Determination of the ovine ovarian reserve during the prenatal and neonatal periods

Objective:To determine the ovine ovarian histomorphology and follicular staging at various age periods in Awassi breed.Methods:Ovaries were collected from prenatal fetuses[gestational age(95±5)days],neonatal(day 0),and prepubertal ewe lambs(two and four months of age);each age group included six animals.Ovaries(n=12,each group)were dissected and processed for hematoxylin and eosin staining.Stained sections(n=24,each group)were imaged and utilized for histomorphology assessment,follicle measurement,and classification.Results:Prenatal ovaries were mainly enriched with primordial follicles accompanied by a lower proportion of primary follicles.In addition to primordial and primary follicles,neonatal ovaries demonstrated a proportion of centrally located multilayered and antral follicles.In comparison with neonatal ovaries,the proportion of multilayered and antral follicles was significantly higher in the ovaries of two-month-old lambs;conversely,the proportion of peripherally situated primordial follicles dramatically declined compared to that of earlier age of lamb.Although there was no statistical variation in the sizes of primordial follicles across groups,the mean diameter of the primary follicle in the prenatal ovaries was substantially smaller than in postnatal ovaries.Compared to the neonatal ovaries,the size of the multilayered and antral follicles in the prepubertal ovaries was substantially larger.Conclusions:The earliest follicular developmental stages were established prenatally whereas the advanced growth stages started in the neonatal period and greatly increased in the prepubertal period.

Protective effect of resveratrol against cadmium-induced toxicity on ovine oocyte in vitro maturation and fertilization

Background:Heavy metal cadmium(Cd)is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility.It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization,through oxidative stress induction.Resveratrol(Res)is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline.Here,we explored whether the addition of Res to in vitro maturation(IVM)medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization.Firstly,we evaluated the effect of supplementing IVM medium with two different Res concentrations(1and 2μmol/L)on nuclear maturation and fertilization of oocytes matured under CdCl2(2μmol/L)exposure.Therefore,the concentration of 1μmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels,mitochondrial(mt)distribution and activity,chromatin configuration,cytoskeleton morphology,cortical granules(CGs)distribution and mRNA expression of genes associated with cellular response to oxidative stress(i.e.SIRT1,SOD 1,GPX1,GSR,CAT)in Cd-exposed in vitro matured oocytes.Results:We found that 1μmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as,Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations.Moreover,we demonstrated that 1μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species(ROS)accumulation,preventing mt dysfunction,maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1,SOD1 and GPX1 genes.Conclusions:Taken together,our findings highlighted the beneficial influence exerted by Res in preventing Cdinduced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes.Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

The characterization of acid and pepsin soluble collagen from ovine bones (Ujumuqin sheep)

Ovine bones are the major by-products after slaughtered. The present study was conducted to extract and characterize acid soluble collagens （ASC） and pepsin soluble collagens （PSC） from ovine bones （Ujumuqin sheep）. Ovine bones collagen were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and liquid chromatography-tan- dem mass spectrometry （LC-MS/MS） as type I collagen. The results of Fourier transform infrared （FTIR） spectra analysis testified the existence of triple superhelical structure in both ASC and PSC, showing pepsin did not disrupt the triple helical structure of ovine bones collagen. Glycine, accounting for one-third of total amino acids, was the major amino acid for ovine bones collagen. Higher imino acid content was responsible for higher thermal denaturation temperature of ovine bones collagen compared to fish collagens. The isoelectric point of ASC was lower than PSC due to the higher content of acidic amino acids. Therefore, this study provides the potential reference for collagen extraction and application of ovine bones by-procduct.

The Identification of the Cryptosporidium ubiquitum in Pre-weaned Ovines from Aba Tibetan and Qiang Autonomous Prefecture in China

Objective Cryptosporidium spp. are prevalent globally and sheep are an important zoonotic reservoir. Little data regarding the rates of Cryptosporidium infections in ovines in China are available. This study assessed the prevalence of Cryptosporidium spp. in pre-weaned ovines from Aba Tibetan and Qiang Autonomous Prefecture in the Sichuan province of China. Methods A total of 213 fecal samples were collected from pre-weaned ovines and were examined microscopically （following modified acid fast staining）. In addition, 18S rRNA genetic sequences were amplified from fecal samples by nested PCR and phylogenetically analyzed. Results The prevalence of Cryptosporidium in the collected samples was at 14.6% （31/213） and four isolates identified by PCR belonged to the Cryptosporidium cervine genotype （Cryptosporidium ubiquiturn） demonstrating that this species was the primary sheep species found in sheep in China. Conclusion The present study suggested that the high incidence of Cryptosporidium in sheep poses a significant public health threat and that surveillance practices must be established to prevent zoonotic disease of humans.

