Rehabilitation protocol consisted of a 20-minute session, 3x/wk for 30 days. The animals were sequentially weighed and euthanized. The femur and tibia were assessed macroscopically and microscopically by scanning electronic microscopy (SEM). The SCI rats gained less weight than Sham-operated animals. Significant reduction of bone mass and periosteal radii was observed in the SCI rats, whereas PS and ES efficiently improved the macroscopic parameters. The SEM images showed less and thin trabecular bone in SCI rats. PS and ES efficiently ameliorated the bone microarchitecture deterioration by thickening and increasing the trabeculae. Based on the detrimental changes in bone tissue following SCI, the mechanical loading through weight bearing and muscle contraction may decrease the bone loss and restore the macro- and microanatomy.To study the effects of mechanical loading on bones after SCI, we assessed macro- and microscopic anatomy in rats submitted to passive standing (PS) and electrical stimulation (ES). The study design was based on two main groups of juvenile male Wistar rats with SCI: one was followed for 33 days with therapies starting at day 3 and the other was followed for 63 days with therapies starting at day 33. Both groups were composed of four subgroups ( Spinal cord injury (SCI) initiates a cascade of systemic effects that disrupts the normal neural, vascular, hormonal, mechanical, and molecular balance, resulting in bone loss . More thThe loss of bone tissue varies according to the anatomic region , and theAccording to our previous experimental study, a significant loss of bone density and strength was found in rats after SCI , which mn = 10). The animals were kept under standard laboratory conditions , housed in individual cages, and fed unrestrictedly with standard laboratory animal chow, containing 1.15% calcium and 0.88% phosphorus, and they were offered water ad libitum. The daily postoperative care consisted of a skin examination, neurological functional assessment, and bladder expression twice daily. All rats were administered (intramuscular) buprenorphine (0.03 mg/kg) twice daily for 5 days for postoperative pain and 10 mL Lactated Ringer's solution subcutaneously once daily for 5 days.Juvenile Wistar rats  were anesthetized by the intraperitoneal (IP) administration of ketamine  and xylazine . Complete SCI was achieved by surgical transection of the cord at the T10 level, as previously described –14. The The experimental protocol and all of the animal care described herein were previously approved by our Institutional Animal Care and Use Committee.n = 10 per group, Eighty rats were divided into 8 groups (The control groups (33 d and 63 d Sham rats) consisted of animals that were able to ambulate normally; these animals were not submitted to physical therapy. The SCI group consisted of SCI animals that were not submitted to physical therapy. The SCI + PS group consisted of SCI animals that began PS treatment either on postoperative day 3 (SCI + PS 33 d) or on postoperative day 33 (SCI + PS 63 d). The SCI + ES group consisted of SCI animals that began electrical stimulation  either on postoperative day 3 (SCI + ES 33 d) or on postoperative day 33 (SCI + ES 63 d). Therapies that were initiated on postinjury day 3 were applied as a preventive physical modality against bone loss caused by the paraplegia. Therapies that were initiated on postinjury day 33 were applied as a therapeutic physical modality to revert the significant bone loss caused by the 30 days of paraplegia . All treThe rats were placed in standing positions held by a custom-built static standing frame that maintained the hind limbs in extension, with the knees straight and the hind paws at a 90° angle with the level surface . Positioμs rectangular pulses delivered at 50 Hz with a 5 s on/15 s off duty cycle. The stimulation amplitude was adjusted (20 to 150 mA) to produce a concentric contraction that could be maintained for the stimulus duration (20 minutes).Electrodes were placed over the motor points of the quadriceps and triceps surae muscles. Stimulation was performed with 300 The goal of the muscle contraction was to cause ankle plantar flexion and knee extension through the range of motion for each joint. Rats were not anesthetized during the therapy but were restrained inside a polyvinyl chloride tube.At the end of each study time-point, the animals were weighed, which allowed the body mass to be compared at each time-point. The rats were then euthanized, and their bones were submitted to both macroscopic analysis and microscopic assessment by scanning electronic microscopy.The bones (tibias and femurs) were cleaned of soft tissues and then subjected to both qualitative and quantitative analyses. Qualitatively, the bones were compared among the groups, and then images were collected. Quantitatively, the bone mass, length, and perimeter were measured.2. The specimens were secured on aluminum stubs with conducting tape, coated with a thin layer of gold in a vacuum Bal-Tec SCD 050 (Fürstentum Liechtenstein) sputter coater, and studied with a Zeiss EVO MA10  scanning electron microscope in the secondary electron image (SEI) mode. Images were captured with an augment of 50x, whence the growth plate was used as the anatomical landmark for the superior limit of image.The distal femur and proximal tibia were sectioned in the coronal plane with a diamond-coated, low-speed saw, which measured 7 mm thick and 10 mm long. Sections were then washed repeatedly in distilled water, ultrasonicated, dehydrated in ascending grades of ethanol, and subjected to critical-point drying in COGeneral linear models were used to compare the mean body weights and macroscopic bone measures at each site between each group. Tukey's correction was applied to adjust for multiple comparisons. The level of significance was set at 5%.P > 0.05). At day 33, we observed that the SCI animals gained weight but not in the same proportion as the control group . The failure of the SCI rats to gain as much weight as the Sham rats persisted at the 63-daytime-point , indicating homogeneity among the animals (P < 0.05). At day 63, the reductions were 33% in the femoral mass, 27% in the femoral neck perimeter, and 25% in the diaphyseal perimeter (P < 0.05).Bones from the paraplegic rats showed deficits at increasing morphological measurements at varying levels, whence these measurements were more pronounced after 63 days of injury . At 33 dP < 0.01). When applied in a chronic stage, to reverse the previously established bone loss, PS significantly increased the femoral mass by 36%, the femoral neck perimeter by 31%, and the femoral diaphysis perimeter by 25% (P < 0.01). The increase in the femoral neck perimeter was sufficient to completely restore its value to that considered to be normal (P > 0.05 in the Sham versus SCI + PS groups).The PS treatment showed significant osteogenic effects not only at preventing bone loss but also at restoring the bone quality of the paraplegic rats. When applied in an acute stage (as a preventive therapy initiated 3 days after injury), PS significantly increased the femoral mass by 9% (P > 0.05) increases of 31% in the femoral mass, 9% in the femoral neck perimeter, and 10% in the diaphysis perimeter were found when ES was applied in a chronic stage.Although ES was not associated with significant prevention or reversion of the morphometric bone losses, nonsignificant . At day 63, the reductions were 34% in the tibial mass and 21% in the diaphyseal perimeter (P < 0.05).Similar to the results observed in the femur, the tibias from the paraplegic rats also failed at increasing the morphological measurements, when compared to the weight-matched controls ; these mNeither PS nor ES increased the morphometric parameters when they were applied as a prevention modality.P > 0.05 in the Sham versus SCI + PS groups). PS increased the tibial mass and the tibial diaphysis perimeter in 46% and 25%, respectively, of the SCI rats (P < 0.01).PS treatment in the chronic stage completely reversed the morphometric losses caused by the paraplegia, and normal values were restored (P > 0.05) increases of 4% in the tibial mass and 15% in the tibial diaphysis perimeter were observed when ES was applied at the chronic stage.Although ES did not significantly prevent or reverse the morphometric bone losses, nonsignificant (Morphological changes in the macroscopic anatomy of the tibia were observed following SCI. SEM images Figures  and 8 shFractures in SCI patients have particular aspects that may differ from typical osteoporotic bone fractures because of the neurological deficit. Lower extremities bones in people with SCI can receive neither compression forces from weight bearing nor traction forces from muscle contraction. Thus, SCI induces substantial bone loss in the regions below the injury , 11, 15.The lack of compression forces produces a greater negative impact at the trabecular bone of the epiphysis and metaphysis compared with the compact lamellar bone of the diaphysis, which is primarily subject to bending and torsional forces , 17. TheIt is known that the bone macroarchitecture influences bone resistance. The bone size and shape directly affect the bone strength, whereby the periosteal radius plays an important role in the bone resistance against bending loads , 9. For Many authors have studied the effects of PS and ES on the bone tissue in SCI patients. Some of these researchers have found that both PS –22 and EWe conclude that spinal cord injury causes detrimental changes in the macroscopic and microscopic anatomy of the bone tissue in paraplegic rats. Additionally, both mechanical loading through weight bearing and muscle contraction through electrical stimulation are efficient techniques at improving the normal anatomy of the long bones.


