Osteomorphological Features Of The Hind Limb Bones In Saiga Antelope (Saiga Tatarica) In Long-Term Captivity And 3D Finite Element Simulative Analysis

The saiga antelope（Saiga tatarica）is a critically endangered species in the world.The wild population of the saiga antelope became extinct in our country in the 1960s and was reintroduced from abroad in the 1980s.For decades,China has protected the species through artificial captive breeding,which aims to help the rare species return to the nature.However,long-term captivity will inevitably lead to wild animal degradation,mainly manifested as the decline of locomotor ability.Locomotor ability is directly related to the survival state of animals in the wild.The hind limb bone is the main motor organ of the good runner.The bone morphology and material properties of hind limb are closely related to locomotor ability.Based on the bone adaptation to mechanical environment,the normal load of animal hind limb bone in different living environments will directly affect its morphology and material composition and other characteristics,so the study on the morphological adaptation characteristics of animal hind limb bone is helpful to predict its locomotor ability.To date,there are few studies on the hind limb bones of saiga antelope.In order to fully understand the bone characteristics of hind limb of saiga antelope and evaluate its locomotor ability,this research took domestic artiodactyls（such as sheep,Ovis aries）and wild artiodactyls（such as forest musk deer,Moschus berezovskii;roan antelope,Hippotragus equinus;mule deer,Odocoileus hemionus hemionus）as controls.The gross anatomical morphology of hind limb bone of saiga antelope was observed by gross anatomy method.Then micro-computed tomography（Micro-CT）,atomic force microscopy（AFM）and scanning electron microscope-energy dispersive X-ray（SEM-EDAX）were used to evaluate and analyze the bone microstructure,mechanical properties and mineral composition of the calcaneus of saiga antelope.The calcaneus of hind limb of saiga antelope was studied by compression mechanics test and three-dimensional finite element simulation.In this study,the structural and morphological characteristics,mechanical properties and mineral composition of the hind limb bone（calcaneus）of saiga were reported for the first time.The changes of calcaneal characteristics and locomotor ability of saiga antelope during growth and development were evaluated from the perspective of morphological,functional and adaptive integration,in order to provide scientific basis and important reference for the conservation and rehabilitation of the species and its future reintroduction.The main research results are as follows:1.Through the observation of anatomical structure of hindlimb bones of adult saiga antelope,it was found that the representative morphological characteristics of other bones of hindlimb of saiga antelope were similar to those of wild artiodactyls,but significantly different from domestic artiodactyls,except calcaneus.The representative characteristics are:deep acetabulum,high coverage,large anterior inclination,developed ischiatic trochosis,prominent gluteus line,deep tibial extensor groove,distal cranial side of the tibia with slender eminence extending along the proximal-distal direction,high intra-limb index of hind limb bone.However,linear indices of representative morphology of calcaneus of saiga antelope were similar to those of domestic artiodactylus,but significantly different from those of wild artiodactyls.Specifically,they are:the transverse width of the cubonavicular facet（LCNF）,the transverse width of body（TWB）,the mid body height（MBH）and length of the body of the calcaneus（LBC）were significantly different from those of the roan antelope（P<0.05）,but not from those of the sheep（P>0.05）.the greatest length of the calcaneus（GLC）and the height of the sustentacular facet（HSF）were significantly different from the roan antelope（P<0.01）.However,there was only significant difference between the two groups（P<0.05）.These results suggest that the plasticity of calcaneus is greater than that of other bones of hind limbs.However,whether calcaneus of saiga antelope will produce morphological response to the captive environment during short-term adaptation（ontogeny）remains to be further studied.2.Micro-CT was used to evaluate and compare the microstructure characteristics of calcaneus of saiga antelope in different age groups.Mule deer and sheep of the same age were used as controls.It was found that calcaneus cortical bone and cancellous bone