Phosphorylation of sarcoplasmic and myofibrillar proteins in three ovine muscles during postmortem ageing

This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum,semitendinosus,and psoas major muscles during postmortem ageing for 5 d.These sarcoplasmic and myofibrillar proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with phosphorous and protein specific stains.Myofibril fragmentation index,pH,the content of lactic acid and the relative activity of μ-calpain in three ovine muscles were measured.These results showed that the relative phosphorylation level of sarcoplasmic and myofibrillar proteins of psoas major muscle were lower compared with longissimus lumborum and semitendinosus muscles(P<0.05).The pH of psoas major muscle was the lowest at 0.5 h postmortem,and the highest after 12 h postmortem(P<0.05).In addition,the relative activity of μ-calpain was higher within 5 d postmortem and myofibril fragmentation index was higher after 1 d postmortem in psoas major muscle than those of longissimus lumborum and semitendinosus muscles(P<0.05).The sarcoplasmic protein phosphorylation may regulate the rate of pH decline to influence the μ-calpain activity and then proteolysis of proteins consequently.This study gives a new perspective of the mechanism of postmortem meat tenderization.

ATP regulates the phosphorylation and degradation of myofibrillar proteins in ground ovine muscle

Phosphorylation post-translational modification plays an important role in postmortem muscle quality traits. Adenosine triphosphate(ATP) is an energy source and a key substrate of phosphorylation which provides the phosphatase groups to proteins in the presence of protein kinases. However, in postmortem muscle, the effects of ATP content on phosphorylation are poorly studied. The study investigated the effect of ATP on protein phosphorylation and degradation in postmortem ovine muscle. The ground muscle with/without additional ATP were treated/control groups and stored at 25 and 4℃, respectively. The ATP content led to different changes of p H value between the ATP-treated and control groups. The phosphorylation level of myofibrillar proteins was higher(P<0.05) in ATP-treated group compared to the control group at both temperatures, which suggested that ATP played a vital role in postmortem protein phosphorylation. A slower degradation rate of μ-calpain, desmin and troponin T was observed in the ATP-treated group which showed that there was a negative relationship between ATP level and the degradation of proteins. These observations clearly highlighted the role of ATP on the development of meat quality by regulating the phosphorylation and degradation of myofibrillar proteins in postmortem ovine muscle.

Effects of Hypoxia on the Growth and Development of the Fetal Ovine Hepatocytes in Primary Culture

Objective To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism.Methods Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia(21% O2) or hypoxia(2% O2).The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry(FCM).The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope.Results The cell purity of hepatocytes was over 95%.Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability.Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm.Conclusion Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics.Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.

Effects of EGF and IGF-I on in vitro Maturation and Cleavage of Ovine Oocytes

The study investigated the effects of epidermal growth factor（EGF） and insulin-like growth factor 1 （ IGF-I）, alone or together, on the in vitro maturation and cleavage of ovine oocytes, aimed to optimize the in vitro maturation conditions for ovine oocytes. The results showed that the maturation and cleavage rates were 71.2% and 45.5% respectively when the medium was supplemented with 50 ng/mL EGF alone, which was significantly higher than other EGF supplemented groups （0, 10, 20, 30, and 40 ng/mL） （P 〈0.05）. The highest maturation and cleavage rates were 72.9% and 45. 7% when the EGF concentration reached 100 ng/mL. The maturation and cleavage rates were 70. 7% and 58. 5% with 40 ng/mL IGF-I supplemented, which were significantly higher than other treatments （0,10,20,60,80, and 100 ng/mL） （P 〈0.05）. The lowest maturation and cleavage rates were 38.8% and 20.0% when the IGF-I concentration reached 100 ng/mL （ P 〈 0.05 ） When 50 ng/mL EGF and 40 ng/mL IGF-I were used concomitantly,the maturation and cleavage rates were 85. 6% and 61. 0% respectively, which were significantly higher than the treatments with EGF or IGF-I alone （ P 〈 0.05 ）.

