Morphometric and histochemical study of involution in the rat uterus after parturition

Morphological changes to and collagen loss from the rat uterus during postpartum involution were investigated. The expression patterns of collagen type III and myeloperoxidase (MPO) were also determined. Morphological changes were studied on days 1, 3, 5, 10, 15, 20, 22 and 25 postpartum. As a control, diestrus rats’ uterine were used. Specimens from the uterine horn were embedded in paraffin, cut into 8 µm coronal sections, and stained with hematoxylin‐eosin. The thickness of the longitudinal and circular smooth muscle layers and of the endometrium were measured. The collagen content was determined using hydroxyproline analysis. Immunostaining was used to examine the expression of collagen type III on days 1, 3, 5, 10, 15 and 20 postpartum; and MPO on days 1, 3, 5, 10 and 22 postpartum. The thickness of the smooth muscle layers was found to decrease rapidly postpartum: the circular smooth muscle layer returned to that of a non‐pregnant, control uterus by day 5 postpartum and the longitudinal smooth muscle layer by day 15 postpartum. Eosinophilic cells were observed in the endometrial stroma adjacent to the myometrium on days 10, 15 and 20 postpartum, and were confirmed as collagenous cells. Immunostaining identified collagen type III positive cells in the vessel‐rich layer adjacent to the placental site on days 1, 3, 5 and 10 postpartum, and these cells were confirmed to be phagocytic. Postpartum reduction in the weight of the uterus was accompanied by decreases in both the collagen content and the thickness of the longitudinal and circular smooth muscle layers. Furthermore, the phagocytic cells were shown to express MPO during postpartum involution of the uterus.     

Growth of rumen papillae in weaned calves is associated with lower expression of insulin‐like growth factor‐binding proteins 2, 3, and 6

This study aimed to characterize the relationship between the growth of rumen papillae in calves and the mRNA expression of insulin‐like growth factor‐binding proteins (IGFBPs) in the rumen papillae. The length of rumen papillae, the mRNA expression of IGFBPs in rumen papillae by quantitative real‐time PCR, and the presence of insulin‐like growth factors I and II (IGF‐I and II) by immunohistochemistry (IHC) were analyzed in nine Holstein calves divided into three groups: suckling (2 weeks, n = 3), milk‐continued (8 weeks, n = 3), and weaned (8 weeks, n = 3). The length of rumen papillae was greater (p < 0.01) in weaned calves than in suckling and milk‐continued calves, whereas the expressions of IGFBP2, IGFBP3, and IGFBP6 genes were lower (p < 0.05) in the rumen papillae of weaned calves than in milk‐continued calves. Thus, rumen papillae length and IGFBP2, 3, and 6 expressions were negatively correlated. The IHC analysis showed that IGF‐I and IGF‐II were present in the rumen epithelium of calves. These results suggested that the growth of rumen papillae after weaning is associated with the induction of IGFs by the low levels of IGFBP2, IGFBP3, and IGFBP6.     

Insulin‐like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3

The objective of our present study was to determine the effects of insulin‐like growth factor I (IGF‐I) on the development of yak (Bos grunniens) embryos after cumulus–oocyte complex (COC) vitrification and warming followed by in vitro fertilization (IVF). In Experiment 1, the yak COCs underwent vitrification and then IVF. Embryos were incubated in synthetic oviductal fluid (SOF) supplemented with four concentrations (0, 50, 100 and 200 ng/ml) of IGF‐I, while the yak COCs without vitrification or IGF‐I supplementation acted as the control group; the BAX, BCL‐2, AQP3mRNA and aquaporin 3 (AQP3) protein expression levels in the five groups of blastocysts were evaluated using quantitative real‐time PCR and immunofluorescence analyses. In Experiment 2, the groups described above were fertilized and incubated. The cleavage rate, blastocyst rate, total cell count per blastocyst and the rate of growth of the inner cell mass (ICM) and trophectoderm (TE) were evaluated. The results were as follows: (1) the AQP3 gene expression and protein expression in the control and 100 ng/ml IGF‐I treatment groups were the highest. (2) The BAX gene expression was the lowest and the BCL‐2 gene expression was the highest in the control and 100 ng/ml IGF‐I treatment groups. (3) The rates of cleavage and blastocysts in the control and 100 ng/ml IGF‐I groups were higher than those in the other three groups. The total cell count per blastocyst in the vitrified and warmed 100 ng/ml IGF‐I group (106.7 ± 4.9) and the control group (107.3 ± 4.2) was higher than that in the vitrified and warmed 0 ng/ml IGF‐I (91.2 ± 3.1), 50 ng/ml IGF‐I (92.3 ± 3.7) and 200 ng/ml IGF‐I (92.4 ± 3.7) groups. Therefore, we conclude that IGF‐I can improve yak blastocyst developmental ability, cytomembrane permeability and formation of the blastocyst cavity after COC vitrification by improving the BAX, BCL‐2 and AQP3 expression levels.     