Salmo salar) precursor cells (aSPCs) from the adipogenic mesenchyme cell lineage to differentiate to the osteogenic lineage, aSPCs were isolated and cultivated under either osteogenic or adipogenic promoting conditions. The results strengthen the hypothesis that aSPCs most likely are predestined to the adipogenic lineage, but they also hold the flexibility to turn into other lineages given the right stimuli. This assumption is supported by the fact that the transcription factor pparγ , important for regulation of adiopogenesis, was silent in aSPCs grown in osteogenic media, while runx2, important for osteogenic differentiation, was not expressed in aSPCs cultivated in adipogenic media. After 2 weeks in osteogenic promoting conditions the cells started to deposit extracellular matrix and after 4 weeks, the cells started mineralizing secreted matrix. Microarray analyses revealed large-scale transcriptome responses to osteogenic medium after 2 days, changes remained stable at day 15 and decreased by magnitude at day 30. Induction was observed in many genes involved in osteogenic differentiation, growth factors, regulators of development, transporters and production of extracellular matrix. Transcriptome profile in differentiating adipocytes was markedly different from differentiating osteoblasts with far fewer genes changing activity. The number of regulated genes slowly increased at the mature stage, when adipocytes increased in size and accumulated lipids. This is the first report on in vitro differentiation of aSPCs from Atlantic salmon to mineralizing osteogenic cells. This cell model system provides a new valuable tool for studying osteoblastogenesis in fish.In order to study the potential plasticity of Atlantic salmon ( Salmo salar) abdominal cavity. We hypothesized that these cells from Atlantic salmon can be stimulated to differentiate into both the adipogenic and osteogenic lineages in vitro, as previously demonstrated for human subcutaneous fat derived stem cells  have the capacity to differentiate into a number of cell types, including osteoblasts, chondroblasts, adipocytes and myocytes . The pluem cells .The microenvironment of stem cells plays an important role in cell behavior, function and pre-determination of cell lineage fate . AdipogeIt is important to understand not only normal regulation of cell fate decisions, but also mis-regulation of mesenchymal differentiation that results in development of disorders. Trans-differentiation and cellular determination has been suggested to be involved in the disease states, such as vertebral malformations in salmon , and in Sparus aurata) calcified tissue and one derived from salmon white muscle precursor cells  are predestined to the adipogenic lineage, but that they also hold the flexibility to turn into other lineages given the right stimuli. In the presented study, we established a cell culture system where aSPCs differentiate into lipid accumulating adipocytes (aSACs) and mineralizing osteoblasts (aSOCs). Using lineage specific molecular markers, microarray and morphological observations, we characterized the differentiation process of aSACs and aSOCs from the common MSC origin aSPCs.Three models of osteoblast-like cell cultures derived from fish tissues have so far been described; two cell lines  and two or cells . HoweverNewly isolated aSPCs were small with a cytoplasm devoid of lipid droplets and morphologically very similar to fibroblasts A. They hpparγ mRNA was absent in cultures given osteogenic media and runx2 transcription was not detected in cultures given adipogenic media at day 9 (results not shown). The transcription of runx2 decreased at day 30, when the cells had high matrix deposition. Simultaneously an increase of alkaline phosphatase , prolyl 4 hydroxylase (p4oh), collagen 1a1 (col1a1), osteocalcin, and osteonectin mRNA levels was observed at day 30. In addition, bmp4 was detected at day 15 and annexin V (anxV) at day 30, however, these two genes were transcribed at very low levels at other stages and relative transcription could not be calculated (results not shown). Compared to aSACs, expression of col1a1, osteonectin (ns), p4oh, anxV and c/ebpb was higher in aSOCs, while level of substance P (sp) was lower, albeit insignificantly , which plays an important role in calcium homeostasis, was up-regulated at day 30. Thyroid receptors (TRs) have been demonstrated in rat, mouse and human osteoblasts  regulators of differentiation  was one of most up-regulated genes at day 30 . Several TFs are known to be involved in osteoblast differentiation. Sp9 or osterix is a runx2 activated TF essential for bone formation . Osteogenic medium also induced several genes coding for transporter proteins, including slc20a1b, ano5b and atp2b3a, which transfer calcium and phosphate and also osteoclast-specific pept1  and increase of osteogenic  mRNA transcription. This indicated that the precursor cells isolated from adipose tissue were pre-determined to the adipogenic lineage. This was further confirmed in aSPCs given only growth media, where the aSPCs started to accumulate lipids in their cytoplasm without being subjected to adipogenic differentiation media. Inactivation of adipogenic genes indicated that precursor cells isolated from adipose tissue differentiated into another lineage upon external cues. Osteogenic markers, such as alp and osteocalcin showed increased transcription in aSOCs, supporting the morphological observations that they were entering the osteogenic lineage. Somewhat unexpectedly, c/ebpβ, an important transcription factor in regulation of adipogenesis had increased mRNA expression in aSOCs. However, the gene product of c/ebpβ is also documented as a trans-activator of osteocalcin and col1a . Along with increased transcription of matrix producing genes, increased matrix secretion was observed in the cultures. All in all, specific gene expression profile along with microscopic examination of cellular morphology and secreted matrix strongly suggests that the aSPCs differentiated into cells belonging to the osteoblast lineage.Differentiation of aSPCs into aSOCs was characterized with loss of adipogenic  with a cost that was quite high. Induction was shown by a number of genes involved in the whole range of processes related to cell communication and proliferation, differentiation and metabolism, formation of cytoskeleton, cellular layers and extracellular matrix. High correlation of expression profiles at days 9 and 15 suggest consistency of changes. It is noteworthy, that major part of responses was transient and their manifestation ceased after visible signs of osteoblast maturation appeared as mineralized nodules in the culture at day 15. Striking magnitude and complexity of transcriptome responses could be partly accounted for by heterogeneity of the primary aSPCs culture: it might include diverse cell types that developed different reactions to the osteogenic medium. A complementary view is that reprogramming of differentiation switched on a search mode and sorting of options. As seen in the end of the experiment, maintenance of novel phenotype required a relatively small number of up-regulated genes and in this respect aSOC and aSAC were similar.in vitro system can be a promising model for studies of osteoblastogenesis in fish.Overall, our results confirmed that the primary culture established in the presented work belongs to cells that qualify as osteoblasts. In the aquaculture industry, numerous problems regarding bone development and mineralization are detected in response to nutritional and environmental challenges. Thus, the established g for 15 min and washed twice with L-15 before digestion with 0,125% collagenase for 60 min at 11°C. Cells were filtered through 250 and 100 µm filters and washed twice in L-15 and once in DMEM-growth media (DMEM-GM)  for 17 min. Cell suspension (2 ml) was gently put on top of the Percoll gradient and centrifuged at 1000 g for 27 min. The upper layer contains the mature cells, while the smaller stem cells reside in the middle layer. The middle layer was collected, centrifuged at 500 g for 10 min, supernatant was removed and pellet was dissolved in growth media. Cells were then seeded out and placed in an incubator, 12°C with 5% CO2. Medium was changed first after 24 h and then every 2–3 days. Cells were grown in triplicate cultures for each time-point on growth media until confluence, and the experiment was repeated 3 times. After reaching confluence, cells were subjected to either osteogenic differentiation media (DMEM-OB)  and visceral fat was harvested and collected in L-15 medium supplemented with 10 ml/l antibiotic/antimycotic . Tissue was minced and centrifuged at 250 DMEM-GM) . Cells wDMEM-OB)  or adipoDMEM-OB)  for 30 dDMEM-OB) . DiffereCells were harvested at day 7 (confluence), day 9 (2 days after addition of differentiation media), day 15 (one week in differentiation media) and day 30 (3 weeks after adding differentiation media). Cells were washed twice with PBS and harvested in DTTT and RLT buffer . Total RNA was isolated using an RNeasy® Mini Kit and QIAshredder columns with on-column RNase-Free DNase set (Qiagen), all in accordance with the manufacturer's protocols. The total RNA concentration and quality were determined by spectrophotometry . 1 µg of total RNA was reverse transcribed to cDNA in a total volume of 50 µl using an oligo(dT) primer and reagents from the TaqMan Gold RT-PCR kit .Primers  were des2O. Oil Red O was used to visualize lipid droplets in aSACs. Briefly, cells were fixated as described above. Oil Red O was added to the wells so that it covered the cells completely for 5 min. Oil Red was removed after 5 min and wells were washed in 60% isopropanol before the nuclei were stained with haematoxylin for 2 min. Cells were rinsed with water, glycerol added and cells microscopically analysed (Leica Biosystems).Differentiation of aSPCs into aSOCs was evaluated by analyzing the deposition of minerals in the extracellular matrix detected by Alizarin Red S staining in cells growing in control or mineralizing media. Medium was carefully aspirated from each well, washed twice with PBS and fixated in ice cold 70% ethanol for 1 hour at room temperature. Alcohol was removed and wells rinsed twice with PBS. 2% Alizarin Red S, pH 4.1–4.3, was added so that it covered the well and incubated at room temperature for 15 min with gentle shaking. Alizarin Red S was removed and wells washed four times with dH2-expression ratios was performed. The regulated genes were selected by expression difference between the time-points (day 7 was set as a reference) and the cell cultures, aSACs and aSOCs (p<0.05 and expression ratio >2-fold).Gene expression profiling was carried out at days 7, 9, 15 and 30. Equal inputs from five aSPCs, aSACs and aSOCs cultures were pooled in each sample; three replicates per culture and time-point were analyzed. Equalized control was prepared by mixing RNA from all samples. Nofima's Atlantic salmon oligonucleotide microarray (GPL10705) and bioinformatic system STARS were used. The platform includes 21,000 unique probes spotted in duplicate. Microarrays were manufactured by Agilent Technologies  and unless indicated otherwise, the reagents and equipment were from the same source. RNA amplification and labeling were performed with a Two-Colour Quick Amp Labelling Kit and a Gene Expression Hybridization kit was used for fragmentation of labeled RNA. Target samples were labeled with Cy5 and Cy3 was used for controls. The input of total RNA in each reaction was 100 ng. After overnight hybridization in an oven , arrays were washed with Gene Expression Wash Buffers 1 and 2 and scanned with a GenePix 4100A . GenePix Pro 6.0 was used for spot to grid alignment, assessment of spot quality, feature extraction and quantification. Subsequent data analyses were performed with STARS. After filtration of low quality spots flagged by GenePix, Lowess normalization of log


Cranial cruciate ligament (CCL) rupture is a very common cause of pelvic limb lameness in dogs. Few studies, using objective and validated outcome evaluation methods, have been published to evaluate long-term (>1 year) outcome after CCL repair. A group of 47 dogs with CCL rupture treated with intracapsular, extracapsular, and osteotomy techniques, and 21 healthy control dogs were enrolled in this study. To evaluate long-term surgical outcome, at a minimum of 1.5 years after unilateral CCL surgery, force plate, orthopedic, radiographic, and physiotherapeutic examinations, including evaluation of active range of motion (AROM), symmetry of thrust from the ground, symmetry of muscle mass, and static weight bearing (SWB) of pelvic limbs, and goniometry of the stifle and tarsal joints, were done.P <0.001) and flexion angles higher (P <0.001) in surgically treated than in contralateral joints, when dogs with no contralateral stifle problems were included (n = 33). In dogs treated using the intracapsular technique, the distribution percentage per limb of peak vertical force (DPVF) in surgically treated limbs was significantly lower than in dogs treated with osteotomy techniques (P =0.044).At a mean of 2.8 ± 0.9 years after surgery, no significant differences were found in average ground reaction forces or SWB between the surgically treated and control dog limbs, when dogs with no other orthopedic findings were included (n = 21). However, in surgically treated limbs, approximately 30% of the dogs had decreased static or dynamic weight bearing when symmetry of weight bearing was evaluated, 40-50% of dogs showed limitations of AROM in sitting position, and two-thirds of dogs had weakness in thrust from the ground. The stifle joint extension angles were lower (The average long-term dynamic and static weight bearing of the surgically treated limbs returned to the level of healthy limbs. However, extension and flexion angles of the surgically treated stifles remained inferior to healthy joints, and impairment of AROM and weakness in thrust from the ground in the surgically treated limbs were frequently present. Ground reaction forces may be inadequate as a sole method for assessing functional outcome after cranial cruciate ligament repair. Cranial cruciate ligament (CCL) rupture is a common cause of canine pelvic limb lameness and stifle joint osteoarthritis. Surgical treatment is advocated to stabilize the stifle joint, alleviate pain, treat any concurrent meniscal pathology, and decelerate the development of osteoarthritis . NumerouThe surgical outcome after CCL surgery has often been evaluated through clinical examination, radiography, and owner assessment. Force plate analysis has been the gold standard for objective evaluation of dynamic weight bearing, and it has also been used to evaluate outcome of CCL repair -12. All In most dogs, despite the surgical technique chosen, osteoarthritic changes progress after CCL surgery ,13-15 anIn veterinary orthopedic patients, the use of rehabilitation by physiotherapists that are specialized in animal physiotherapy has increased substantially during the last decade. Animal physiotherapists often use goniometric measurements, subjective or objective evaluation of muscle atrophy, and evaluation of weight bearing, posture, and thrust from the ground to evaluate the patient’s functional condition and response to given treatment. In cruciate disease research, stifle joint goniometric measurements and thigh circumference measurements have been used to evaluate surgical outcome in several studies ,16,18-23The primary purpose of our study was to evaluate surgical outcome at a minimum of 1.5 years after CCL surgery. This was done by combining force plate analysis, clinical orthopedic and physiotherapeutic evaluations, and radiography. The secondary purpose was to compare surgical techniques used to repair CCL rupture. Our hypothesis was that in the long term, the function of the surgically treated limb would remain inferior to the healthy limb.Based on a questionnaire study  and an aTwo additional subgroups were formed. Subgroup 1 dogs were used when force plate, static weight bearing, active range of motion and muscle atrophy results were evaluated. For these evaluations, only study group dogs with no signs of other orthopedic disease were included to minimize the effect of other conditions on the test results. On the other hand, when goniometric results in surgically treated limbs were compared to contralateral limbs, the study group dogs that did not have any findings in the contralateral stifle/tarsal joints, i.e. subgroup 2, were included. Subgroup 2 dogs were allowed to have hip dysplasia, elbow OA etc. because these findings were regarded to have no effect on the results of stifle or tarsal goniometry.For the force plate and physiotherapeutic evaluation, a group of 21 clinically healthy Labrador Retrievers and Rottweilers, voluntarily presented by their owners, was used . InclusiWith permission from the owners, the surgical records were reviewed to ascertain the surgical date, technique used, and the surgically treated limb and to obtain information about the meniscal tear and possible complications or revision surgery needed.A full orthopedic examination was done to all dogs and included lameness evaluation on a scale from 0 to 4 (no/mild/moderate/severe weight bearing/non-weight bearing)  and palpThe stifle, hip, and elbow joints bilaterally as well as the lumbar spine of the study group dogs were radiographed under sedation. The amount of osteoarthritis in stifle joints was evaluated from mediolateral and craniocaudal views (no/mild/moderate/severe)  in hip j2.Gait was evaluated using a piezoelectric force plate  embedded in a 14 m runway and computer-based software program . Three pairs of photoelectric cells, positioned 1 m apart from each other, were used to measure the velocity and acceleration of the dogs. Dogs were guided over the force plate by the owner or a member of the research team at a trotting velocity of 2.10-2.50 m/s and a maximum acceleration of ±0.5 m/sSTPL is the PVFs recorded in the surgically treated limbs, PVFCPL in the contralateral pelvic limbs, PVFITL in the ipsilateral thoracic limbs and PVFCTL in the contralateral thoracic limbs. The DPVFs were calculated similarly for contralateral pelvic and thoracic limbs of surgically treated dogs as well as thoracic and pelvic limbs of control dogs.Data from five valid trials for each side were normalized for the dog’s body mass and averaged. Peak vertical forces (PVFs) and vertical impulses (VIs) in thoracic and pelvic limbs were evaluated. In addition, distribution percentages per limb of peak vertical forces (DPVFs) was calculated using the equationCPL/L is the value recorded in the contralateral (study group) or left (control group) pelvic limb and XSTPL/R is the value recorded in the surgically treated (study group) or right (control group) pelvic limb. In surgically treated dogs, a symmetry index value of 0 indicates perfect symmetry; a positive value indicates decreased dynamic weight bearing on the surgically treated limb and a negative value decreased dynamic weight bearing on the contralateral pelvic limb. The symmetry indices were calculated similarly for control group dogs, but absolute values of the symmetry indices were used. Thus, only the magnitude of the symmetry index value was evaluated, while side (right or left pelvic limb) of deviation from perfect symmetry was disregarded. The control group values were used to determine the cut-off value for differentiation between normal and lame dogs by using the equation: Mean + Standard deviation. Based on the cut-off value, the surgically treated dogs were assigned to have normal gait symmetry if SI was between the negative and positive cut-off values, lame on the surgically treated limb if SI was greater than cut-off value, or lame on the contralateral pelvic limb if SI was lower than negative cut-off value.A symmetry index (SI)  was calcA physiotherapeutic examination was done by a physiotherapist specialized in veterinary physiotherapy, as previously described . The fulWhen evaluating functional AROM and symmetry of thrust, the dog was led over a 20-m distance and asked to sit and sit-to-move 3 times, and similarly lie-down and lie-to-move 3 times within equal distances. The position of pelvic limbs during sitting and lying was observed, and possible functional limitations or compensations of the pelvic limbs in sitting or lying position  or weakness of pelvic limbs in thrust from sitting or lying positions were recorded . SubjectCPL is the value recorded in the contralateral pelvic limb and SWBSTPL the value recorded in the surgically treated limb [Quantitative measurement of SWB in pelvic limbs was done using digital bathroom scales, as previously described . Two ideted limb . Based oted limb , the surPROM was measured in un-sedated dogs from both stifle and tarsal joints in extension and flexion using a universal goniometer with a five-degree scale. The measurements were taken in lateral recumbency three times and averaged . In addiStatistical analysis was done using the SPSS software program . Kolmogorov-Smirnov and Shapiro-Wilk tests were applied to determine whether the data were normally distributed. Results from nominal and ordinal data , symmetry index and DiffSWB% are reported as frequencies and percentages. For continuous data , the results are summarized as mean ± SD, and for age, body mass, and follow-up also as range.P <0.05 was considered significant.The Kruskall-Wallis test with pairwise comparisons was used to evaluate differences in age, follow-up, stifle joint palpation findings, and amount of radiographic osteoarthritis between surgical techniques, and Independent samples t-test or one-way ANOVA was used to evaluate differences in body mass between study group and control dogs or surgical techniques. The Mann-Whitney rank sum test for non-normally distributed and Independent samples t-test for normally distributed force plate, SWB%, and PROM data were used to evaluate differences between surgically treated limbs and contralateral limbs  or control dog limbs . Similarly, the Kruskall-Wallis test with pairwise comparisons for non-normally distributed and one-way ANOVA with Bonferroni and Tukey post hoc analyses for normally distributed force plate, SWB%, and PROM data were used to evaluate differences between surgical techniques. From the 278 questionnaire results, 47 dogs  with owners willing to bring their dog to the evaluation fulfilled the inclusion criteria. The breeds of these dogs were Labrador Retriever (15), Rottweiler (6), mixed breed (4), Golden Retriever (3), Bernese Mountain Dog (2), Doberman Pincher (2), Newfoundland Dog (2), Nova Scotia Duck Tolling Retriever (2), and one each of Beauceron, Black Russian Terrier, Bordeaux Dog, Bullmastiff, Collie, Dalmatian, Giant Schnauzer, Karelian Bear Dog, Short-Haired German Pointer, South African Boerboel and Wheaten Terrier. The mean ± SD (range) age of the dogs and their body mass at the time of evaluation were 6.9 ± 2.7 (1.8-13.0) years and 38.2 ± 9.5 (17.5-60) kg, respectively.P =0.001) or extracapsular  technique groups.Of the pelvic limbs , 19 limbs (40.4%) had been surgically treated using a modification of the original intracapsular technique , with anP <0.001). No significant difference was found in body mass between the healthy control group and study group dogs.The mean ± SD age and body mass of the healthy control dogs  were 3.2 ± 1.6 years and 35.5 ± 8.2 kg, respectively. The healthy control dogs were significantly younger than the dogs in the study group . One dog treated with the intracapsular technique had grade 2/4 lameness. In addition, 7 dogs had signs of lameness in the contralateral pelvic limb  and 2 dogs in both front limbs (grade 1/4). The gait could not be evaluated in one surgically treated dog due to lack of cooperation. The 21 healthy control dogs did not show any signs of orthopedic disease or lameness.P =0.030) or intracapsular (P =0.028) group, and amount of periarticular swelling was significantly lower in the osteotomy than in the extracapsular group (P =0.047). No significant differences were found between surgical techniques in pain response to stifle flexion/extension. Of 12 dogs with positive drawer sign, 5 had been treated with intracapsular and 7 with osteotomy techniques. Tibial compression test was negative in all stifle joints. The amount of radiographic osteoarthritis was lower in the osteotomy group than in the intracapsular group (P =0.007).Orthopedic and radiographic findings in the surgically treated stifle joints are presented in Table In the contralateral stifle joints clinical findings were recorded in 9 dogs, including crepitation in all dogs, periarticular swelling in 8, pain in 3, and decreased ROM in 2 dogs. Radiographic signs of osteoarthritis were seen in 11 dogs, and drawer sign was positive in 3 dogs. Concurrent clinical findings were also seen uni- or bilaterally in hip joints of 17 dogs, elbow joints of 8 dogs, and shoulder joints of 6 dogs. Pain on palpation of the spine was found in 19 dogs. Radiographic findings of osteoarthritis were seen uni- or bilaterally in the hip joints of 21 dogs, elbow joints of 4 dogs, and the lumbosacral spine of 13 dogs.Based on orthopedic and radiographic examinations, 21 dogs were included in subgroup 1 and 33 dogs in subgroup 2.2, respectively. Body mass, trotting velocity or acceleration did not differ significantly between the study group or subgroup 1 and the control group dogs, between the surgically treated and the contralateral limbs or between the surgical technique groups.In 6 dogs the force plate analysis could not be performed due to lack of cooperation or inability to maintain adequate trotting speed. Mean ± SD trotting velocity and acceleration of the study group (n = 41), subgroup 1 (n = 21) and control group dogs (n = 21) were 2.25 ± 0.07, 2.27 ± 0.07 and 2.26 ± 0.06 m/s, and - 0.05 ± 0.12, - 0.09 ± 0.13 and - 0.05 ± 0.14 m/sWhen force plate results for the study group were evaluated, no significant differences emerged when comparing the surgically treated limb with the contralateral pelvic limb or with the pelvic limbs of control dogs.P =0.040) and DPVF (P =0.005) than contralateral pelvic limbs, but no significant differences were found when surgically treated limbs were compared with control dog limbs . The dogs that had been treated with the intracapsular technique (n = 7) had significantly lower VI (P =0.035) and DPVF (P =0.018) in the surgically treated limb than in their contralateral pelvic limb. No significant differences were seen when limbs treated with the osteotomy technique (n = 11) were compared with their contralateral pelvic limbs. The number of dogs treated with the extracapsular technique (n = 3) was too low to be analyzed.When force plate results of different surgical technique groups were evaluated, no significant differences were found in study group between intracapsular, extracapsular and osteotomy techniques, or either in study group or subgroup 1 when surgically treated limbs in each technique group separately were compared with pelvic limbs in control dogs. Subgroup 1 dogs that had been treated with the intracapsular technique had significantly lower DPVF in the surgically treated limb than the dogs treated with osteotomy techniques  , decreased flexion of the stifle joint in 10 dogs (47.6%) , and abduction of the limb in 9 dogs (42.9%) . Except for one dog, the positive findings were always localized in the surgically treated limbs. Thrust from the sitting position was weaker in the surgically treated limb in 14 dogs (66.7%) . When evaluating muscle atrophy in subgroup 1, altogether 16 dogs (76.2%) had decreased muscle mass of the surgically treated pelvic limb on palpation , while only 4 dogs (19.0%) had symmetrical muscle mass , and one dog (4.8%) had decreased muscle mass in the contralateral pelvic limb.P =0.010 and P =0.006, respectively), but no significant differences were found when surgically treated limbs were compared with control dog limbs  , but in the intracapsular group (n = 7) the SWB% in the surgically treated limb was significantly lower than that in the contralateral limb (0) Table . The numP <0.001) were significantly lower and stifle flexion (P <0.001) and tarsal flexion (P =0.002) angles were significantly higher (loss of flexion) in the surgically treated limb (Table P =0.045) were significantly higher (loss of flexion) than in contralateral limbs. No significant differences were found between the surgical techniques.The PROM was not evaluated in 3 dogs due to lack of cooperation and in one dog due to concurrent orthopedic findings in the tarsal joint. When PROMs of surgically treated limbs in subgroup 2 were compared with contralateral limbs, stifle extension angles  between the surgically treated limbs and the pelvic limbs of control dogs as well as in VI between the surgically treated and the contralateral limbs. However, the PVF of surgically treated limbs was significantly lower than the corresponding value for the contralateral pelvic limbs. The mean (SD) difference in PVF between the surgically treated and contralateral limbs was 4.7% (7.9), but it must be taken into account that load distribution from the injured to the contralateral pelvic or other limbs often occurs and artificially increases the difference between limbs . FurtherWhen the differences are interpreted, the clinical importance of changes must be considered. Our results indicate that the mean differences between the surgically treated and healthy limbs were minor and dynamic weight bearing of the surgically treated limbs had returned to the level of healthy limbs. Similar long-term studies have not been reported before, but these results are in accordance with those of previous short- and mid-term force plate studies where the function of the surgically treated limb was at the level of the contralateral or control dog limbs 7 months after an extracapsular repair , or 4 anAlthough the average differences in ground reaction forces between surgically treated and healthy limbs were minor, the SIs for PVF and VI were greater than cut-off values in almost one-third of dogs, indicating clinically relevant problem of weight bearing in these dogs and revealing considerable variation between the evaluated dogs. By using the SIs, more differences were revealed than what was discovered when the mean ground reaction forces between the surgically treated and healthy limbs were compared.A physiotherapeutic examination concentrating on the stifle joints and performed by a veterinary physiotherapist was included in our study. To our knowledge, evaluation of AROM, thrust of pelvic limbs, or SWB has not been used to evaluate surgical outcome after CCL repair. Interestingly, compensations of AROM were seen in 40-50% of the dogs. In addition, the compensations of AROM were almost always localized to the surgically treated limb, not to the contralateral healthy limb.Static weight bearing of the pelvic limbs was measured using commercial bathroom scales, and validity study of their results has been reported elsewhere . The meaGoniometry of the stifle joints has been evaluated after CCL repair in several studies ,16,18-23In addition to loss of extension and flexion of the stifle joint, also flexion of the tarsal joint was significantly decreased in the surgically treated limbs. Loss of flexion in the tarsal joint might be explained by the anatomical fact that in order to measure full flexion of the tarsus a full flexion of the stifle joint is required and thus the limitations in the stifle joint flexion also may affect ipsilateral tarsal joint flexion angles.An interesting aspect in the long-term assessment of CCL repair is the occurrence of pain. In our study, in almost one-third of dogs the symmetry indices for PVF and VI indicated lameness and the DiffSWB% indicated decreased weight bearing in the surgically treated limb, likely not only reflecting the functional outcome, but also pain in the surgically treated limb. Moreover, in the orthopedic examination, lameness was seen in almost 20% and pain response on flexion and/or extension of the surgically treated stifle joints in one-third of dogs. These findings resonate well with our previous report of chronic pain after CCL surgery, which is based on a validated questionnaire filled out by 253 dog owners and includes the 42 dogs serving as subjects here. In that report, almost one-third of the dogs had signs of chronic pain at a mean of 2.7 years after CCL surgery . RecogniThere are limitations in our study. Our patient material was very heterogeneous, with many confounding factors. Due to the retrospective study design we were unable to control such variables as conformation and age of the dog, duration of clinical signs before surgery, completeness of the cruciate ligament tear, meniscal pathology, and treatment or postoperative rehabilitation and medications. However, our patient material can be used to assess general surgical outcome and it better reflects a real-life situation with heterogeneity in patient material, surgical procedures, and postoperative treatments. The confounding factors may nevertheless have caused bias in the comparison of surgical techniques. Therefore the results must be interpreted with some caution.The number of patients was unfortunately low. Due to the limitations set by the force platform, only dogs with a body mass exceeding 17 kg were included. For force plate and physiotherapeutic evaluation, we only included dogs with no other concurrent orthopedic problems (subgroups 1 and 2) in order to minimize the effect of concomitant problems on dynamic or static weight bearing, AROM, and PROM. Although only the dogs with unilaterally treated CCL rupture and no other owner-reported significant orthopedic or neurologic problems were originally included in the study group, during the evaluation surprisingly many of them were diagnosed with bilateral stifle joint pathology or other concurrent orthopedic problems, resulting in a low number of dogs for final evaluation. To detect a statistical significance in PVF between the surgically treated and control dogs using the data generated from this study, 400 dogs would have been needed (using α =0.05 and 80% power). On the other hand, it may be speculated that even if a statistical significance had been shown, a difference of 1.6% in PVF between the surgically treated and control dog limbs might anyway not be clinically significant.In force plate analysis, we decided to compare the ground reaction forces to both contralateral and control dog limbs, both of which have some disadvantages. Due to weight redistribution to the contralateral pelvic limb or thoracic limbs, the use of the contralateral pelvic limb as a reference has been questioned . On the In the physiotherapeutic examination, only a subjective assessment of muscle mass symmetry was used when evaluating muscle atrophy. We could have improved the test by measuring atrophy with a tape measure, thus quantifying the measurement and increasing the objectivity of the method. On the other hand, in a recent study , the subThe postoperative treatment did not include any special rehabilitation program and only one of the dogs had had postoperative rehabilitation by a physiotherapist. Previous studies have shown that postoperative rehabilitation significantly improves the surgical outcome , and nowThe dogs treated with osteotomy techniques had less crepitation in the surgically treated limb than dogs treated with intra- or extracapsular techniques. In addition, the amount of radiographic osteoarthritis was lower in the osteotomy group than in the intracapsular group. These results must be interpreted with caution because of potential initial preoperative differences in the orthopedic or radiographic examination between the surgical technique groups. On the other hand, it could be speculated that the preoperative differences in radiographic osteoarthritis between groups might level off in the long run. Also, in two recent studies the preoperative amount of osteoarthritis was reported to correlate negatively with the progression of osteoarthritis postoperatively, meaning that dogs with severe pre-existing osteoarthritis would have less potential for osteoarthritis to worsen, compared with dogs with only minor pre-existing osteoarthritis ,41.The low number of dogs treated with extracapsular technique did not allow comparison of ground reaction forces or SWB of this group to other technique groups. The ground reaction forces and SWB% in the osteotomy group had a tendency to be higher than in the intracapsular technique and to reach control dog values. The dogs that had been treated with the intracapsular technique had significantly lower DPVF in the surgically treated limb than the dogs treated with the osteotomy technique. No other significant differences emerged between surgical technique groups when dynamic and static weight bearing or goniometric results were evaluated. No significant differences between surgically treated and contralateral or control dog limbs were found in the osteotomy group, whereas in the intracapsular group statistically significant differences in VI, DPVF and SWB between the surgically treated and contralateral limbs were achieved. Parallel results have been reported also in two previous force plate studies. Conzemius et al.  stated tThe average long-term dynamic and static weight bearing of the surgically treated limbs returned to the level of healthy limbs. However, when symmetry of weight bearing was evaluated using cut-off values, approximately 30% of dogs had decreased static or dynamic weight bearing in the surgically treated limb, and almost one-third of dogs showed pain in stifle joint palpation. In addition, the extension and flexion angles of the surgically treated stifles remained inferior to healthy joints, and many dogs showed impairment of AROM and weakness in thrust from the ground in the surgically treated limbs. In sum, measurement of ground reaction forces may be inadequate as a sole method for assessing functional outcome and thus more objective outcome assessment methods should be included in evaluation of CCL repair.


This study evaluated the associations between lymphatic and vascular invasion of oral cavity squamous cell carcinoma (OSCC) and clinicopathological manifestations, as well as their impact on patient outcomes after treatment.In total, 571 patients with primary OSCC who underwent surgery with or without adjuvant therapy were enrolled.P < 0.001 and P = 0.020, respectively), tumor stage , nodal metastasis (both P < 0.001), extracapsular spread (both P < 0.001), perineural invasion (both P < 0.001), bone invasion , depth of invasion , and pathologic differentiation . In the analysis of adverse events during follow-up, neither lymphatic nor vascular invasion was statistically associated with local recurrence, neck recurrence, and distant metastasis. Although lymphatic invasion exhibited significant associations with poorer overall survival (P < 0.001), disease-specific survival (P < 0.001), and disease-free survival (P = 0.01), it was not demonstrated to be an independent prognostic factor in all multivariate analyses.Lymphatic and vascular invasion were found in 28 (5%) and 16 (3%) patients, respectively. Significant associations were found between lymphatic and vascular invasion and overall stage (Although both lymphatic and vascular invasion are associated with many clinicopathological manifestations, neither affects the occurrence of locoregional recurrence and distant metastasis in patients with OSCC after treatment. Numerous studies have demonstrated that tumor size, histological differentiation, nodal stage, and perineural invasion can predict recurrence and survival in patients with OSCC.5 However, representing an important step in tumor progression toward regional and distant metastasis, the impact of lymphatic and vascular invasion on prognosis and survival has not yet been fully clarified. Although they have long been considered important histopathological features for locoregional recurrence or even distant metastasis, currently available cohort studies have not fully addressed the role of lymphatic and vascular invasion in terms of clinicopathological factors and treatment outcomes.Oral cavity squamous cell carcinoma (OSCC) is the most common oral malignancy, representing 90% of all malignant neoplasms of the oral cavity.To the best of our knowledge, no cohort study examined lymphatic and vascular invasion separately in terms of the prognosis and survival of patients with OSCC. The aim of this cohort study was to examine the associations of lymphatic and vascular invasion with other clinicopathological manifestations, such as perineural invasion and extracapsular spread, the failure patterns after OSCC treatment, and the outcome of patients with OSCC who were treated by surgical resection as the primary modality with or without the use of adjuvant treatment. The current study also evaluated whether lymphatic and vascular invasion may provide supplemental prognostic value in addition to the current TNM system. In addition, the prognostic value of lymphatic and vascular invasion for predicting locoregional control, distant metastasis, and overall survival was also evaluated.Between August 2002 and December 2012, 571 consecutive patients who were diagnosed with OSCC and who underwent surgery as the primary modality of therapy at Chang Gung Memorial Hospital  were enrolled in the study. All patients were followed up for at least 18 months or until death. Patients with at least one of the following conditions were excluded from the study: any prior history of malignancy, unresectable disease, other primary cancer (synchronous or metachronous), recurrent cancer, distant metastasis at presentation, initial treatment with neoadjuvant therapy, or a medical contraindication for surgery. Lesions diagnosed as carcinoma in situ, verrucous carcinoma, or a histologically basaloid subtype were also excluded from the study. This study was approved by the hospital Institutional Review Board.6 After surgical treatment, the pathological tumor and nodal staging (TNM) staging was performed according to the AJCC Cancer Staging 2010 guidelines to determine whether adjuvant treatment was indicated.7Patients in the study underwent standard preoperative workups according to institutional guidelines, including a detailed history, complete physical examination, computed tomography or magnetic resonance imaging, chest radiographs, bone scan, and abdominal ultrasonography. The primary tumors were excised with adequate surgical margins, and the tumor margin tissues were sent for cryosectioning and examination intraoperatively to ensure that the surgical margins were tumor free. Various types of neck dissection were performed according to the primary tumor site and clinical lymph node status.Postoperative radiotherapy was performed on patients with pathologic T4 tumors and positive lymph nodes within 6 weeks following surgery. Patients with any pathologic findings such as metastasis in multiple neck lymph nodes, extracapsular spread, positive surgical margins, nodal dissemination at level 4 or 5, and perineural invasion received adjuvant concurrent chemoradiotherapy. The chemotherapy was a cisplatin-based regimen, and the total radiation dose was 66 Gy. The prescribed dose was delivered in fractions of 1.8–2 Gy per day for 5 days per week. All patients completed regular follow-up visits every 2 months for the first year after discharge, every 3 months for the second year, and every 6 months thereafter.11The histological differentiations were determined on the basis of the original pathology reports examined by multiple pathologists at this single institute. Two pathologists (L-YL and YH) were invited to review the specimens again independently for quality control of these pathological diagnoses. For each patient, we recorded the following data: anatomic subsite of the oral cavity, histopathological findings of tumor differentiation, bone invasion, perineural invasion, tumor depth, tumor status, lymph node status, overall stage, extracapsular spread status, lymphatic invasion, and vascular invasion. Lymphatic and vascular invasion were reported in the whole series based solely on hematoxylin and eosin staining Figure . Lymphatχ2 test, Fisher exact test, Wilcoxon test, or the Kruskal–Wallis test, where appropriate. The factors evaluated included sex, age, TNM staging, and other pathological findings. The time to events, including local recurrence, neck recurrence, distant metastasis, and death, from the date of surgical removal of the primary tumor was calculated for each patient. Multivariate regression analyses were applied to define specific risk factors for overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS). Concerning the patients’ survival, the survival rate is estimated by Kaplan–Meier plotting and compared by log-rank test. For Kaplan–Meier plotting, in OS, the event is defined as the patient death directly from OSCC or from an unrelated cause. In DSS, the event is specified only as the death related to OSCC. In DFS, the event is defined as the patient death or any tumor relapse occurring loco-regionally or distantly. Statistical analyses were performed using SAS software . All patients received follow-up consultations at our outpatient clinic until July 2014 or death. All P values were 2-sided with the significance level set at P < 0.05.Patient and OSCC tumor characteristics were stratified according to various clinicopathological factors and evaluated using the P = 0.842) or age group (P = 0.894). The same finding was noted for vascular invasion regarding sex (P = 0.693) and age (P = 0.218). The patient characteristics that correlated with the lymphatic and vascular invasion of OSCC are listed in Table The age at diagnosis for the 571 patients with OSCC ranged from 21.9 to 86.8 years . The study population consisted of 516 men and 55 women. The tumor subsites were the buccal mucosa (209 patients), gum (83), hard palate (15), lip (18), floor of the mouth (35), and tongue (211). Lymphatic invasion was reported in 28 (4.9%) patients, and vascular invasion was reported in 16 (2.8%) patients. Lymphatic invasion was not found to be associated with a particular sex (P = 0.009), nodal metastasis (P < 0.001), extracapsular spread (P < 0.001), perineural invasion (P < 0.001), bone invasion (P = 0.004), depth of invasion (P < 0.001), and pathologic differentiation (P = 0.002). Regarding vascular invasion, significant associations were noted with T classification (P = 0.025), nodal metastasis (P < 0.001), extracapsular spread (P < 0.001), perineural invasion (P < 0.001), depth of invasion (P = 0.001), and pathologic differentiation (P < 0.001), whereas no significant association was observed with bone invasion (P = 0.327). Overall, these associations indicated that the histopathological findings of lymphatic and vascular invasion in the primary OSCC tumors were associated with positivity for cervical metastasis, extracapsular spread, perineural invasion, poor differentiation, and deeper tumor depth.Table In the analysis of adverse events affecting survival during follow-up, including local recurrence, neck recurrence, and distant metastasis, neither lymphatic nor vascular invasion was found to correlate with any of these variables. In addition, there was no difference in the duration from treatment to local recurrence, neck recurrence, and distant metastasis in the same comparison between the 2 groups Table .P < 0.001; Figure P = 0.511; Figure P < 0.001; Figure P = 0.247; Figure P = 0.01; Figure P = 0.452; Figure P = 0.002, 0.010, and 0.048, respectively; Table P = 0.133) and vascular invasion (P = 0.730) were not predictive of OS. Similar analyses also revealed that lymphatic invasion is also not an independent predictor of DSS and DFS .The association of lymphatic and vascular invasion with patients’ survival, including OS, DSS, and DFS, was further evaluated. Survival analysis revealed that the 5-year OS rates for patients with and without lymphatic invasion were 49.3% and 70.3%, respectively. These differences in OS were significant as compared by the log-rank test .15 Other investigators examined lymphovascular invasion in T1-T2 OSCC and reported an incidence in the range of 8% to 28%.17 The incidence of overall lymphovascular invasion in the current study was compatible with that in previous reports as lymphatic invasion was found in 28 (5%) patients, whereas vascular invasion was found in 16 (3%) patients. To the best of our knowledge, the current study contains the largest cohort of patients with OSCC reported to date in an attempt to analyze lymphatic and vascular invasion as 2 separate entities for examining their relationship with other clinicopathological in OSCC tumors and their impact on the survival and treatment outcome of the patients.Tumor invasion into lymphatic and/or blood vessels has long been postulated to be an important pathologic factor. However, its impact on tumor locoregional control and recurrence remains to be elucidated. An assessment of the presence and extent of lymphovascular invasion was proposed by Jakobbson et al as a part of the multifactorial grading system.18 Other studies reported the association of lymphovascular invasion with tumor site, thickness, perineural invasion, and status of resection margin.20 Additionally, many previous studies also found an association between lymphovascular invasion and cervical nodal metastasis,25 but a recent series did not find such association in T1-T2 cases of OSCC patients.26 The current study represents the first cohort study examining lymphatic and vascular invasion, respectively, in relation to clinicopathological characteristics. Lymphatic and vascular invasion were both significantly associated with T classification, N classification, perineural invasion, extracapsular spread, depth of invasion, and degree of differentiation.Poleksic et al first linked vascular invasion to aggressive tumor characteristics in head and neck SCC.27 This finding was also supported by the observation of Chen et al in disease control of early-stage OSCC and of Tai et al in locoregional control of early-stage tongue SCC.28 However, other investigators have linked lymphovascular invasion with locoregional recurrence or distant metastasis.31 In the current study, the first examining the lymphatic and vascular invasion independently, we found that neither lymphatic nor vascular invasion impacts locoregional recurrence or distant metastasis after treatment. Furthermore, although some previous reports demonstrated an association of lymphovascular invasion with poorer patient survival,32 other reports showed no such association.28 In our study, vascular invasion was not found to affect patient survival, whereas lymphatic invasion was associated with worse OS, DSS, and DFS. In further evaluation of the prognostic significance by multivariate survival analysis, lymphatic invasion was not identified as an independent factor. These results suggest that lymphatic invasion is not an independent determinant of patient survival after controlling for all clinicopathological factors, whereas the depth of primary tumor invasion, nodal stage, and extracapsular spread are.The relation of lymphovascular invasion with the treatment outcome of OSCC remains unclear. Regarding the effect of lymphovascular invasion on treatment failure, Fagan et al reported that vascular and lymphatic invasion were not significantly associated with local recurrence in 142 patients with head and neck SCC.33 Indeed, offering adjuvant radiotherapy to patients with low-stage OSCC tumors and concomitant risk factors would potentially expose a subpopulation to unnecessary, harmful toxicities, and this overtreatment would have negative effects on patient quality of life resulting from late adverse sequelae, such as neck soft tissue fibrosis, dysphagia, xerostomia, radionecrosis, or radiation-induced malignancies.34 Therefore, the precise identification of low-stage patients who are at high risk of disease progression or recurrence would represent a tremendous advantage in the management of this disease.35 Besides, according to the current study, lymphatic and vascular invasion both have no influence on the adverse events affecting locoregional control or distant metastasis during follow-up. Moreover, lymphatic invasion was not an independent prognostic factor in survival analysis. A large, prospective cohort study will be necessary to determine the efficacy of using vascular or lymphatic invasion as criteria for adjuvant chemotherapy and/or radiotherapy.Efforts have been centered upon modalities for the postoperative management of patients with OSCC in light of their outcomes on an individual basis. Postoperative adjuvant radiotherapy is offered for patients with head and neck malignancies with large primary tumors or nodal metastasis. With evidence emerging from randomized control trials on the use of postoperative chemoradiotherapy in head and neck cancers, most centers administer postoperative chemoradiotherapy for patients with head and neck cancers and high-risk factors.Because lymphatic and vascular invasion were discovered to be strongly associated with various pathological factors mentioned above Table , we foun5 Some investigators used immunohistochemistry as a routine method of staining and reported a higher incidence of lymphatic and vascular invasion separately in OSCC using immunohistochemistry techniques , but the incidence of both lymphatic and vascular invasion was also remarkably variable, as the incidence of lymphatic invasion was reported as 54% and 25%, respectively, and that of vascular invasion was reported as 29% and 49%, respectively.37 Moreover, this method is currently not commonly used by most pathologists, and there is no current consensus on the usage of any specific antibodies over than the traditional histopathological examination via hematoxylin and eosin staining for diagnosis or a standardized protocol for immunohistochemical staining. Consequently, these immunohistochemical methods are still considered investigational techniques and not included as part of the routine pathological evaluation.38The major limitations of this study are its retrospective design and dependence solely on traditional histopathological examination using hematoxylin and eosin staining, making it difficult to exclude the true absence of lymphatic and vascular invasion in OSCC tumors. Although the criteria for 2 parameters are quite consistent, there may be significant variability in the processing of the specimens in the routine pathological practice. The thorough and serial examination of the whole cancer specimens is not usually possible in the routine pathological examination of most hospitals. These facts might affect or even underestimate the true incidence of lymphatic/vascular invasion of the OSCC tumors. Although lymphatic/vascular invasion is commonly reported on routine examination using hematoxylin and eosin staining, immunohistochemistry appears more effective for detecting lymphatic and vascular invasion.Although both lymphatic and vascular invasion are associated with many clinicopathological manifestations, neither affects the risk of locoregional recurrence and distant metastasis in posttreated OSCC. Furthermore, in the survival analyses, we found that neither lymphatic nor vascular invasion is an independent prognostic indicator. Based on these results, pathological findings of either lymphatic or vascular invasion might not necessarily be an indicator for postoperative adjuvant therapy; thus, a larger prospective cohort study to investigate the utility of the sole finding of lymphatic or vascular invasion as the indication for postoperative adjuvant radiotherapy or chemoradiotherapy for patients with primary OSCC tumors is mandatory in the future.