LH Plays a Role to Enhance the Nuclear and Cytoplasm Synchronization During In vitro Maturation of Ovine Oocytes

This study is aimed to investigate the effect of luteinizing hormone （LH） on maturation and fertilization in vitro of ovine oocytes. The ovine oocytes were cultured in vitro in medium with or without LH, and then checked by confocal laser scanning microscope to observe the distribution of cortical granules stained with FITC-LCA during different maturation periods. Similarly, some in vitro matured oocytes were also fertilized in vitro for analysis of their developmental potentiality further. After being cultured in vitro for 4 h, there were significant differences about the rate of germinal vesicle break down （GVBD） between the treatment （with LH） and the control groups （without any hormones） （36.76% vs 18%, P〈0.05）. Further, there were also significant differences of the cleavage and blastocyst rates between these two groups （67.15% vs 42.37%, 21.9% vs 12.71%, P〈0.05, respectively）. The distribution of cortical granules appeared to spread from the edges to the central site of sheep oocytes following their delaying durations of maturation in vitro. It can be concluded that LH may play a role to delay the occurence of GVBD, prolong the maturation duration of cytoplasm, and enhance the nuclear and cytoplasm synchronization of ovine oocytes matured in vitro and finally improve their in vitro developmental potentiality.

Construction and Preliminary Analysis of Ovine Oocytes Proteomic 2-DE Map

[Objective] To obtain ovine oocytes 2-dimensional gel electrophoresis （2-DE） maps. [Method] Ovine oocytes were solubilized in lysis buffer, and protein concentrations were measured using the method of Bradford and Ramagli. Then the samples were subjected to two-dimensional polyacrylamide gel electrophoresis. The 2-DE maps of ovine oocytes were analyzed through PDQuest 8.0. [Resait] The Ramagli method could re- flect concentration of oocyte protein more actually and could optimize the quantity of samples to get a 2-DE map with higher quality. The protein concentration measured by the Bradford method was higher than its actual value. [Coclusion] Each 2-DE map loading 700 oocytes is more reasonable.

Ovine Progressive Pneumonia Virus Is Transmitted More Effectively via Aerosol Nebulization than Oral Administration

A new method of experimental infection of ovine progressive pneumonia virus (OPPV), aerosol nebulization (Nb), was compared to intravenous (IV) and oral (PO) methods of experimental infection. Seven month old lambs were given 3.5 × 107 TCID50 of Dubois OPPV LMH19 isolate using IV, PO, or Nb methods and were monitored for infection using cELISA and OPPV quantitative (q) PCR for 35 weeks. Four out of four sheep in the IV group, six out of six sheep in the Nb group, but only two out of six sheep in the PO group became infected by OPPV;whereas the uninoculated controls (n = 2) and a sentinel control (n = 1) remained uninfected during the course of the study. The time to a cELISA or OPPV qPCR positive result in the Nb group was quicker and statistically different from the time to a cELISA or OPPV qPCR positive result in the PO group (cELISA P value = 0.0021 and OPPV qPCR P value = 0.0007). When the Nb and IV groups were compared, sheep became cELISA and OPPV qPCR positive at similar times (cELISA P value = 0.6 and OPPV qPCR P value = 0.1). In addition, sheep became OPPV qPCR positive prior to cELISA in both the IV and Nb groups (IV P value = 0.027 and Nb P value = 0.007). Aerosol nebulization is a more natural experimental method of transmitting OPPV and may be valuable for testing potential vaccines or specific host genetics.

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm

Background： In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up（SU） or swim up ＋ zona pellucida（SU ＋ ZP） binding.Results： Experiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation（precense of one PN）. Treatments showed similar results（54, 47, 42 %, respectively） but statistically differents（P 0.05）.Conclusions： Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU ＋ ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU ＋ ZP were significantly different than frozen-thawed sperm,but between sperm treatments no significant differences were obtained.