Three-dimensional reconstruction of intramuscular collagen networks of bovine muscle: A demonstration by an immunohistochemical/confocal laser-scanning microscopic method

We undertook a three‐dimensional reconstruction of intramuscular collagen networks of bovine muscle using an immunohistochemical/confocal laser‐scanning microscopic method. By immunohistochemical staining, type I and III collagens were observed mainly in the perimysium, while type IV collagen was observed in the endomysium. On the other hand, type V and VI collagens were observed in both the perimysium and endomysium. By confocal laser‐scanning microscopy, the collagen observed in the perimysium was three‐dimensionally reconstructed as plate‐shaped layers whereas the collagen observed in the endomysium surrounded myofibers. The three‐dimensionally reconstructed observations using immunohistochemical/confocal laser‐scanning microscopic method is useful for investigating collagen networks in muscle.     

Effect of a low‐voltage electrical stimulation on yak meat tenderness during postmortem aging

This study evaluates the effect of a low‐voltage electrical stimulation (ES) on the tenderness of yak longissimus muscle (LM). Samples from 16 yak bulls were divided into four treatment groups: normal chilling (NC), ES and chilling (ES & C) for 72 s (ES &C 72 s), ES & C for 90 s (ES & C 90 s), and ES & C for 108 s (ES & C 108 s). The temperature, the pH, the glycogen content, the Warner‐Bratzler shear force (WBSF), the myofibril fragmentation index (MFI), and the muscle ultrastructure were determined during the course of postmortem aging. ES caused a rapid decrease in the pH to form a high‐temperature and low‐pH environment. The glycogen content gradually decreased with aging. The WBSF value of the ES & C groups was significantly lower than for the NC group (p < .05). The MFI values of ES & C groups after 24 hr postmortem aging were significantly higher than for the NC group. We concluded that ES improved yak meat tenderness during postmortem aging and that the different duration time by ES indicated different effects, and its affect was remarkable in the ES & C 90 s.     

Effect of skeletal muscle decorin on collagen fibrillogenesis in vitro

The effect of skeletal muscle decorin on collagen fibrillogenesis was investigated, in order to provide background for understanding the functions of decorin in skeletal muscle. The self‐assembly of type I and III collagen with the addition of decorin or the core protein of decorin from bovine neonatal skeletal muscle was monitored using a spectrophotmeter. Time course changes in the absorbance of collagen solutions showed typical sigmoidal curves composed of three phases. The time of the initial phase was not different between the collagen solution with decorin and that without decorin. The increase rate of the absorbance in the second phase decreased with concentration of decorin added in collagen solutions. Similar effects on fibrillogenesis of type I and III collagens were observed when the core protein of decorin was added in collagen solutions. These results suggest that regulation of collagen fibrillogenesis by decorin depends on its core protein. The networks of reconstructed collagen fibrils with decorin were looser than those without decorin. Bovine skeletal muscle decorin could participate in the regulation of collagen fibrillogenesis and in the arrangement of collagen fibrils in the intramuscular connective tissue.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

Maternal dietary linoleic acid supplementation promotes muscle fibre type transformation in suckling piglets

