[Analysis on personal protection in occupational population at high risk for brucellosis and influencing factor in China].

Objective: To understand the current status of personal protection in occupational population at high risk for brucellosis in China and provide evidence for the evaluation of implementation of National Brucellosis Prevention and Control Plan (2016-2020). Methods: Four counties in Shanxi Province and Xinjiang Uygur Autonomous Region were selected to conduct a questionnaire survey in occupational population at high risk for brucellosis from December 2019 to July 2020 by using cross-sectional survey methods. Results: A total of 2 384 persons at high risk for brucellosis were surveyed, and the standardized utilization rate of personal protective equipment (PPE) was 20.13% (480/2 384). The utilization rate of glove, mask, rubber shoe, and work cloth were 38.26% (912/2 384), 31.80% (758/2 384), 32.01% (763/2 384) and 30.87% (736/2 384),respectively. There were significant differences in the utilization rate and standardized utilization rate of the four types of PPE among populations in different age, occupation, educational level and area groups (all P<0.001). The utilization rate and standardized utilization rate of PPE were lower in people over 60 years old, women, farmers, and those with lower educational level. The results of multivariate analysis showed that occupation and area were the influencing factors for the standardized utilization of PPE, the standardized utilization rates of PPE were higher in herdsmen and veterinarians. The standardized utilization rate of PPE in Yanggao County and Huocheng County was significantly higher than that in Zuoyun County and Hunyuan County. Conclusions: The utilization rate of the four types of PPE in occupational population at high risk for brucellosis was not high in China, and the standardized utilization rate was low, lower than the requirement in National Brucellosis Prevention and Control Plan, and there were significant differences among different areas. It is urgent to distribute PPE to occupational population at high risk for brucellosis and carry out health education about PPE utilization. Meanwhile, it is necessary to strengthen information exchange or sharing among different areas.

Genome-wide analysis reveals genetic diversity, linkage disequilibrium, and selection for milk production traits in Chinese buffalo breeds.

The water buffalo is an important dual-purpose livestock that is widespread throughout central and southern China. However, there has been no characterization of the population genetics of Chinese buffalo. Using an Axiom buffalo genotyping array (Thermo Fisher Scientific, Wilmington, DE), we analyzed the genetic diversity, linkage disequilibrium pattern, and signature of selection in 176 Chinese buffaloes from 13 breeds. A total of 35,547 SNP passed quality control and were used for further analyses. Population genetic analysis revealed a clear separation between swamp and river types. Ten Chinese indigenous breeds were clustered into the swamp group, the Murrah and Nili-Ravi breeds were clustered into the river group, and the crossbred breed was closer to the river group. Genetic diversity analysis showed that the swamp group had a lower average expected heterozygosity. Linkage disequilibrium decay distance was much shorter in the swamp group compared with the river group, with an average square of correlation coefficient value of 0.2 of approximately 50 kb. Analysis of runs of homozygosity indicated extensive remote and recent inbreeding within swamp and river groups, respectively. Moreover, one genomic region under selection was detected between the river and swamp groups. Our findings contribute to our understanding of the characterization of population genetics in Chinese buffaloes, which in turn may be used in buffalo breeding programs.

Genome-wide association study identifies loci linked to serum electrolyte traits in Chinese Holstein cattle.

We aimed to identify QTL for serum electrolyte traits by performing a GWAS of calcium, chloride, sodium, potassium and magnesium ion concentrations in Chinese Holstein cattle. We detected five SNPs that had significant associations with calcium ion concentrations and identified GATA2 from Bos taurus chromosome (BTA)22 as having the highest significance. Among the genes with significant results, we speculate that TMEM123 might be related to calcium channel proteins according to the functions of the TMEM family.

[Occupational characteristics and clinical manifestations of 245 cases of occupational brucellosis].