Use of Acellular Fish Skin for Dura Repair in an Ovine Model: A Pilot Study

Recently the use of biologic materials as dura mater repair patches has been increasing. The purpose of this study is to assess the basis for efficacy and safety of using a novel fish derived acellular dermis (Kerecis Omega3 DuraTM). In an ovine model a craniotomy under general anaesthesia was performed. A defect was produced in the dural covering of approximately 1 × 2 cm and closed with an onlay technique, with Kerecis Omega3 Dura. The bone defect was covered with the bony flap and the overlying tissues closed in layers. At 2, 5, 8 and 11 weeks the sheep underwent MRI scanning followed by euthanasia, necropsy and histological assessment. MRI images taken at 2, 5, 8 and 11 weeks showed initially moderate inflammatory response, which diminished over time, and at 11 weeks no evidence of inflammation existed. There was evidence of cerebrospinal fluid leakage at no time point. Necropsy revealed some adhesions at 5 and 8 weeks, in particular at 5 weeks, but at 11 weeks there were no adhesions found. From 2 - 11 weeks, there was evidence of initially an inflammatory reaction followed by neodura formation at the defect site through cellular ingrowth and remodeling of the acellular fish skin. Histology showed a histiocytic foreign body reaction initially that subsided over time. As early as 8 weeks there was evidence of neodura formation and by 11 weeks there was a minimal inflammatory response with an intact neodura formed. In this pilot study the Kerecis Omega3 Dura patches performed in a safe and efficacious manner. This new material needs to be fully assessed and compared with other products that are currently on the market in a larger scale animal study.

Analysis and PCR detection of antigen compositions of ovine progressive pneumonia virus

Ovine progressive pneumonia virus (OPPV) was proliferated utilizing sheep foetal lung cells, and the cytopathic effect (CPE) of the virus was investigated. OPPV was purified with a 10%-sucrose cushion and then with 20%-55% discontinuous sucrose density gradient centrifugation. The structural proteins and antigen compositions of OPPV were analysed by SDS-PAGE and Western blotting. Besides, the OPP proviral cDNAs of the virus-infected cell cultures and the peripheral blood monocytes from sheep infected by the virus were detected using polymerase chain reaction (PCR). The results show that the CPE of sheep foetal lung cells infected by OPPV is typical of the disease. The purified virions are intact and of high purity when observed with an electron microscope. OPPV proteins consist of 18 polypeptide bands and the molecular weights range from 18 to 120kd. Among these, 3 were glycoproteins (designated by gp120, gp50 and gp47). The appearance and peak time of the p28 antibody from sheep inoculated with OPPV

Dental pulp stem cells and BonelikeVR for bone regeneration in ovine model

Development of synthetic bone substitutes has arisen as a major research interest in the need to find an alternative to autologous bone grafts.Using an ovine model,the present pre-clinical study presents a synthetic bone graft(BonelikeVR)in combination with a cellular system as an alternative for the regeneration of non-critical defects.The association of biomaterials and cell-based therapies is a promising strategy for bone tissue engineering.Mesenchymal stem cells(MSCs)from human dental pulp have demonstrated both in vitro and in vivo to interact with diverse biomaterial systems and promote mineral deposition,aiming at the reconstruction of osseous defects.Moreover,these cells can be found and isolated from many species.Non-critical bone defects were treated with BonelikeVR with or without MSCs obtained from the human dental pulp.Results showed that BonelikeVR and MSCs treated defects showed improved bone regeneration compared with the defects treated with BonelikeVR alone.Also,it was observed that the biomaterial matrix was reabsorbed and gradually replaced by new bone during the healing process.We therefore propose this combination as an efficient binomial strategy that promotes bone growth and vascularization in non-critical bone defects.

Phosphate,calcium,and vitamin D signaling,transport,and metabolism in the endometria of cyclic ewes