As meat quality is basically dependent on muscle fibre characteristics, it is important to know how muscle fibres are regulated and transformed. This study aimed to investigate the effect of maternal dietary supplementation on muscle fibre types using 3% saturated fatty acid (palmitic acid, PA) or 3% unsaturated fatty acid (linoleic acid, LA) from 80 days of gestation to the weaning of offspring (25 days post‐natal). The results indicated that higher mRNA levels of MyHCI type genes were found in the soleus muscles of piglets that suckled from LA‐supplemented sows than from PA‐supplemented sows. In addition, LA treatment increased the gene expression of the type I muscle fibre marker troponin I (p < 0.01), suggesting that LA promoted muscle fibre type transformation to type I fibres. Moreover, PGC‐1α (p < 0.01) and MEF2c (p < 0.05) mRNA levels were higher in the piglets from the LA treatment group than in those from the PA treatment group. Furthermore, LA supplementation also significantly increased AMP‐activated protein kinase (AMPK) mRNA levels (p < 0.05), which is an upstream regulator of PGC‐1α. Collectively, these findings demonstrated that maternal dietary LA supplementation promoted muscle fibre transformation to type I fibre and that this process may be mediated through an AMPK‐dependent pathway.     

Meat quality traits as a function of cow maturity

To investigate the physico‐chemical and sensory properties of striploin muscles, 90 Hanwoo carcasses (QG 1⁺) were randomly selected within six maturity levels (4 to 9 according to age in months). Results demonstrated that the protein contents at maturity levels 4 and 5 were significantly higher than 9. No significant difference in fat, moisture and collagen contents were found at different maturity levels (P > 0.05). The quantity of collagen type I and ratio of type I to III were observed at higher maturity levels; collagen type III showed significantly high levels (P > 0.05) at low maturity and decreased with increase in maturity levels. Warner‐Bratzler shear force (WBSF) was significantly lower in groups 4 to 6, whereas water holding capacity (WHC) was significantly higher than maturity level 8 and 9 groups (P < 0.05). There were no significant differences in cooking loss among the maturity level groups (P > 0.05). Color properties, L* values of striploin muscle from maturity level 4 were significantly different from level 9 (P < 0.05). Sensory evaluation at level 4‐6 groups had significantly higher tenderness and overall likeness scores than level 9 (P < 0.05). The maturity levels were significantly correlated with age, fat, protein content, WHC, WBSF, cooking loss, CIE L* values and sensory properties like tenderness, juiciness, flavor‐likeness and overall likeness.     

In Vitro Expression of the Extracellular Matrix Components Aggrecan,Collagen Types I and II by Articular Cartilage‐Derived Chondrocytes

The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan‐based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long‐term results of therapeutic procedures including cell‐based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage‐derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT‐qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells.     

Relationships among muscle fiber type composition,fiber diameter and MRF gene expression in different skeletal muscles of naturally grazing Wuzhumuqin sheep during postnatal development

The aim of this study was to determine the relationships among muscle fiber‐type composition, fiber diameter, and myogenic regulatory factor (MRF) gene expression in different skeletal muscles during development in naturally grazing Wuzhumuqin sheep. Three major muscles (i.e. the Longissimus dorsi (LD), Biceps femoris (BF) and Triceps brachii (TB)) were obtained from 20 Wuzhumuqin sheep and 20 castrated rams at each of the following ages: 1, 3, 6, 9, 12 and 18 months. Muscle fiber‐type composition and fiber diameter were measured using histochemistry and morphological analysis, and MRF gene expression levels were determined using real‐time PCR. In the LD muscle, changes in the proportion of each of different types of fiber (I, IIA and IIB) were relatively small. In the BF muscle, a higher proportion of type I and a 6.19‐fold lower proportion of type IIA fibers were observed (P < 0.05). In addition, the compositions of type I and IIA fibers continuously changed in the TB muscle (P < 0.05). Moreover, muscle diameter gradually increased throughout development (P < 0.05). Almost no significant difference was found in MRF gene expression patterns, which appeared to be relatively stable. These results suggest that changes in fiber‐type composition and increases in fiber size may be mutually interacting processes during muscle development.     

