Fluoride concentration in teeth of the roe deer (Capreolus capreolus) and red deer (Cervus elaphus) from areas of Poland industrially uncontaminated with fluoride compounds.

The last biomonitoring study in Poland on intoxication with fluoride compounds of deer was conducted almost two decades ago. Given the fact that fluoride level in air and water is not widely monitored in Poland, it is justified to undertake monitoring of F- levels in people and other long-lived mammals. This paper provides the assessment of the present level of fluoride accumulation in mineralized tissue of large herbivorous mammals. The aim of the present study was to determine fluoride concentration in teeth of deer inhabiting the areas of Poland which are industrially uncontaminated with fluoride compounds, to establish possible correlations between the analysed parameters, and to provide a comparison of the present results with those obtained in other studies. Mean concentration of fluoride in all analysed samples amounted to 231.0 F mg/kg, with the minimum value of 22.0 F mg/kg and the maximum of 935.0 F mg/kg. This results from the development of industry and a widespread use of fluoride-supplemented caries prevention products which contributes to an intense accumulation of these substances in vertebrates, predominantly in mineralized tissue.

TaqMan real-time quantitative PCR for identification of antlers in tradition Chinese medicine.

In this study, a method was established for discriminating the true Cervus antlers from its counterfeits using TaqMan real-time quantitative PCR. The method combines the use of true Cervus antlers-specific primers, that amplify a 226 bp fragment from true Cervus antlers DNA, and mammalian-specific primers amplifying a 146 bp fragment from mammalian species DNA, which are used as endogenous control. A TaqMan probe that hybridizes in the 'Cervus antler' and also in the 'mammalian' DNA fragments is used to monitor the amplification of the target gene. The Cervus antler mitochondrial DNA was used as target gene to design the primers and TaqMan probes. The data revealed that the TaqMan real-time PCR-based assay can be used for identification of the true Cervus antlers from counterfeits in a single step. The limit of detection (LOD) was lower than 1 pg of DNA per reaction.

Identification of velvet antler and its mixed varieties by UPLC-QTOF-MS combined with principal component analysis.

Many species of velvet antler have been used as traditional medicine for thousands of years; however, as medicinal materials, velvet antler derived from different animals have different clinical effects. To distinguish the differences and homologies, ultra-performance liquid chromatography-quadruple-time of flight mass spectrometry (UPLC-QTOF-MS) coupled with principal component analysis (PCA) was developed and applied to identify these antler samples derived from Cervus nippon Temminck, Cervus elaphus Linnaeus and Rangifer tarandus Linnaeus, which were first tested and compared at the molecular level of protein. The UPLC-MS data of the trypsin digested samples were subjected to PCA, and the potential markers based on peptide were depicted to illustrate their differences. With the integrated strategy combining UPLC-QTOF-MS with PCA, the results from this study indicated that the proposed methods could be successfully applied to distinguish reindeer antler from sika deer antler and red deer antler, which were prescribed in the Chinese Pharmacopoeia (2015 edition).

Comparison of anti-inflammatory effect and protein profile between the water extracts from Formosan sambar deer and red deer.

Velvet antler (VA), the unossified antler from members of the family Cervidae, has been used in traditional Chinese medicines and health foods for over 2000 years in enhancement of kidney function and treatment or prevention of cardiovascular, immunological and gynaecological disease. The aim of this study was to investigate the anti-inflammatory effect of velvet antler water extracts from Formosan sambar deer (Rusa unicolor swinhoei, SVAE) and red deer (Cervus elaphus, RVAE). Results indicated that both SVAE and RVAE significantly reduced the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) productions in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells at concentrations above 200 µg mL-1. SVAE seems to demonstrate a better anti-inflammatory effect than that of RVAE in vitro. Both SVAE and RAVE also enhanced the anti-inflammatory cytokine IL-10 production in LPS-stimulated RAW 264.7 cells. The results of MTT assay indicated that SVAE and RVAE did not exhibit any cytotoxicity in LPS-stimulated RAW 264.7 cells. Two-dimensional (2D) gel electrophoresis analysis revealed that the levels of 6 specific proteins were different between these two velvet antlers samples. Furthermore, the storage period was the major factor affecting the anti-inflammatory activity of SAVE. In this study, we demonstrated the difference of anti-inflammatory effect and the protein profile between SVAE and RVAE. SVAE showed better anti-inflammatory potential than RVAE. In the future, the anti-inflammatory active components and their related mechanisms should be further investigated.

Genetic diversity, genetic structure and diet of ancient and contemporary red deer (Cervus elaphus L.) from north-eastern France.

In north-eastern France, red deer (Cervus elaphus L.) populations were rebuilt from a few hundred individuals, which have subsisted in remote valleys of the Vosges mountains, and to a lesser extent from individuals escaped from private enclosures; at present times, this species occupies large areas, mainly in the Vosges Mountains. In this study, we examined the population dynamics of red deer in the Vosges Mountains using ancient and contemporary mitochondrial DNA (mtDNA) from 140 samples (23 ancient + 117 modern) spanning the last 7'000 years. In addition, we reconstructed the feeding habits and the habitat of red deer since the beginning of agriculture applying isotopic analyses in order to establish a basis for current environmental management strategies. We show that past and present red deer in the Vosges Mountains belong to mtDNA haplogroup A, suggesting that they originated from the Iberian refugium after the last glacial maximum (LGM). Palaeogenetic analysis of ancient bone material revealed the presence of two distinct haplotypes with different temporal distributions. Individuals belonging to the two haplotype groups apparently occupied two different habitats over at least 7'000 years. AM6 correlates with an ecological type that feeds in densely forested mountain landscapes, while AM235 correlates with feeding in lowland landscapes, composed of a mixture of meadows and riverine, herb-rich woodlands. Our results suggest that red deer of north-eastern France was able to adapt, over the long term, to these different habitat types, possibly due to efficient ethological barriers. Modern haplotype patterns support the historical record that red deer has been exposed to strong anthropogenic influences as a major game species.

[Identification of Cervus nippon, C.elaphus and their hybridize samples based on COI and SRY gene].

For rapid identification of Cervus nippon, C. elaphus and their hybridize samples, the specific PCR for mutual authentication of them was established based on the SNPs in COI and SRY sequence. C. nippon, C. elaphus and their hybridize samples were collected from different origins, total DNA of 24 identified samples were extracted, and the COI and SRY gene was seqenced. SNPs in the COI and SRY sequences of the samples were found by Clustul X 2.1 program. Primers for identifying C. nippon and C. elaphus were designed according to the SNP site, two multi-PCR reaction system were established to identify them. In addition, 24 samples which were randomly collected in different herbal medicine market were identified. The band special for C. nippon (232 bp)and band special for C. elaphus (518 bp) based on COI sequence,and the band special for C. nippon (803 bp)and band special for C. elaphus (425 bp) based on SRY sequence, were found using multi-PCR reaction, and three of the twenty-four samples were identified as the hybridize samples. The multi-PCR reaction system could be used to identify C. nippon, C. elaphus and their hybridize samples.

[Molecular identification of hairy antler by analysis of high resolution melting].

High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization.

[Research progress on molecular genetics of forest musk deer].

Forest musk deer is one of the large-scale farming musk deer animals with the largest population at the same time. The male musk deer can secrete valuable medicines, which has high medicinal and economic value. Due to the loss of habitat and indiscriminate hunting, the numbers of wild population specie and the distribution have been drastically reduced. Therefore, in-depth understanding of the molecular genetics progress of forest musk deer will pave a way for musk deer protection and breeding. In this review, the progress associated with the molecular marker, genetic classification, artificial breeding, musk secretion and disease in past decades were reviewed, in order to provide a theoretical basis for subsequent molecular genetic researches in forest musk deer.

Utilization of real-time PCR to detect Rangifer Cornu contamination in Cervi Parvum Cornu.

Cervi parvum cornu (CPC) is a well-known ethnopharmacological source, whereas Rangifer cornu (RC) is not considered to be a major source. CPC is distributed in sliced form. Addition of RC to CPC has become an issue in CPC distribution because the appearance of sliced RC is not different from sliced CPC. Therefore, a real-time polymerase chain reaction (PCR) method was developed in this study to detect contaminating RC in CPC. The C-VIC and R-FAM primer/probe sets were designed to specifically amplify CPC and RC DNA, respectively. The specificities and sensitivities of real-time PCR using two primer/probe sets and the applicability of the real-time PCR to powder mixtures, which involved mixtures of powdered CPC and powdered RC in diverse ratios, were evaluated. Real-time PCR using C-VIC and R-FAM primer/probe sets specifically and sensitively amplified both CPC and RC DNA. Furthermore, real-time RCR sensitively detected RC DNA in the powder mixtures of CPC and RC. These results indicate that this real-time PCR method using two primer/probe sets is sufficiently applicable for the detection of contaminant RC in CPC.

[Content comparison of polyamines compounds in Penis et Testis Cervi].

OBJECTIVE: To compare the content of the main polyamines compositions in 10 samples of Penis et Testis Cervi from different species and habitats. MEHTODs: The putrescine spermidine and spermine in Penis et Testis Cervi were determined by HPLC. RESULTS: The contents of total Polyamine and 3 kinds of polyamines compositions in all samples were different. CONCLUSION: The content of the main polyamines compositions can be used to control the quality of Penis et Testis Cervi.

Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies.

As a direct and indirect consequence of human activities, only two subspecies, Cervus nippon sinchuanicus and Cervus nippon kopschi, currently subsist in the wild of China. However, a large population of Cervus nippon hortulorum and Cervus nippon nippon is raised in order to gain deer parts for Chinese traditional medicine. According to Chinese Wild Animal Conservation Law, hunting, capturing and trading of the wild sika deer are strictly banned, however, raising and trading of the domestic individual are permitted. Thus, it is very necessary to identify the subspecies of sika deer in China in forensic tests. In our study, we used mitochondrial DNA control region sequence analysis and phylogenetic analysis to identify the subspecies of sika deer. Mitochondrial DNA control region sequences analysis revealed that two haplotypes came from the unknown samples. One is the same as the haplotype that came from the samples of wild population of C. n. kopschi. Phylogenetic analysis indicated that the two haplotypes of unknown samples clustered with the haplotypes of C. n. kopschi, and had significant difference from the haplotypes of the other subspecies. These results together revealed that the unknown samples came from two individuals that belong to the wild population of C. n. kopschi living in the Qinglingfeng State Natural Reserve of Zhejiang province. Therefore, the results provide forensic evidence of illegal wild animal hunting.

[The resource conservation and breeding of wild Hydropotes inermis in China].

There have been four larger populations of wild water deer (Hydropotes inermis) in China, but all the populations lie in the endangered status. Most of populations are very small, and their distrbution ranges have already been broken up into many isolated patches. Furthermore, their habitats have been deteriorating. So it is very necessary to enhance the protection of habitat in the first place. Some endangered and small populations of wild water deer can be saved by inflows of domestic health deer. It is a valid way between conservation and utilization.

[Research on the identification of penis et testis cervi with molecular taxonomy].

OBJECTIVE: To make the kit with witch to identify Penis et Testis Cervi with molecular taxonomy. METHOD: The mtDNA of sika and red deer from different areas was amplified by PCR and sequenced. Compared with the mtDNA of bovine and horse from witch the false medicines were made, characteristic segments of deer were found. We selected one as the species distinctive PCR primer of deer. RESULT: The kit made up with this primer and related reagents could be used to discern Penis et Testis Cervi from the false medicine. CONCLUSION: It is a scientific, steady, accurate and convenient way to identify Penis et Testis Cervi with molecular taxonomy.

