-Pep groups and a control group. Col-Pep was administered by mixing in with commercial food. On day 49, the ITL muscles were analyzed by morphological observation and the textural property test. The width of the perimysium in the 0.3% Col-Pep group was significantly larger than other two groups. Although scanning electron microscopic observations did not reveal any differences in the architecture of the endomysium, elastic improvement of the ITL muscle was observed as suggested by an increase of the width of perimysium and improved rheological properties. Our results indicate that ingestion of Col-Pep improves the textural property of ITL muscle of chickens by changing structure of the perimysium.

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

Influences of protein ingestion on glucagon-like peptide (GLP)-1-immunoreactive endocrine cells in the chicken ileum.

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P<0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine.

Immunoelectron microscopic observation of chicken glucagon-like peptide (GLP)-1-containing cells in tissues derived from thin section, paraffin block and conventional method.

The purpose of the present study was to investigate the possibility of immunoelectron microscopic observation of endocrine cells in paraffin-embedded tissues. The procedure, which involves reprocessing from sliced tissues and immunohistochemical staining by colloidal-gold immunolabeling of paraffin sections from paraffin blocks, was able to reveal the fine characteristics of secretory granules containing glucagon-like peptide-1. Morphometric analyses of the secretory granules showed no significant differences between the reprocessing procedure and a conventional post-embedding procedure, which was performed as a control. The reprocessing procedure has some advantages besides providing information on secretory granules containing the amino acid peptide. For example, the same cell can be observed under both a light microscope and the electron microscope. In addition, the high-electron densities of silver-enhanced gold particles are easily recognized, and the boundary between the profile of the granules and the immunogold labeling is clearly shown at the electron microscopic level. Furthermore, the procedure, which is inexpensive and does not require special devices, can effectively use precious samples that are already paraffin-embedded and unable to be obtained twice, such as the case for endangered animals and rare pathological tissues. To the best of our knowledge, the present study is the first to report the advantages of the reprocessing method for sliced paraffin sections of gut endocrine cells.

Distribution of glucagon-like peptide (GLP)-2-immunoreactive cells in the chicken small intestine: antigen retrieval immunohistochemistry.

An antigen retrieval method for immunohistochemical staining of glucagon-like peptide (GLP)-2-immunoreactive cells was investigated in the chicken small intestine. GLP-2-immunoreactive cells were observed as open-typed endocrine cells in the villous epithelium and crypts on both antigen retrieval agent-treated and untreated preparations. No obvious differences were detected in morphological features of GLP-2-immunoreactive cells between treated and untreated preparations. The frequencies of occurrence of GLP-2-immunoreactive cells, however, were significantly different in treated and untreated preparations: in the proximal and distal regions of jejunum and ileum obtained from untreated preparations, the frequencies of occurrence were 0.5 ± 0.2, 0.7 ± 0.1, 0.9 ± 0.2 and 1.5 ± 0.3, respectively (cell numbers per mucosal area: cells/mm(2), mean ± SD), whereas those from treated sections were 14.7 ± 2.3, 19.8 ± 2.3, 23.5 ± 4.7 and 34.6 ± 4.9 cells/mm(2), respectively. These data indicate that this antigen retrieval method is able to make immunoreactive GLP-2 available for detection and that GLP-2 may act as one of the common hormones secreted by L cells in the chicken small intestine.

Ultrastructural study on colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in chicken intestinal L-cells.

Colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in L-cells was investigated in the chicken ileum by using double immunofluorescent and immunocytochemical techniques. Ultrastructural features of L-cells were also clarified in this study. L-cells showing immunoreactivity for both GLP-1 and GLP-2 were distributed in the whole ileum. They showed comma-like or flask-like shape and were located in epithelium of crypts and lower part of intestinal villi. L-cells showing GLP-1-immunoreactivity only were found in epithelium of lower and middle parts of intestinal villi. Transmission electron microscopy indicated that L-cells identified by colloidal gold-labeled immunocytochemistry were covered apically with microvilli, open-type and contained many secretory granules in their perikarya. These secretory granules without halo were round to oval in shape and showed moderate electron density. The longest and shortest diameters of secretory granules were 355 ± 62 nm (mean ± SD) and 287 ± 48 nm, respectively. Double labeling immunocytochemistry using two different sizes of particles (6 and 12 nm in diameter) of colloidal gold revealed that GLP-1 colocalized with GLP-2 in the same secretory granules. This study advances new morphological data about the endocrine system of the chicken small intestine.

Concerted and adaptive alignment of decorin dermatan sulfate filaments in the graded organization of collagen fibrils in the equine superficial digital flexor tendon.

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small-diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large-diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small- and large-diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface-to-surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center-to-center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT.