Cranial suture closure pattern in water deer and implications of suture evolution in cervids

Interspecific diversity exists in cranial suture closure patterns among mammalian species. Suture closure patterns partly reflect phylogenesis, but it has also been claimed that it is influenced by biomechanical factors. Here we provide the first report on the cranial suture closure pattern in water deer. Among cervids, the water deer is an exceptional species, as it is the only one without antlers. Instead, the male water deer possess prominent canines, which are utilized during male-male combats. This unique morphological setting allowed us to examine whether the presence of prominent canines affects cranial suture closure. We found that the only sexual difference in cranial suture closure pattern is the closure of the interfrontal suture in males, which is found in some other cervid species as well. Except for the interfrontal suture closure, timing and sequence of cranial suture closure were comparable between both sexes, suggesting that the presence of prominent canines in males has no influence on cranial suture closure pattern. Our results indicate that interfrontal suture closure in males is a phylogenetic pattern, i.e. an apomorphy for Capreolini, Hydropotini and Alceini.     

Old and new hypotheses about the homology of the compound bones from the cheek and otico-occipital regions of the anuran skull

We studied the larval development of compound bones from the otico-occipital and cheek regions in species of the neobatrachian genera Batrachyla, Hylorina, Leptodactylus, Odontophrynus and Pleurodema. Comparisons were made using a set of Ambystoma spp. (Caudata) and Ceratophrys ornata (Anura; Ceratophryidae) larvae. As suggested by previous studies, we verified the compound nature of the exoccipital (two centers, anurans only), frontoparietal (one center, most anurans and Ambystoma; three centers, some anurans), and squamosal (two centers, all anurans and Ambystoma) bones. We discuss old and new homology hypotheses for each of the compound bone centers in the context of the most widely accepted scenario of lissamphibian origins and relationships, i.e., monophyletic Lissamphibia that includes the clade Batrachia (Caudata + Anura) and the most divergent Gymnophiona. Our findings have a direct impact on our understanding of the composition of the skull in Lissamphibia. We recognized the presence of the following bones: (i) opisthotic (fused to the exoccipital) and tabular (fused to the squamosal) in Batrachia (Anura + Caudata) and (ii) supratemporal (fused to the parietal portion of the frontoparietal) in Anura. Separate centers of the parietal were found only in Pleurodema.     

Gestational losses in a rabbit line selected for growth rate

Prenatal death can occur due to several genetic and environmental factors which alter normal embryo development, maternal environment to support normal fertilisation, development of embryos, placenta and foetus, or affect the necessary relationship between embryo and endometrium. The aim of this work was to study gestational losses and progesterone, 17 β-estradiol and IGF I serum levels in a rabbit line selected for growth rate (paternal line). In this study, a maternal line well characterised in previous studies was used as a reference line. A total of 211 laparoscopies were carried out, and the number of corpora lutea and implanted embryos at 12^th days, total born and live born were recorded per female. To analyse the endocrine levels, blood serum was collected from 54 females with implanted embryos at 12^th and 24^th day of gestation (27 from each line). The paternal line showed the lowest ovulation frequency, number of implanted embryos, total born and live born (0.70, 11.3, 7.4, and 6.4 vs 0.86, 12.8, 11.1 and 10.6 for maternal line, respectively) and consequently, the highest implantation, gestational, foetal and perinatal losses (0.31, 0.60, 0.40, and 0.15, respectively). Progesterone serum levels at 12^th days of gestation were similar between lines; however, progesterone serum level at 24^th day of gestation was significantly lower in the paternal line (4.8 vs 8.2 ng/mL). Serum levels of 17β-estradiol and IGF-I at 12^th days of gestation were different between lines (14.6 vs 26.5 pg/mL, 237 vs 149 ng/mL for paternal and maternal lines respectively). These higher gestational losses of the paternal line could be explained by differences in 17 β-estradiol level at 12^th days of gestation and the possible effect on low progesterone serum levels at 24^th days of gestation. Further studies in steroid production and bioavailability have to be done during oestrus and pregnancy related with metabolic activity of this line.     

Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate

Fibroblast growth factor 21 (FGF21) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF21 during chronic food restriction is associated with reduced bone growth and growth hormone (GH) insensitivity. In light of the inhibitory effects on growth plate chondrogenesis mediated by other FGFs, we hypothesized that FGF21 causes growth inhibition by acting directly at the long bones' growth plate. We first demonstrated the expression of FGF21, FGFR1 and FGFR3 (two receptors known to be activated by FGF21) and β-klotho (a co-receptor required for the FGF21-mediated receptor binding and activation) in fetal and 3-week-old mouse growth plate chondrocytes. We then cultured mouse growth plate chondrocytes in the presence of graded concentrations of rhFGF21 (0.01-10 μg/ml). Higher concentrations of FGF21 (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF21 in a concentration-dependent manner. In addition, FGF21 reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF21 on GH action were all prevented, supporting a specific effect of this growth factor in chondrocytes. Our findings suggest that increased expression of FGF21 during food restriction causes growth attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the growth plate. In addition, high concentrations of FGF21 may directly suppress growth plate chondrocyte proliferation and differentiation.     

Effects of manganese-supplemented diets on growth performance,blood biochemistry,nitrogen metabolism and skeletal development of rex rabbits

(Background) Manganese (Mn) is an essential mineral, although its effects on rabbits is not clear. (Research Purpose) This study was conducted to investigate the effects of the level of supplementation of dietary manganese on growth performance, blood biochemistry, nitrogen metabolism and skeletal development of growing Rex rabbits. (Methods) Two hundred 3-month-old healthy Rex rabbits with similar body weights were randomly divided into 5 groups (A, B, C, D, E), with 40 replicates in each group. The rabbits in the 5 groups were fed a basal diet containing 0, 5, 10, 20 and 40 mg/kg manganese (in the form of manganese sulfate), respectively. The trial included 7 days for adaptation and 29 days of testing. Seven days before the end of feeding, eight rabbits from each group were transferred into a metabolic cage for metabolic testing. (Results) The results showed that supplemental dietary manganese levels did not significantly influence final body weight (FBW) or average daily feed intake (ADFI) (P＞0.05). Average daily gains (ADG) were significantly higher in the 20 mg/kg manganese group than in the other groups, and the ratio of feed to body weight gain (F/G) was significantly affected by manganese level (P < 0.05). No significant differences were found in the digestion coefficients among the groups (P > 0.05). Regarding carcass traits, the thymus index and total fat were significantly different (P < 0.05) among the groups, but there were no other significant differences (P > 0.05). The addition of manganese had no significant effect on the intake of nitrogen (IN), fecal nitrogen (FN), digestible nitrogen (DN) or the apparent digestibility of nitrogen (NAD). Compared to the other groups, urinary nitrogen (UN) was lower in the 20 mg/kg group, although nitrogen deposition (RN), nitrogen utilization (NUR) and the biological potency of nitrogen (NBV) were higher in this group (P < 0.05). As the amount of manganese added to the diet increased, serum triglycerides decreased (P < 0.05). Serum Mn-SOD was significantly lower in the 5 mg/kg manganese group than in the other groups (P < 0.05). The results of this study demonstrate that a diet with supplemented manganese can improve Rex rabbit growth performance and increase RN, NUR and NBV. There were no significant effects of different dietary levels of Mn on the ratio of bone to meat (P > 0.05) or bone strength (P < 0.05). (Conclusion) In conclusion, we determined that the optimal level of manganese supplementation in the diet of growing Rex rabbits was 20 mg/kg, which was also found to reduce nitrogen emissions into the environment.     