would respond to the mechanical environment.The Cortical bone thickness ratio（CTR）,Percent cortical area（%CA）,Trabecular number（Tb.N）and Trabecular thickness（Tb.Th）,Bone volume fraction（BV/TV）and Connection density（Conn.D）were sensitive to external mechanical incentives and can change rapidly in response to changes in external mechanical environment during short-term adaptation（ontogeny）.CTR,%CA,Tb.N and Tb.Th in the mid-shaft of the calcaneus and Tb.N,BV/TV and Conn.D in the distal shaft of the calcaneus significantly decreased from sheep with growth and development（P<0.05）,while the differences of these index between saiga antelope and mule deer increased significantly with growth and development（P<0.05）.These indicated that the calcaneus of saiga antelope adapted to the low activity level of the routine load in captivity,and the adaptive changes of calcaneal microstructure are more likely to appear in the mid-shaft of calcaneal bone.3.Micro-CT,AFM and SEM-EDAX techniques were used to observe and compare the micro-nano structure,bone mineral density,nano mechanical properties and mineral composition of calcaneal trabecular bone of saiga antelope in different age groups.Sheep of the same age were used as the control.It was found that the degree of anisotropy（DA）,Calcium to phosphorus ratio（Ca/P）,Ca and Mn of trabecular bone in mid-shaft of calcaneus of saiga antelope were decreased with growth and development compared with sheep.Principal component analysis（PCA）showed that trabecular bone morphology was mainly related to DA,BMD,Ca/P,Ca,Mn and Fe,and the characteristics of trabecular bone in the dorsal calcaneal mid-shaft region of adult saiga antelope were similar to those of adult sheep.The above results indicated that the trabecular characteristics of calcaneal mid-shaft of saiga antelope were similar to those of sheep with growth and development to a certain extent.In addition,correlation analysis found that the five indexes of DA,BMD,Ca,Fe and Elastic modulus（E）of trabecular bone were significantly correlated with each other（P<0.05）,indicating that the structure,mineral composition and mechanical properties of trabecular bone were closely related in the process of trabecular bone reconstruction.4.Using biomechanical test and finite element analysis method,the compression failure test and 3D simulation reconstruction of the calcaneal bone of saiga antelope and sheep were carried out,and the macroscopic E（Saiga antelope=4747.51±914.42MPa,Sheep=2855.47±627.76 MPa）and Ultimate stress(σ_max)（Saiga=108.72±34.44MPa,Sheep=95.84±16.40 MPa）were much higher than that of cancellous bone(E _{saiga antelope}=452.42±123.46 MPa,E _sheep=459.46±58.90 MPa,σ_{max saiga antelope}=15.43±5.98MPa,σ_{max sheep}=22.40±2.62 MPa).It indicates that cortical bone is the main bearing structure.According to the measured mechanical parameters of the calcaneus of saiga antelope,the bone material attributes of the model were assigned.Through the simulation and analysis of the stress distribution and fracture risk of the acceleration model of hind limb of saiga antelope,it was found that the stress on the mid-shaft of the calcaneus of saiga antelope was significantly greater than that on the distal shaft,namely,mid-shaft of the calcaneus was the concentrated area of stress.The stress（126.91-252.46 MPa）on the dorsal and plantar side of the mid-shaft of the calcaneus is much greater than the measuredσ_max（108.72 MPa）of the calcaneal cortex of the saiga antelope,indicating that the mid-shaft of the calcaneus is prone to fracture injury when the saiga antelope runs at full speed.In conclusion,the shape plasticity of calcaneus of saiga antelope is greater than that of other bones of hind limbs.In the process of short-term adaptation（ontogenetic development）,the adaptive changes of calcaneal bones of saiga antelope tend to occur in the mid-shaft region where the forces are relatively concentrated,and CTR,%CA,Tb.N,Tb.Th,DA,Ca/P,Ca and Mn have obvious changes,which may lead to the reduction of mechanical properties of calcaneal mid-shaft.This increases the risk of fracture of the mid-shaft of the calcaneus.To a certain extent,this indicates that long-term confinement can cause calcaneal degeneration and locomotor ability decline of saiga antelope.This study provides reference for the population rejuvenation and artificial breeding management of saiga antelope,suggesting that some targeted training should be carried out in the breeding and management of saiga antelope,in order to avoid its bone degradation and improve its locomotor ability.