[Study on allele-specific diagnostic PCR of the traditional Chinese medicines of the deers].

AIM: To develop a convenient and accurate method of DNA molecular marker for the identification of traditional Chinese medicines made of deers, consisting of pilose antler, penis and testis, tendon and foetus. METHODS: Based on the analysis of DNA sequence of mitochondrial Cyt b gene from original animals of both genuine crude drugs, Cervus nippon and Cervus elaphus, and adulterants, a pair of allele-specific primers named as ILu01-L and ILu01-H were designed for distinguishing geniune crude drugs of deers from their adulterants. RESULTS: The results of diagnostic PCR annealing at 64 degrees C for original animals showed that a 365 bp fragment was only amplified from DNA templates of Cervus nippon and Cervus elaphus. For the identification of medicinal materials total of 43 samples from 6 packages were tested under the same reaction conditions except for DNA templates extracted from these crude drugs. Only 9 samples mentioned above was shown to generate positive amplificon. The result indicate that of 8 samples from 1 package of pilose antler and only 1 sample of deer tendon was genuine crude drug. After that, 3 amplified fragments selected randomly were performed with sequencing analysis with the purpose of verifying the results from diagnostic PCR. Data from sequencing confirmed the reliability of diagnostic PCR identification. CONCLUSION: The diagnostic primers designed in the present study were highly specific for Cervus nippon and Cervus elaphus, and they could be used for the authentication of traditional Chinese medicines made from the deer. The quality of the crude drugs of the deer in the current market is a problem and more effective quality control for these traditional Chinese medicines is urgently needed.

[A comparison of chemical composition and bioactivity of polypeptides from velvet antlers of Cervus nippon Temminck and Cervus elaphus Linnaeus].

OBJECTIVE: To compare the chemical composition and bioactivity of polypeptides(PPs) isolated from velvet antlers of sika deer (Cervus nippon Temminck) and red deer (Cervus elaphus Linnaeus). METHOD: The two kind of polypeptides were isolated from the above mentioned velvet antlers with same technology. The chemical composition was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF mass spectrometry. Stimulant activity of cells proliferation was measured by [3H] TdR incorporation into DNA. RESULT: The graphs of SDS-PAGE and MALDI-TOF MS of velvet antler polypeptides (VAPPs) from Chinese and New zealand red deer were very similar, but there were obvious difference in respect of graph between sika deer and red deer. VAPPs 25-50 mg.L-1 showed marked proliferation-promoting activity for rabbit costed chondrocytes, either sika deer or red deer. However, the activity of sika deer VAPPs 12.5 mg.L-1 for epidermal cells was weaker than that of red deer (12.5 mg.L-1). CONCLUSION: The chemical property and bioactivity of VAPPs from sika deer and red deer are significantly different.

[Character identification of 12 kinds of pilose antler medicinal materials].

In this paper, commercial medicinal materials of 12 kinds of pilose antler, Cervus nippon, C. elaphus, C. albirostris, C. unicolor, C. eldihainanus, C. timorensis C. porinus, Dama dama, Rangifer farandus, Alces alces, Elaphurus davidianus, Capreolus capreolus were compared and identified. A key and simple character illustration were listed.

Characterization of the erythrocyte superoxide dismutase allozymes in the deer Cervus elaphus.

The cDNA sequences for Cu,Zn superoxide dismutase from two Cervus elaphus subspecies, North American wapiti and European red deer, were determined. The derived amino acid sequences showed two differences: residue 8 was Leu in wapiti and Met in red deer and residue 25 was His in wapiti and Asn in red deer. The extra positive charge at position 25 in the wapiti isoform accounted for its greater mobility towards the cathode during non-denaturing electrophoresis, a procedure widely used in the genetic analysis of deer. There was no difference in specific activity between the two Cu,Zn superoxide dismutase isoforms, but the wapiti isoform was slightly more susceptible to heat denaturation.