Comparison of collagen content, distribution and architecture among the pectoralis, iliotibialis lateralis and puboischiofemoralis muscles with different myofiber composition in Silky cocks

The total amount of collagen, the relative distributions of types I and III collagens in perimysium and endomysium, and the collagen fiber architecture were compared among the pectoralis (PT), iliotibialis lateralis (ITL) and puboischiofemoralis (PIF) muscles in Silky cocks. All of the myofibers in the PT muscle were type IIB, the myofibers in the ITL muscle were divided into type IIA, 41.7% and IIB, 58.3%, and the PIF muscle was composed of type I, 24.6%; IIA, 64.6%; and transitional, 10.8%. The total amount of collagen differed significantly among the PT (2.92 mg/g), PIF (4.20 mg/g) and ITL (8.06 mg/g) material, where only the PIF was a whole muscle with epimysium. On the image analysis of the immunohistochemical preparations, the percentage area of perimysial collagen to the total area in each type differed significantly among the PIF, PT and ITL muscles, where it was 26.8, 50.0 and 74.4% for the type I collagen and 27.4, 32.9 and 61.7% for the type III collagen, respectively. In the scanning electron micrography of the perimysium in macerated preparations, thick bundles of collagen fibers were observed in the ITL muscle, thinner but broad platelets in the PT muscle, and a coarse tissue of thinner collagen fibers in the PIF muscle. However, the endomysial fabric of collagen fibrils was similar among the muscles. Small, transverse collagen fibers, which branched off from the thicker perimysia, occupied narrow interendomysial spaces and separated the primary myofiber fasciculi. The results indicate that the ITL muscle, localized in the distorted and overextended part of the leg and subject to strong external forces, had highly developed perimysial collagen fiber bundles, but the ITL endomysial collagen architecture was similar to that of the PT and PIF muscles.     

Proportion of types I and III collagen in longissimus collagen from bulls and steers

The proportion of types I and III intramuscular collagen in longissimus muscles of Simmental bulls (n = 8) and steers (n = 8) 17 mo of age was studied. Longissimus samples taken 7 d after slaughter were evaluated for total collagen, types I and III collagen, heat-soluble collagen, sensory panel traits and Warner-Bratzler shear force. Intramuscular collagen (IMC) was isolated and digested with cyanogen bromide, and peptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Percentage of type III IMC was calculated from the total of types I and III collagen as determined from the peak area of densitometric scans of the cyanogen bromide peptides alpha 1(I)CB8 and alpha 1(III)CB8. Longissimus muscles from steers had lower (P less than .05) Warner-Bratzler shear values, less (P less than .05) sensory panel-detectable connective tissue and more (P less than .05) tender panel ratings for muscle fiber tenderness and overall tenderness. Muscles from steers had more (P less than .05) heat-soluble collagen than those from bulls, but no differences (P greater than .05) were found for total collagen and percentage of type III collagen. Some intramuscular-collagen characteristics may have contributed to the less tender muscle of bulls. However, the proportion of types I and III collagen did not account entirely for the tenderness difference between steer and bull muscles. Because there were differences in collagen solubility in muscles from steers and bulls, other collagen characteristics such as crosslinking or fiber size may have been more important than collagen type.     

不同CBI日粮对牦牛瘤胃发酵参数、血清生化指标和养分消化率的影响

本试验旨在探究不同碳水化合物平衡指数(Carbohydrate balance index，CBI)日粮对牦牛瘤胃发酵参数、血清生化指标和养分消化率的影响，选取3种不同CBI(1.17，2.45，5.67)日粮，采用3×3拉丁方试验设计，通过一系列指标测定，明确牦牛最优日粮CBI水平。结果表明：不同CBI牦牛日粮瘤胃发酵参数均不显著；CBI=1.17与CBI=5.67牦牛日粮的白蛋白/球蛋白差异不显著，但均显著高于CBI=2.45的牦牛日粮(P<0.05)；CBI=2.45牦牛日粮干物质消化率、蛋白质消化率显著高于CBI=1.17和CBI=5.67(P<0.05)；CBI=2.45与CBI=5.67牦牛日粮的中性洗涤纤维消化率差异不显著，但均显著高于CBI=1.17(P<0.05)。综上所述：牦牛日粮的CBI=2.45更有利于其瘤胃发酵和养分消化利用。     