Objective: To analyse epidemiological, clinical characteristics and laboratory examination results of 245 occupational brucellosis form 2008 to 2018, which providing theoretical basis for prevention and control of occupational brucellosis. Methods: Based on the China Center for Disease Control and Prevention Information System, a database of occupational brucellosis cases in HunlunBuir from January 2008 to July 2018 was established. The Epidemiological characteristics, Clinical manifestation, laboratory examination of 245 occupational brucellosis and 359 without occupational brucellosis were comparatively analyzed about the same period. Results: Among the 245 patients, 219 were males, 254 in 359, malese were significantly higher than control group (χ(2)=21.331, P<0.05) . Fever, fatigue, hyperhidrosis and splenomegaly are common in patients with occupational brucellosis and non occupational brucellosis. Arthralgia (54.3%/44.8%) and CRP (81.2%/71.3%) were significantly also higher than control group (χ(2)=5.193, P<0.05; χ(2)=7.704, P<0.05) Fever, hyperhidrosis, fatigue and splenomegaly were common clinical manifestation and signs in the two groups. Brucellosis can cause a variety of complications, including osteoarticular hematological system and hepatic involvement, some patients with multiple system damage. Conclusion: The incidence of occupational brucellosis in HulunBuir is concentrated in agriculture and animal husbandry. Veterinarians are the main occupational groups, Occupational health interventions should be strengthened for key occupational hazards, regular occupational health examination, avoid chronic brucellosis and protect the health of key occupational groups.

Comparative proteomic analysis of different developmental stages of swamp buffalo testicular seminiferous tubules.

With ageing, many protein components change markedly during mammalian spermatogenesis. Most of these proteins have yet to be characterized and verified. Here, we have employed two-dimensional electrophoresis coupled to tandem mass spectrometry to explore the different proteins from pre-pubertal, pubertal and post-pubertal swamp buffalo testicular seminiferous tubules. The results showed that 25 protein spots were differentially expressed among developmental stages, and 13 of them were successfully identified by mass spectrometry. Of which four proteins were up-regulated and three proteins were down-regulated with age, and the remaining six proteins were fluctuated among developmental stages. Bioinformatics analysis indicates that these proteins were probably related to cellular developmental process (53.8%), cell differentiation (53.8%), spermatogenesis (15.4%), apoptotic process and cell death (30.8%). Expression profiles of calumenin (CALU) and galectin-1 (LGALS1) were further verified via Western blotting. In summary, the results help to develop an understanding of molecular mechanisms associated with buffalo spermatogenesis.

Synonymous single nucleotide polymorphisms in the MC4R gene that are significantly associated with milk production traits in water buffaloes.

Melanocortin-4 receptor (MC4R) is associated with feed intake, growth, fatness, and carcass composition in many domestic animals. The aim of this study was to evaluate the association of single nucleotide polymorphisms (SNPs) in MC4R with milk production traits in water buffalo. Eight SNPs were identified by direct polymerase chain reaction sequencing of samples from 18 buffaloes. SNPs were then genotyped using the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) method in 332 buffaloes. Two of eight SNPs were not in Hardy-Weinberg equilibrium. Based on the SNP data, seven haplotypes were constructed. Three SNPs H1 (AGT), H2 (GAT), and H3 (GAC) accounted for 93.0% of the total individuals. Statistical analysis indicated that the SNP g.1104C>T was significantly associated with milk yield, protein, and fat percentage (P < 0.05). In conclusion, these results provide evidence that polymorphisms in the buffalo MC4R gene are associated with milk production traits and might be potential biomarkers for water buffalo breeding.

Associations between polymorphisms of the gene and milk production traits in water buffaloes.

Signal transducer and activator of transcription 1 () is an important regulator of mammary gland differentiation and cell survival that has been regarded as a candidate gene affecting milk production traits in mammals. Therefore, this study was conducted to evaluate significant associations between SNP of the gene and milk production traits in buffaloes. Here, 18 SNP were identified in the buffalo gene, including 15 intronic mutations and 3 exon mutations. All the identified SNP were then genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry methods from 192 buffaloes. All the SNP were in Hardy-Weinberg equilibrium, and 2 haplotype blocks were successfully constructed based on these SNP data, which formed 5 and 3 major haplotypes in the population (>5%), respectively. The results of association analysis showed that only SNP13 located in exon 10 was significantly associated with the milk production traits in the population ( < 0.05). Single nucleotide polymorphism 2, SNP5, SNP8, and SNP9 were associated with protein percentage, and SNP4 and SNP10 were associated with 305-d milk yield ( < 0.05). Our results provide evidence that polymorphisms of the buffalo gene are associated with milk production traits and can be used as a candidate gene for marker-assisted selection in buffalo breeding.

Cloning and sequencing of the rDNA gene family of the water buffalo (Bubalus bubalis).