Background Recent evidence suggests important roles for progesterone(P4)and interferon tau in the regulation of calcium,phosphate,and vitamin D signaling in the uteri of pregnant sheep.However,the effects of P4 and estradiol(E2),with respect to the expression of their receptors PGR and ESR1,respectively,in uterine epithelia on mineral signaling during the estrous cycle has not been investigated.Estrous cycles of mature Suffolk ewes were synchronized,prostaglandin F2αwas administered,and ewes were observed for estrus(designated as Day 0)in the presence of vasectomized rams.On Days 1,9,or 14 of the estrous cycle,hysterectomies were performed.Results 25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1(P<0.05).Expression of fibroblast growth factor receptor 2(FGFR2),a disintegrin and metalloprotease 17(ADAM17),and parathyroid hormone-related protein(PTHrP)mRNAs was greater in endometria on Day 9 compared to Days 1 and 14(P<0.01).Similarly,expression of transient receptor potential cation channel subfamily V member 6(TRPV6)mRNA was greater in endometria on Day 9 than Day 1(P<0.05).ATPase plasma membrane Ca^(2+)transporting 4(ATP2B4)and S100 calcium binding protein G(S100G)mRNA expression was greater in endometria on Day 14 than on Days 1 and 9(P<0.01).In contrast,endometrial expression of vitamin D receptor(VDR)mRNA was lower on Days 9 and 14 than Day 1(P<0.01).Expression of klotho(KL)(P<0.05)and cytochrome P450 family 24 subfamily A member 1(CYP24)(P<0.01)mRNAs was lower on Day 14 than Days 1 and 9.ADAM17,FGF23,CYP2R1,CYP27B1,KL,and VDR proteins immunolocalized to the uterine myometrium,blood vessels,and uterine luminal(LE),superficial glandular(sGE),and glandular(GE)epithelia.S100A9 protein was weakly expressed in the uterine myometrium,LE,sGE,and GE.Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14.In contrast,S100G protein was expressed exclusively by GE,and immunoreactive S100G protein was less on Day 9.S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels,but not by uterine epithelial cells.Conclusion Collectively,these results implicate E2,P4,and PGR in the regulation of phosphate,calcium,and vitamin D signaling in cyclic ewes.

应用Y—DNA特异片段的PCR技术鉴定牛和绵羊胚胎性别及应用研究

本研究采用国际八十年代中后期研究成功的特定ＤＮＡ片段扩增技术—聚合酶链反应（ＰＣＲ）技术，进行牛和绵羊胚胎性别鉴定。采用删除富集法，构建富含牛和绵羊Ｙ—ＤＮＡ文库，筛选出特异克隆，并根据其序列，分别设计合成引物，用ＰＣＲ技术分别扩增牛Ｙ染色体上约１４０ｂｐ的片段，绵羊Ｙ染色体上约１３０ｂｐ的片段。对已知性别的牛和绵羊血液样品进行检测，准确率均为１００％（１０／１０；１１／１１）。应用已建立的简易、快速胚胎取样方法及防污染措施，以及设计合成的引物和ＰＣＲ检测胚胎性别的技术方法，奶牛胚胎经性别鉴定后移植，产犊牛７头（４头公犊，３头母犊），准确率为１００％（７／７）。在内蒙牧区生产现场，对绵羊胚胎经性别鉴定后移植，共移植成功１８只受体母羊（每只母羊移植２－４枚同性别胚胎），产羔２５只（８只公羔，１７只母羔），准确率为８０％（２０／２５）。

High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVEP)were vitrified at either early(EBs:n=74)or fully expanded blastocyst stage(FEBs:n=195),using a new device named"E.Vit",composed by a 0.25-m L straw with a 50-μm pore polycarbonate grid at one end.Embryos at each stage(EBs and FEBs)were vitrified by either Two-step(TS)or Multi-step(MS;6 different concentrations of vitrification solutions)protocol.Non-vitrified embryos(n=102)were maintained in in vitro culture as a control.Warming consisted of placing the straws directly into 1.5 m L tubes containing a TCM-199 solution with three decreasing concentrations of sucrose.Blastocyst re-expansion,embryo survival and hatching rate were evaluated at2,24 and 48 h post warming.The number of apoptotic cells was determined by TUNEL assay.Results:Blastocyst re-expansion(2 h)after warming was higher(P<0.05)in FEBs group,vitrified with the MS and TS methods(77.90%and 71.25%,respectively)compared with the EBs group(MS:59.38%and TS:48.50%,respectively).Survival rates of vitrified FEBs after 24 h IVC were higher(P<0.001)in both methods(MS and TS)than vitrified EBs(MS:56.25%;TS:42.42%)and was higher(P<0.05)in the MS method(94.19%)compared with those in TS(83.75%).After 48 h of culture the hatching rate for FEBs vitrified in MS system(91.86%)was similar to control(91.89%),but higher than FEB TS(77.5%)and EBs vitrified in MS(37.5%)and TS(33.33%).Number of apoptotic cells were higher in EBs,irrespective of the system used,compared to FEBs.The number of apoptotic cells in FEBs vitrified with MS was comparable to the control.Conclusions:A high survival rate of IVP embryos can be achieved by the new"E.Vit"device with hatching rates in vitro comparable with control fresh embryos.This method has the potential for use in direct embryo transfer in field conditions.