Increased collagen III in culled chicken meat after feeding dietary wood charcoal and vinegar contributes to palatability and tenderness

We comprehensively evaluated meat quality in chickens fed a diet consisting of wood charcoal and vinegar (WCV) using food scientific and histological approaches. In culled hens, lipid and fatty acid in Musculus semimembranosus, cooking loss and sensory tests of whole thigh meat, and meat texture of breast meat were observed. In male broilers, cross section of M. semimembranosus was used for observations on muscle area, perimysium, non‐collagen total protein and total collagen content, and anti‐collagen I and III reactions. In frozen male broilers, conventional morphology of M. semimembranosus as well as chicken anti‐collagen III reaction to selected muscles of thigh meat and breast meat were compared between the control and WCV‐fed birds. Increased lipid and fatty acids, decreased cooking loss, high score in total evaluation for sensory test of thigh meat, and decreased meat texture values were observed for culled hens fed WCV. The higher values of muscle area, total collagen and collagen III were observed for broilers fed WCV. No perimysium collapse for M. semitendinosus or increased collagen III reactions of M. tensor fasciae latae, the flexor muscle group and M. pectoralis superficialis were observed for frozen muscles in the WCV group. These total results suggest that WCV produces palatable and tender meat by increasing collagen III.     

Differences in monocarboxylic acid transporter type 1 expression in rumen epithelium of newborn calves due to age and milk or milk replacer feeding

The aim of the present study was to investigate whether besides age and solid feed intake, monocarboxylic acid transporter type 1 (MCT1) expression in the rumen epithelium of calves is affected by liquid feed type [whole milk (WM) or milk replacer (MR)]. Thirty bull calves at the mean age of 5 days were randomly allocated to five experimental groups (six calves/group). Six calves were slaughtered immediately after allocation to the trial (5 days of life), eighteen calves were fed MR and slaughtered at week intervals (on 12, 19, 26 days of life respectively), and six calves were fed WM and slaughtered at the 26 days of life. MCT1 protein abundance and the MCT1 mRNA level were investigated in the dorsal and ventral sack of the rumen. Solid feed intake and short‐chain fatty acids (SCFA) concentration in the rumen fluid increased linearly with calves' age. The amount of the MCT1 protein and mRNA in the dorsal sac of rumen as well as the amount of MCT1 protein in the cranial ventral sac of rumen also increased linearly with calves' age. Calves fed WM had greater solid feed intake in the last week of the study as compared to calves fed MR, but SCFA concentration in the rumen fluid was not different. MCT1 mRNA expression in the cranial dorsal sac of rumen and protein MCT1 expression in both dorsal and ventral cranial sack of the rumen were higher in calves fed WM as compared to calves fed MR. This study confirmed age‐dependent changes of MCT1 expression in the rumen epithelium of newborn calves and showed that its expression might be affected by liquid feed type.     

Molecular cloning of type I collagen cDNA and nutritional regulation of type I collagen mRNA expression in grass carp

Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean‐fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059‐bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α‐helixes, eight β‐sheets, 16 regions of triple helical repeats, 21 low‐complexity regions, 10 function domains and two zinc‐binding sites; however, no calcium‐binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi‐quantitative RT‐PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real‐time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean‐fed grass carp.     

Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma

The changes in the levels of carbonic anhydrase isozyme III (CA‐III) in swine plasma and urine have not been previously determined or reported. CA‐III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA‐III from swine muscle tissues and determined CA‐III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA‐III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1–5 months) and adult female pigs (age, 2–3 years) were determined using the ELISA system for swine CA‐III. The mean (± SD) levels of CA‐III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA‐III in the plasma samples from 2‐ to 3‐year‐old pigs was 18 ± 60 ng/ml. CA‐III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far‐western blotting to clarify the cause of the observed decrease in CA‐III in plasma. Our results demonstrated that CA‐III is bound to the transferrin and albumin. In addition, we determined that the levels of CA‐III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.