Both polycystic ovary syndrome (PCOS) and obesity are associated with specific reproductive health complications, including lower oocyte quality and clinical pregnancy rates in assisted conception cycles, which may be a result of metabolism-induced changes in the oocyte through the microenvironment of follicular fluid. Free fatty acids (FFAs) are important biomedical indicators of abnormal lipid metabolism and have pronounced effects on cells, leading to changes in metabolism, cell growth, and differentiationOur objective was to determine the effect of FFA metabolism in plasma and follicular fluid on oocyte quality in the women with PCOS undergoing in vitro fertilization.Ninety-three women undergoing in vitro fertilization treatment, including 55 with PCOS and 38 age-matched controls, were recruited. PCOS patients were divided into obese and nonobese subgroups on the basis of their body mass index.Embryo quality was morphologically assessed, and serum sex hormone and insulin levels were measured. FFAs in plasma and follicular fluid were measured using gas chromatography-mass spectrometry.P < .05). Obese women with PCOS had a significantly higher total T level, FAI, fasting insulin, insulin resistance index as determined by homeostasis model assessment for insulin resistance, and lower SHBG levels than the nonobese women with PCOS (P < .05). The embryo fragmentation score was significantly positively correlated with the oleic acid concentration in all PCOS patients .PCOS was found to be associated with significantly higher LH/FSH, total T, free androgen index (FAI), and lower SHBG levels, independent of obesity(Our findings clearly demonstrated that PCOS is associated with significantly higher FAI and insulin resistance levels and decreased plasma SHBG levels, independent of body mass index. Obese PCOS patients had higher palmitoleic acid and oleic acid levels in both the plasma and follicular fluid than did the control subject and nonobese PCOS patients. Our results indicated that developmental competence is associated with oleic and stearic acid concentrations, which may contribute to the poor pregnancy outcomes in patients with PCOS. Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, with a prevalence of 5%–10% . Hyperan3–6–10–Studies on animal models of maternal diabetes provide evidence that abnormal maternal physiology affects oocytes and results in abnormal pregnancy outcomes. Oocytes from diabetic, insulin-resistant, and obese mice show delayed maturation, smaller size, and increased granulosa cell apoptosis –15. Thes–Therefore, the present study aimed to provide insights into the effects of FFA metabolism in the plasma and follicular fluid on oocyte quality in women with PCOS undergoing in vitro fertilization (IVF). We also examined the relationship between insulin sensitivity and FFA metabolism in the follicular fluid of PCOS patients.2), non-PCOS; 2) lean PCOS; 3) obese (BMI > 30 kg/m2), non-PCOS; and 4) obese (BMI > 30 kg/m2), PCOS. The non-PCOS subjects were selected from women who visited the same reproductive center to receive IVF treatment owing to fallopian tube disorders.Ninety-three women undergoing IVF treatment at the Reproductive Center of Ruijin Hospital affiliated to Shanghai Jiaotong University Medical School were recruited to this study. Of these, 55 women had PCOS and 63 were age- and BMI-matched controls. According to the 2003 Rotterdam criteria, the presence of two or more of the following signs were considered to be diagnostic of PCOS: oligoovulation and/or anovulation, clinical and/or biochemical signs of hyperandrogenism, and polycystic ovaries after exclusion of other etiologies . Women who had received any hormonal treatment or insulin-lowering agent within the last 3 months were excluded from the study. Patients were divided into four groups: 1) lean  from day 6 of stimulation. Stimulation was monitored using estradiol concentrations, together with ultrasound measurements of follicle numbers and diameters. Ovulation was induced using human chorionic gonadotropin when the leading follicle reached 18–20 mm in diameter.Baseline blood samples were collected between days 3 and 5 of the menstrual cycle in the control group and 3–5 days after a spontaneous bleeding episode in patients with PCOS after an overnight fast of 10–12 hours. In women with PCOS who did not have a spontaneous bleeding episode for 90 days, 60 mg progesterone was administered to induce a bleeding episode, and blood samples were collected afterward. Plasma was obtained from whole-blood samples collected in tubes containing ethylenediamine tetraacetic acid as the anticoagulant. The plasma samples were stored at −80°C until gas chromatography (GC) analysis.g for 30 minutes. The supernatant was purified on a cation-exchange resin and used for subsequent GC analysis as described below.Preovulatory ovarian follicular fluid was collected during transvaginal ultrasound-guided oocyte retrieval. Only follicular fluid samples that were found to be free of blood upon macroscopic analysis were retained for further analyses. Samples were obtained when the follicles measured 18–22 mm in diameter. The follicular fluid samples were collected in capped disposable polypropylene tubes, mixed with an equal volume of 0.5 M trichloroacetic acid, and centrifuged at 15 000 × Embryo morphology was evaluated on day 3 by assessing the embryo cell number and the percentage of fragmentation [embryo fragment score (EFS)]. EFS was graded on the basis of the percentage of fragmentation as follows: 5% or less fragmentation, score 4; 5%–10% fragmentation, score 3; 11%–25% fragmentation, score 2; 26%–50% fragmentation, score 1; and 51% or greater fragmentation, score 0 .Serum sex hormones  and the serum insulin level were analyzed using commercially available kits from the Unicel DXI 800 Access immunoassay system (Beckman Coulter). The inter- and intraassay coefficients of variation were all less than 10%. SHBG was analyzed by an ELISA (USCNLIFE). Intra- and interassay variations were 5.2% and 7.5%, respectively. The concentrations of glucose and lipoproteins  were measured using Unicel DXC 800 Synchron (Beckman Coulter). The inter- and intraassay coefficients of variation of LDL and HDL were 4.8%, 5.1% and 5.2% and 5.6%, respectively.BMI was calculated by dividing the weight in kilograms by the square of the height in meters to assess obesity. The free androgen index (FAI) was calculated as the ratio of total T levels (nanomoles per liter) to SHBG levels (nanomoles per liter) × 100 (percentage). The IR index as determined by homeostasis model assessment (HOMA-IR) was calculated to estimate the insulin sensitivity index as fasting insulin (milliunits per milliliter) × fasting plasma glucose (millimoles per liter)/22.5, as described before , two replicates of 200 μL each of the supernatants were transferred to a GC autosampler vial, and 20 μL myristic-d27 acid (Sigma Aldrich) was added to each aliquot. The samples were then used as internal standards for retention time lock . Finally, the mixture was lyophilized overnight at room temperature. To prevent batch effect, all the assays were conducted in a random and double-blind manner. Then 200 μL of the reference standard was transferred to 2.0-mL Eppendorf tubes, derivatized with the procedure illustrated above, and prepared for GC/MS analysis as described below.GC-MS analysis was performed according to Agilent's specifications . Plasma Each 1-μL aliquot of the derivatized solution or reference standard was injected in splitless mode into an Agilent 7890A GC system equipped with an Agilent DB-5MS capillary column  and an Agilent 5975C Series autosampler (Agilent Technologies). Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The temperatures of the inlet, transfer line, ion source, and quadrupole were maintained at 270°C, 260°C, 200°C, and 150°C, respectively. The temperature protocol was set as follows: isothermal heating at 80°C for 2 minutes, followed by oven temperature ramps at the rate of 10°C/min to 180°C, 5°C/min to 240°C, and 10°C/min to 26°C, and a final maintenance at 260°C for 8 minutes. Data acquisition was achieved using MS in electron impact mode and full-scan monitoring mode over a mass to charge ratio range of 30–600 with an acquisition rate of 20 spectra/sec. The solvent delay time was set at 5 minutes.After GC/MS analysis, each sample or reference standard was represented by a GC/MS total ion current chromatogram. Among the detected peaks, a multidimensional vector was manually constructed to characterize the compounds. Each vector was normalized to the total sum of vector intensity, thereby partially accounting for the different concentrations due to the different sample sizes used. The peaks occurring due to column bleed and derivatization reagent were removed. Only peaks that were consistently detected in at least 80% of the samples were included in the analysis. All the detected peaks were identified by the National Institute of Standards and Technology mass spectral library  and customized reference mass spectral libraries , 24.The mass spectra obtained were manually investigated, and molecules with matching probability of greater than 80% were considered. Within each sample, the retention time and mass to charge ratio data pairs were used as the identifier for each peak, and the ion intensities for each detected peak were then normalized to the sum of the peak intensities in that sample. To account for any differences in concentration between samples, all data were normalized to a total value of 100.2 test. GC-MS variables were rank transformed before being subjected to two-way ANOVA because most of them did not follow a normal distribution. Primary analyses were performed using the Statistical Package for the Social Sciences version 14.0 (SPSS Inc), with a value of P < .05 considered to be statistically significant.Results are presented as means ± SD or SEM. Continuous clinical variables were analyzed by two-way ANOVA, and categorical variables were analyzed by the χ2), whereas 30 women had a BMI greater than 30 kg/m2 . PCOS was associated with significantly higher LH to FSH ratio, total T, FAI, and lower SHBG levels, independent of obesity. Women with PCOS also had significantly higher HOMA-IR ratio independent of obesity. Among the women with PCOS, obese women had significantly higher total T, FAI, fasting insulin, HOMA-IR, and lower SHBG levels than those with normal BMI. Furthermore, obese patients with PCOS showed significantly higher fasting insulin, triglycerides, and LDL levels and lower HDL levels than the control group. In non-PCOS patients, obese women had higher fasting insulin, HOMA-IR, LDL, and lower SHBG level than lean women.In the total study population, women with and without PCOS were comparable in age . Of the P < .05). Through Spearman correlation analysis, we also discovered a weak correlation between the total FFA levels in the follicular fluid and the plasma triglyceride levels among PCOS patients (P < .05). In lean PCOS patients, there was also a weak correlation between the total FFA levels in the follicular fluid and IR. No other significant correlations between the total FFAs in the follicular fluid and BMI, FAI, total cholesterol (TC), or IR were noted  and obese PCOS patients . In the nonobese PCOS patients, no meaningful correlations were detected between embryo blastomere score and the levels of other specific FFAs in the follicular fluid. In obese PCOS patients; however, a significant negative correlation was observed between the blastomere score and stearic acid concentration in follicular fluid .To further investigate the effects of FFA levels in the follicular fluid on oocyte quality, correlations between embryo fragmentation or blastomere score and specific FFA levels in the follicular fluid were measured . The embThe present study was performed to elucidate the characteristics of FFA metabolism in the plasma and follicular fluid and determine its effects on oocyte quality in women with PCOS undergoing IVF.In PCOS, the defective glucose metabolism observed at the systemic level can also be expected at the follicle and ovary level, which may be reflected in the follicular fluid composition. Based on the research of hyperinsulism, it has been postulated that reduced availability of glucose in the oocytes and follicular cells may be caused by defective transportation of glucose can provoke various alternative energy pathways, which would then result in altered levels of various biomolecules in the follicular fluid, such as ketone bodies, lipids, and amino acids .Our findings clearly show that PCOS is associated with significantly higher FAI and IR levels and decreased plasma SHBG levels, independent of BMI. In the subgroup analysis, obese women with PCOS had higher total T and fasting insulin levels than both the control subjects and nonobese women with PCOS. IR is a common but not universal feature of PCOS, and it is not always associated with an increased BMI. Indeed, many studies have shown that both lean and obese women with PCOS have IR , althoug28–In terms of lipid metabolism, obese women with PCOS had higher triglyceride and LDL levels and a lower HDL level than the control group; these changes were manifested as lipid disorders and dyslipidemia in the women with PCOS. Our results are partly in line with the findings of other investigators who evaluated the effect of obesity and PCOS on glucose, lipid, and insulin metabolism –34. Mori–Our findings demonstrated that oleic, palmitic, stearic, undecanoic, nutmeg bean, and linoleic acids are the predominant FFAs in the plasma and ovarian follicles. Although linoleic acid is an essential FFA obtained from the diet, the others are synthesized by the body. The predominant FFAs in the ovarian follicles were consistent with those found in the plasma, and the FFA concentrations in the follicular fluid were found to be positively correlated with the FFA concentrations in the plasma  and 3. TIn our study, the metabolic profiles of both plasma and follicular fluid indicated the dramatically increased levels of two long-chain fatty acids  in PCOS patients compared with the control subjects, irrespective of obesity. Moreover, we also found higher concentrations of linoleic acid in the follicular fluid (but not in the plasma) of obese PCOS patients than in that of the control subjects and nonobese PCOS patients. These results suggest that cells of the ovarian follicle metabolize specific FFAs at different rates or perhaps that certain FFAs are transported preferentially into the ovarian follicle. In line with our findings, several published studies have reported increased levels of plasma long-chain fatty acids such as linoleic and oleic acids as well as increased levels of palmitoleic acid in obese women irrespective of PCOS, suggesting that increased lipolysis was possibly secondary to impaired insulin action in adipose tissue , 39.P = .04) and IR calculated using HOMA-IR . In the IVF cycle, associations between elevations in total follicular FFAs and poorer COC quality have been found, suggesting that excess FFAs adversely influence ovarian follicular function  concentration was increased in the plasma but not in the follicular fluid of obese PCOS patients as compared with the concentrations in the other groups. Importantly, our results indicated that oocyte developmental competence is associated with oleic and stearic acid concentrations, which may contribute to the mechanisms of poor pregnancy outcomes in patients with PCOS. We propose that further studies with a larger number of patients be conducted to validate our study's findings and to identify novel treatments that may improve fertility in these affected women.