A New Model to Study Healing of a Complex Femur Fracture with Concurrent Soft Tissue Injury in Sheep

High energy bone fractures resulting from impact trauma are often accompanied by subcutaneous soft tissue injuries, even if the skin remains intact. There is evidence that such closed soft tissue injuries affect the healing of bone fractures, and vice versa. Despite this knowledge, most impact trauma studies in animals have focussed on bone fractures or soft tissue trauma in isolation. However, given the simultaneous impact on both tissues a better understanding of the interaction between these two injuries is necessary to optimise clinical treatment. The aim of this study was therefore to develop a new experimental model and characterise, for the first time, the healing of a complex fracture with concurrent closed soft tissue trauma in sheep. A pendulum impact device was designed to deliver a defined and standardised impact to the distal thigh of sheep, causing a reproducible contusion injury to the subcutaneous soft tissues. In a subsequent procedure, a reproducible femoral butterfly fracture (AO C3-type) was created at the sheep’s femur, which was initially stabilised for 5 days by an external fixator construct to allow for soft tissue swelling to recede, and ultimately in a bridging construct using locking plates. The combined injuries were applied to twelve sheep and the healing observed for four or eight weeks (six animals per group) until sacrifice. The pendulum impact led to a moderate to severe circumferential soft tissue injury with significant bruising, haematomas and partial muscle disruptions. Posttraumatic measurements showed elevated intra-compartmental pressure and circulatory tissue breakdown markers, with recovery to normal, pre-injury values within four days. Clinically, no neurovascular deficiencies were observed. Bi-weekly radiological analysis of the healing fractures showed progressive callus healing over time, with the average number of callus bridges increasing from 0.4 at two weeks to 4.2 at eight weeks. Biomechanical testing after sacrifice showed in- creasing torsional stiffness between four and eight weeks healing time from 10% to 100%, and increasing ultimate torsional strength from 10% to 64% (relative to the contralateral control limb). Our results demonstrate the robust healing of a complex femur fracture in the presence of a severe soft tissue contusion injury in sheep and demonstrate the establishment of a clinically relevant experimental model, for research aimed at improving the treatment of bone fractures accompanied by closed soft tissue injuries.

Development of a surgical procedure for removal of a placentome from a pregnant ewe during gestation

Background: In recent decades, there has been a growing interest in the impact of insults during pregnancy on postnatal health and disease. It is known that changes in placental development can impact fetal growth and subsequent susceptibility to adult onset diseases;however, a method to collect sufficient placental tissues for both histological and gene expression analyses during gestation without compromising the pregnancy has not been described. The ewe is an established biomedical model for the study of fetal development. Due to its cotyledonary placental type, the sheep has potential for surgical removal of materno-fetal exchange tissues, i.e., placentomes. A novel surgical procedure was developed in well-fed control ewes to excise a single placentome at mid-gestation.Results: A follow-up study was performed in a cohort of nutrient-restricted ewes to investigate rapid placental changes in response to undernutrition. The surgery averaged 19 min, and there were no viability differences between control and sham ewes. Nutrient restricted fetuses were smaller than controls(4.7 ± 0.1 kg vs. 5.6 ± 0.2 kg;P < 0.05), with greater dam weight loss(-32.4 ± 1.3 kg vs. 14.2 ± 2.2 kg;P < 0.01), and smaller placentomes at necropsy(5.7 ± 0.3 g vs. 7.2 ± 0.9 g;P < 0.05). Weight of sampled placentomes and placentome numbers did not differ.Conclusions: With this technique, gestational studies in the sheep model will provide insight into the onset and complexity of changes in gene expression in placentomes resulting from undernutrition(as described in our study),overnutrition, alcohol or substance abuse, and environmental or disease factors of relevance and concern regarding the reproductive health and developmental origins of health and disease in humans and in animals.