Relationships between undernutrition,infection, and growth and development

Interactions between undernutrition, infection, and growth and development are complex, and are reviewed in this article. Anthropometry is a common means of nutritional assessment, but the relationship between food availability and anthropometric status is at best very loose, at least at the national level. This suggests that anthropometric assessment is less a measure of nutritional status than of the totality of environmental factors that influence growth, including infectious disease. The effects of diet, nutrition and infection on the nutritional status of a child can vary according to the disease ecology, the age of the child, patterns of feeding and types of food consumed. There are two possible ways in which this relationship can begin; one in which poor nutritional status leads to impaired immunocompetence and reduced resistance to infection, and the other in which exposure to infectious disease can lead to appetite loss and anorexia, malabsorption, and elevated metabolism of energy and other nutrients. Once started, the interactions between these two major environmental stressors becomes increasingly complex, with the nature of the disease ecology influencing the balance of immunoparesis and adaptive immunity its effect on subsequent disease experience, and the extent, if any, of anorexia, fever, and malabsorption during infectious episodes which has an impact on nutritional status. Specific nutritional deficiencies can subsequently influence immune status and responsiveness, and adaptive immunity. In addition, cultural factors can influence patterns of disease management and sickness behaviour, which can in turn affect the incidence, severity and duration of infection, and their effects on nutritional status, while deficiencies of vitamins and trace elements can have major effects on immune responsiveness.     

Postnatal sutural growth of the calvarium: a survey with special reference to the rabbit

The postnatal development of the cranial vault is reviewed with special emphasis upon the rabbit in respect to longitudinal, volumetric, and kinematic growth. Also, the calvarial growth relative to long bone growth is discussed. The cranial vault suture is a multifunctional syndesmosis with variable structure and a rich variability of local behavior.     

Biomechanics of the rostrum and the role of facial sutures

The rostrum is a large diameter, thin-walled tubular structure that receives loads from the teeth. The rostrum can be conceptualized both as a rigid structure and as an assemblage of several bones that interface at sutures. Using miniature pigs, we measured in vivo strains in rostral bones and sutures to gain a better understanding of how the rostrum behaves biomechanically. Strains in the premaxillary and nasal bones were low but the adjacent maxillary-premaxillary, internasal, and intermaxillary suture strains were larger by an order of magnitude. While this finding emphasizes the composite nature of the rostrum, we also found evidence in the maxillary and nasal bones for rigid structural behavior. Namely, maxillary strain is consistent with a short beam model under shear deformation from molar loading. Strain in the nasal bones is only partially supported by a long beam model; rather, a complex pattern of dorsal bending of the rostrum from incisor contact and lateral compression is suggested. Torsion of the maxilla is ruled out due to the bilateral occlusion of pigs and the similar working and balancing side strains, although it may be important in mammals with a unilateral bite. Torsional loading does appear important in the premaxillae, which demonstrate working and balancing side changes in strain orientation. These differences are attributed to asymmetrical incisor contact occurring at the end of the power stroke.     

微生态制剂对断奶仔兔生产性能及免疫的影响

目的研究微生态制剂对断奶仔兔生产性能、免疫功能的影响,为开发应用新型添加剂提供依据。方法选择64只35日龄、体重相近的断奶仔兔,随机分为4组,每组设2个重复,每个重复8只。实验期为40 d。对照组Ⅰ饲喂基础饲粮,实验组Ⅱ、Ⅲ、Ⅳ在基础日粮中分别添加0.1%、0.3%、0.5%的微生态制剂。测定日增重、饲料报酬、发病率、死亡率、胸腺指数、脾脏指数及T淋巴细胞ANAE阳性细胞百分率等指标。结果实验组Ⅱ、Ⅲ、Ⅳ日增重分别比对照组I提高了10.6%（P〈0.05）、23.967%、27.0%（P〈0.01）,饲料报酬分别提高了6.8%、14.3%、15.1%,发病率分别降低了12.5%、18.7%、18.7%,死亡率分别降低了12.5%、12.5%、18.7%,脾脏指数分别提高了11.5%（P〈0.05）、29.5%、34.4%（P〈0.01）,胸腺指数分别提高了2.3%、15.9%、17.9%,T淋巴细胞阳性率分别提高了17.7%（P〈0.05）、26.9%、28.4%（P〈0.01）。结论在断奶应激的情况下,饲料中添加微生态制剂可提高仔兔的生产性能和免疫功能。     

Desmosomes: Their occurrence between adjacent primary oocytes in polyovular follicles in the rabbit

Summary In polyovular, primordial follicles in the rabbit, desmosomes are found between apposing oocyte surfaces. Morphologically these desmosomes correspond closely to those described in epithelia of vertebrates. The desmosomes alternate with other junctions which are probably gap junctions.This work was supported in part by the G.R.S.T. Grant n 75.7.1313     

