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.

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.

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.

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 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.

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 ）.

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.

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.

Characterization of Bovine and Ovine WPC Obtained by Different Membrane Configuration Processes

The objective of this stud）, was to valorize bovine and ovine cheese whey resulting from small and medium cheese manufacture plants, by producing liquid and dr）＂ whey protein concentrates （LWPC and WPC）. The flexibility implemented by batch ultrafiltration （UF） and diafiltration （DF） allowed for the production and ovine WPC with 61 -87% （dry basis）. Diatiltration, performed in of liquid bovine WPC with 43-66% protein contents （dry basis） sequential dilution mode （DFsdm） did not improve significantly the composition of WPC liquid products comparing to the results achieved by conventional UF. However, using DF in volume reduction mode （DFvrm） protein content was increased more than 20% comparing to conventional UF. Ovine products showed higher protein levels （62-84% dry basis） what makes their manufacture attractive. Protein profiles varied with the whey origin and with the concentration process. Using batch DFvrm was possible to obtain richer protein products free of low MW compounds.

Conversion of Dermal Proteins of an Algerian Ovine to an Antimicrobial Agent for Skin Lesion

The aim of this study was the conversion of the ovine dermal proteins as a natural product that has an antimicrobial power which used for tissue repair and skin lesion. After dehairing, fleshings, deliming the animal tissues （ovine） and purification, the essential oils of Lavandula officinalis, Eucalyptus globules and Eugenia caryophyllata had been supplied and fixed. The product obtained is a spongy material communicating with open voids between the fibers. With a transparent light yellow color, it has a remarkable antibacterial power and a very strong inhibitory activity on all the bacterial strains tested whose average diameter of the inhibition zones exceeds 16 mm. It is also characterized by a smell which can be aromatic, pleasant and spicy or fresh and spicy depending on the essential oil used.

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.

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.

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 are earlier than those of the p94 antibody to OPPV. Besides, the strength of immune reaction of p28 antibody is greater than that of p94 antibody. With PCR, it is demonstrated that the initial time for OPP proviral cDNAs to be integrated into the sheep foetal lung cells and the peripheral blood monocytes in sheep were 24 h and 9 d after inoculation, respectively.

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）。

Expression of Endogenous Beta Retroviruses and Hyal-2 mRNA in Immune Organs of Fetuses and Lambs

Endogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV),this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). The aim of this study was to clarify the function of enJSRV and the immunological mechanisms of its corresponding antibody, that is undetectable in JSRV-infected ovine serum. The expression of enJSRV envelope protein and Hyal-2 mRNA in immune organs and lungs of ovine fetuses and lambs were analyzed by Real-Time reverse transcription PCR and In Situ Hybridization using specific probes. In Situ Hybridization results indicated that the enJSRV envelope protein and Hyal-2 mRNA were expressed in thymus, spleen, mesenteric lymph nodes and lungs at different times, while no positive signals were detected in the negative controls. On the other hand, results from Real-Time reverse transcription PCR analysis showed that in 130d fetuses and 3d newborn lambs the enJSRV mRNA levels were much higher in organs associated with the immune system than that in lungs, especially in the thymus and spleen, but levels of Hyal-2 mRNA expression was not significantly different in all collected tissue. These results provided evidence from an immunology point of view to understand why the circulating antibodies against exJSRV are undetectable in JSRV-infected ovine, and will help to unravel the pathogenesis of JSRV-infected ovine.

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses (enJSRV)

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame (orf-x) that overlaps the 3' end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain (Accession No. AF153615) and USA JSRV21 strain (Accession No. AF105220). The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.