Consistent results were obtained from each, showing little difference between Ig and Fab in terms of antigen binding and in vitro neutralization, but with relatively large differences in protection of animals. We also studied whether importing Ab into the cell by Fc receptors enhanced the intracellular neutralization of ricin toxin. We found that the imported Ab was found in the ER and Golgi, a compartment traversed by ricin, as it traffics through the cell, but intracellular Ab did not contribute to the neutralization of ricin. These results indicate that the Fc region of antibody is important for in vivo protection, although the mechanism of enhanced protection by intact Ig does not appear to operate at the single cell level. When using xenogeneic antibodies, the diminished immunogenicity of Fab/F(ab’)2 preparations should be balanced against possible loss of protective efficacy.We have studied the role of the antibody (Ab) Fc region in mediating protection from ricin toxicity. We compared the  It is clear that neutralization of toxins by Ab plays a major role in protective immunity. Critical vaccines  and passive Ab therapies are based upon this fact, which represents one of the few generally-agreed upon “truths” in the field of human vaccinology. Yet how exactly Abs protect us from toxins is not fully understood. We generally teach our students that Ab functions by preventing attachment and internalization of the toxin to target cells ,2, sugge2 fragments, with the intent to limit immunogenicity and risk of “serum sickness” in such preparations  × 100.Cell viability was measured by MTS dye reduction assay at 48 h. Ab-mediated neutralization of ricin was performed as detailed elsewhere . Ricin aOutbred NIH Swiss/Webster female mice 4–6 weeks of age were obtained from Charles River Laboratories . All experiments were done in compliance with federal laws and regulations and have been approved by the IACUC at The Research Institute for Children (OLAW assurance A-4336-01), following protocols described elsewhere . Ricin . After each incubation, cells were washed 3× with PBA. Ten thousand cells underwent analysis using an LSR II flow cytometer  and analyzed with FlowJo software . Histograms displayed are based on cells gated first for SSC and FSC.4 cells were seeded into 35 mm culture dishes with a glass bottom of 0.17 mm thickness . Cells were cultured in 5% CO2 at 37° in RPMI 1640 containing no phenol red and 10% FCS. The following day, cells were placed into RPMI 1640, 1% bovine serum albumin (BSA), and 10 mM Hepes, and then transferred to the microscope stage for imaging. Experimental details for each study are provided in the figure captions. The brightness and contrast of confocal images shown in this manuscript have been adjusted to enhance visualization. Quantitative analyses were performed on the raw data.Qualitative and quantitative confocal microscopy was performed on live cells using an inverted Zeiss LSM 510 microscope, with a heated stage and 63× 1.4 NA oil-immersion heated objective. Zeiss LSM and Axiovision software were used to obtain and analyze images. Full details of the experimental protocols, mathematical analyses, as well as appropriate controls, are provided elsewhere [in vitro at 10-fold lower concentrations than did the mAbs, but similar in vivo protection from ricin challenge was observed for both mAb and pAb. In comparing the function of intact Ig vs. Fab preparations of these two different sets of anti-ricin Abs, we found similar, and thus generalizable, results. In vitro functions  were unaffected by cleavage into fragments, although Fabs had lower avidity than F(ab’)2, likely due to decreased valence. However, in vivo there was a marked difference in protection, with intact Ig outperforming Fab and F(ab’)2. Moreover when tested in mice, murine mAb RAC18 outperformed its chimeric counterpart carrying human C regions, highlighting the importance of a homologous Fc.In this report, we study the role of the Fc region of neutralizing Abs in protection from the toxic effects of ricin administration. We used both mAbs and Fabs, which differed in several ways. The mAbs were A chain specific, whereas the pAbs reacted with both A and B chains. The pAbs neutralized toxin in vivo observations showed the importance of the Fc region; (2) a study which used FcRγ knockout mice to demonstrate the requirement for FcRs in protection toxin [We next studied the role of FcRγ on protection of cells from ricin toxicity because: (1) our on toxin ; and (3)on toxin  led us tin vivo, the presence of Fc enhances protective efficacy. We cannot say whether this effect as mediated by FcR, as suggested by others [vs. Fab, decreased avidity of Fab fragments, or by some other completely different Fc-mediated mechanism. When using xenogeneic antibodies for passive immune therapy, Fab/F(ab’)2 preparations may be less likely to provoke an immune response and induce serum sickness. However, this advantage should be weighed against the potential loss of in vivo protective activity, compared to intact Ig.Overall, our findings indicate that neither the Fc region of the Ab nor the presence of FcR on the target cell has an influence on Ab-mediated protection of individual cells. However, y others , by the


It is generally accepted that domestication of pigs took place in multiple locations across Eurasia; the breeds that originated in Europe and Asia have been well studied. However, the genetic structure of pig breeds from Russia, Belorussia, Kazakhstan and Ukraine, which represent large geographical areas and diverse climatic zones in Eurasia, remains largely unknown.This study provides the first genomic survey of 170 pigs representing 13 breeds from Russia, Belorussia, Kazakhstan and Ukraine; 288 pigs from six Chinese and seven European breeds were also included for comparison. Our findings show that the 13 novel breeds tested derived mainly from European pigs through the complex admixture of Large White, Landrace, Duroc, Hampshire and other breeds, and that they display no geographic structure based on genetic distance. We also found a considerable Asian contribution to the miniature Siberian pigs (Minisib breed) from Russia. Apart from the Minisib, Urzhum, Ukrainian Spotted Steppe and Ukrainian White Steppe breeds, which may have undergone intensive inbreeding, the breeds included in this study showed relatively high genetic diversity and low levels of homozygosity compared to the Chinese indigenous pig breeds.This study provides the first genomic overview of the population structure and genetic diversity of 13 representative pig breeds from Russia, Belorussia, Kazakhstan and Ukraine; this information will be useful for the preservation and management of these breeds.The online version of this article (doi:10.1186/s12711-016-0196-y) contains supplementary material, which is available to authorized users. Sus scrofa) is an important farm animal, as well as a relevant biomedical model for humans. The pig was independently domesticated in China and Europe approximately 9000 years ago [The pig , one breed from Belorussia , one breed from Kazakhstan , and six breeds from Ukraine   values were calculated using the ADZE v1.0 software [o) and runs of homozygosity (ROH) were computed with the PLINK v1.9 software [2) between pairwise SNPs was calculated by the commands –r2 and –ld-window-r2 in PLINK v1.9 [2. Effective population sizes (Ne) were computed using the equation of Herrero-Medrano et al. [T is effective population size at T generation ago, T is calculated by T = 1/2c [2 between SNPs separated by large and small genetic distances reflect recent and ancient Ne, respectively [Allelic richness . The admixture analysis suggests that the Minisib breed contains ancestries from Large White, Landrace and Luchuan or Tibetan pigs from China Fig. a, K = 6.We ran the admixture analysis from K = 2 to 26 and found that K = 19 was the optimal value, with the smallest cross-validation errors  and observed heterozygosity (Ho) were calculated to provide measures of the genetic diversity of the 13 populations  for the past 20 years was estimated for each population using the linkage disequilibrium and recombination rate data. The breeds with the largest Ne included the Breitov 148.7), Belorussian pork swine (145.8), and Red White Belted (142.8) breeds, whereas the Murom (86.2), Minisib (99.5), and Ukrainian pork swine (115.4) breeds had the smallest Ne  and neighbor-joining tree analyses, many of the 13 pig breeds investigated in this study have retained unique identities and are differentiated from mainstream commercial breeds such as the Large White and Landrace breeds. This can be attributed to several factors, including differences in the origin of the populations, a long period of genetic isolation, and differences in climate and locally available feed between West Russia, Belorussia, Ukraine and west Europe.We observed widespread Asian ancestries in all 13 pig breeds. Introgression of Asian haplotypes into European breeds, such as the Large White and Landrace breeds, have been reported in several previous studies , 31. TheA previous comparison of genomic diversity parameters based on whole-genome sequence (WGS) and 60K SNP data showed that while 60K SNP and WGS data provided similar results for European breeds, 60K SNP data underestimated the genetic diversity of the Asian populations due to ascertainment bias . This stThis is the first genetic survey of pig breeds from Russia, Belorussia, Kazakhstan and Ukraine using 60K SNP genotype data. We confirmed that these breeds were mainly of European origin, since they formed a separate group from Chinese breeds but were close to commercial breeds, including the Large White, Landrace, Duroc and Hampshire breeds. The Minisib and Urzhum breeds have been subject to severe inbreeding and consequently have limited genetic diversity. In contrast, most of the other breeds have a greater level of genetic diversity and a lower level of ROH because of their crossbreeding histories. This study provides the first genomic survey of the population structure and evolutionary history of pig breeds in Russia, Belorussia, Kazakhstan and Ukraine, which will contribute to conservation and breeding programs of these breeds.