Relationships between larval nutritional experience, larval growth rates, juvenile growth rates, and juvenile feeding rates in the prosobranch gastropod Crepidula fornicata

Planktonic larvae experiencing short periods of starvation or reduced food supply often grow and develop more slowly, have poor survival, fail to metamorphose, metamorphose at smaller sizes, or grow slowly as juveniles. In this study, we examined the impact of short periods of food limitation at various stages of larval development on larval and juvenile growth in Crepidula fornicata. In addition, we considered whether juveniles that were stressed as larvae grew poorly because of reduced rates of food collection due to impaired gill function. For 5 experiments, larvae were either starved for several days beginning within 12 h of hatching or were starved for the same number of days following 1 or more days of feeding at full ration (cells of the naked flagellate Isochrysis galbana, clone T-ISO, at 18×10⁴ cells ml⁻¹). In one experiment, larvae were transferred for 2 or 4 days to seawater with extremely low phytoplankton concentration (1×10⁴ cells ml⁻¹). In all experiments, larvae were returned to full ration following treatment. Control larvae were fed full ration from hatching to metamorphosis. When larvae reached shell lengths of about 900 μm they were induced to metamorphose and then reared individually at full ration in glass bowls, with phytoplankton suspension replenished daily. Larval and juvenile growth rates were determined by measuring changes in shell length (longest dimension) over time. Juvenile feeding rates were determined by monitoring changes in phytoplankton concentration over 2–3 h at the end of the growth rate determinations. In general, larval growth rates for the first 2 days after the resumption of feeding were inversely proportional to the length of time that larvae were starved. However, larval growth rates ultimately recovered to control levels in most treatments. Starving the larvae caused a significant reduction in initial juvenile growth rates (first 3–4 days post-metamorphosis) in most experiments even when larval growth rates had recovered to control levels prior to metamorphosis. Juvenile growth rates were not significantly reduced when larvae were subjected to reduced food availability (1×10⁴ cells ml⁻¹), even for treatments in which larval growth rates were compromised. Mean weight-specific filtration rates for juveniles were significantly reduced (p<0.05) following larval feeding experience in only one or possibly 2 of the 4 experiments conducted. Our data suggest that although larvae of C. fornicata may fully recover from early nutritional stress, the resulting juveniles may exhibit poor initial growth due to impaired gill function, reduced digestive capability, or reduced assimilation efficiency.     

Jagged1 functions downstream of Twist1 in the specification of the coronal suture and the formation of a boundary between osteogenic and non-osteogenic cells

The Notch pathway is crucial for a wide variety of developmental processes including the formation of tissue boundaries. That it may function in calvarial suture development and figure in the pathophysiology of craniosynostosis was suggested by the demonstration that heterozygous loss of function of JAGGED1 in humans can cause Alagille syndrome, which has craniosynostosis as a feature. We used conditional gene targeting to examine the role of Jagged1 in the development of the skull vault. We demonstrate that Jagged1 is expressed in a layer of mesoderm-derived sutural cells that lie along the osteogenic-non-osteogenic boundary. We show that inactivation of Jagged1 in the mesodermal compartment of the coronal suture, but not in the neural crest compartment, results in craniosynostosis. Mesodermal inactivation of Jagged1 also results in changes in the identity of sutural cells prior to overt osteogenic differentiation, as well as defects in the boundary between osteogenic and non-osteogenic compartments at the coronal suture. These changes, surprisingly, are associated with increased expression of Notch2 and the Notch effector, Hes1, in the sutural mesenchyme. They are also associated with an increase in nuclear β-catenin. In Twist1 mutants, Jagged1 expression in the suture is reduced substantially, suggesting an epistatic relationship between Twist1 and Jagged1. Consistent with such a relationship, Twist1-Jagged1 double heterozygotes exhibit a substantial increase in the severity of craniosynostosis over individual heterozygotes. Our results thus suggest that Jagged1 is an effector of Twist1 in coronal suture development.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Geometric morphometrics of the skull of Tinamidae (Aves, Palaeognathae)

The Tinamidae comprise exclusively Neotropical palaegnathous birds, with homogeneous body morphology and no sexual dimorphism. The goal of this work was to explore the variation in skull morphology between taxa and its possible correspondence with features such as diet or gender using geometric morphometric tools. Eleven landmarks were analyzed in 53 skulls of 4 genera that inhabit grasslands: Nothoprocta, Eudromia, Nothura and Rhynchotus. Intrageneric and intergeneric variability was analyzed. The genera studied here can be distinguished based on the geometric shape of their skull, with prenarial region length and neurocranium shape as the most outstanding features. In the genus Eudromia, males and females could be differentiated, while in the genus Nothoprocta, the species differentiated according to their trophic habits. This study allows establishing that genera and, in some cases, the gender of the Tinamidae can be differentiated based on cranial shape.     

Biochemical differentiation between the Wild rabbit (Oryctolagus cuniculus L.), the Domestic rabbit and the Brown hare (Lepus europaeus Pallas)

Twenty five specimens of the brown hare and fourteen specimens of the wild rabbit were examined for genetic differentiation and polymorphism in 28 enzyme systems by means of horizontal starch gel electrophoresis and isoelectric focusing in polyacrylamide gels. Indices of genetic variation show values similar to those averaged over numerous mammalian species. A D-value of 0, 0288 between wild and domestic rabbits suggests a high amount of gene frequency changes at enzyme loci during domestication. Evolutionary time of divergence between the rabbit and the hare (about 2430000 years) calculated from electrophoretic data is in good agreement with palaeontological findings.     

Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture

Auricular cartilage is an attractive potential source of cells for many tissue engineering applications. However, there are several requirements that have to be fulfilled in order to develop a suitable tissue engineered implant. Animal experiments serve as important tools for validating novel concepts of cartilage regeneration; therefore rabbit auricular chondrocytes were studied. Various parameters including isolation procedures, passage number, rate of proliferation and gene expression profile for major extracellular matrix components were evaluated in order to assess the potential use of elastic chondrocytes for tissue engineering. Chondrocytes were isolated from rabbit ear cartilage and grown in monolayer cultures over four passages. Yields of harvested cells and proliferation were analysed from the digestion step to the fourth passage, and changes in phenotype were monitored. The proliferation capacity of cell cultures decreased during cultivation and was accompanied by enlargement of cells, this phenomenon being especially evident in the third and fourth passages. The expression of cartilage specific genes for collagen type II, aggrecan and cartilage non-specific collagen type I was determined. The mRNA levels for all three genes were obviously lower in the primo culture than immediately after isolation. During subsequent cultivation the expression of collagen type II decreased further, while there were only slight changes in expression of aggrecan and collagen type I. This study provides a valuable basis for testing of different tissue engineering applications in rabbit model, where auricular chondrocytes are considered as cell source.     

Assessment of the role of sutures in a lizard skull: a computer modelling study

Sutures form an integral part of the functioning skull, but their role has long been debated among vertebrate morphologists and palaeontologists. Furthermore, the relationship between typical skull sutures, and those involved in cranial kinesis, is poorly understood. In a series of computational modelling studies, complex loading conditions obtained through multibody dynamics analysis were imposed on a finite element model of the skull of Uromastyx hardwickii, an akinetic herbivorous lizard. A finite element analysis (FEA) of a skull with no sutures revealed higher patterns of strain in regions where cranial sutures are located in the skull. From these findings, FEAs were performed on skulls with sutures (individual and groups of sutures) to investigate their role and function more thoroughly. Our results showed that individual sutures relieved strain locally, but only at the expense of elevated strain in other regions of the skull. These findings provide an insight into the behaviour of sutures and show how they are adapted to work together to distribute strain around the skull. Premature fusion of one suture could therefore lead to increased abnormal loading on other regions of the skull causing irregular bone growth and deformities. This detailed investigation also revealed that the frontal-parietal suture of the Uromastyx skull played a substantial role in relieving strain compared with the other sutures. This raises questions about the original role of mesokinesis in squamate evolution.     

BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long‐term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long‐term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.