The rDNA genes coding for ribosomal RNA in animals are complicated repeat sequences with high GC content. We amplified water buffalo rDNA gene sequences with the long and accurate (LA) PCR method, using LA Taq DNA polymerase and GC buffer, based on bioinformatic analysis of related organisms. The rDNA genes were found to consist of 9016 nucleotides, including three rRNA genes and two internal transcribed spacers (ITS), which we named 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA. We tested and optimized conditions for cloning these complicated rDNA sequences, including specific rules of primer design, improvements in the reaction system, and selection of the DNA polymerase.

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer.

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.

Study on the inter-subspecies nuclear transfer of river buffalo somatic cell nuclei into swamp buffalo oocyte cytoplasm.

The objective of this study was to explore the feasibility of inter-subspecies somatic cell nuclear transfer (SCNT) of river buffalo (50 chromosomes) somatic cell nuclei into swamp buffalo (48 chromosomes) oocyte cytoplasm. The enucleated swamp buffalo oocytes were fused with four different types of river buffalo cells: freshly thawed ear fibroblasts, serum-starved ear fibroblasts, cumulus cells and ear fibroblasts from a cloned buffalo calf. As a result, the developmental competence of embryos reconstructed with freshly thawed ear fibroblasts was the poorest (P<0.01), while those of the other three types were not different from each other. Furthermore, the efficiency of swamp-swamp buffalo, swamp-river buffalo and bovine-buffalo SCNT were also compared. The results showed that the blastocyst rate of swamp-river reconstructed embryos was not different from swamp-swamp embryos, while significantly higher than that of bovine-buffalo embryos (P<0.01). A total of thirty cloned blastocysts derived from freshly thawed ear fibroblasts were transferred into thirteen recipient buffaloes, four recipients established pregnancy, while three of them aborted on Days 65, 75 and 90 of gestation, respectively. One cross-bred buffalo (Murrah x swamp, 49 chromosomes) receiving three embryos delivered a 39 kg female calf on Day 335 of gestation. These results indicate that the inter-subspecies SCNT is feasible to produce swamp-river buffalo embryos, and these can develop to full term and result in live buffalo calves.

An inter-subspecies cloned buffalo (Bubalus bubalis) obtained by transferring of cryopreserved embryos via somatic cell nuclear transfer.

The aim of this study was to explore the feasibility of cryopreservation of inter-subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum-starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross-bred buffalo (Murrah × Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13-month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter-subspecies cloned embryos is feasible to produce buffalo offspring.

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up.

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.

Birth of twins after in vitro fertilization with flow-cytometric sorted buffalo (Bubalus bubalis) sperm.

Flow-cytometric sorting of mammalian sperm for production of offspring with the desired sex is one of the most important new biotechnologies available for livestock industry. The objectives of this study were: (i) to sort the sperm into X- and Y-enriched populations and (ii) use the sorted sperm for in vitro fertilization (IVF) to produce sex-preselected embryo and offspring. The results revealed that the accuracy of sorted X- and Y-sperm was 94% and 89%, respectively. There was a decrease in blastocyst development rate in IVF with sorted sperm comparing to unsorted sperm, but the percentage of blastocysts on D6-D8 was not statistically different. Transplantation of the presumed X-embryos derived from IVF into a recipient resulted in the birth of female twins. These results indicated the feasibility of sperm sorting by flow cytometry and in vitro production of sex-preselected embryos and offspring in buffalo.

Safety and efficacy of percutaneous injection of polyurethane elastomer (MPU) plugs for vas occlusion in man.

This report describes the safety and efficacy of the procedure for percutaneous injection of medical-grade polyurethane elastomer (MPU) to form plugs in the vas deferens. The injection of 0.16-0.22 ml MPU in 53 men resulted in occlusion and azoospermia in 85% of the men after 12 months; 96% achieved azoospermia by 2 years. The success rate of the method and the rate of sperm suppression to azoospermia depended on the shapes of the plugs; this was determined by palpation after insertion. There were few complications and the results are discussed in terms of the reversibility potential of this method.

PIP: In China, physicians performed percutaneous injection of medical-grade polyurethane (MPU) in 53 men at the National Research Institute for Family Planning in Beijing and followed them for 2 years to evaluate the safety and efficacy of MPU vas occlusion. The injection of 1% procaine solution was used to kill residual spermatozoa in the distal reproductive tract. 1 year and within-subject fructose levels did not fluctuate greatly. 85% of the men reached azoospermia within 1 year after vas occlusion. 96% were azoospermic at 18 months. 2 men still had spermatozoa in the semen after 2 years (8-48 million ml-1). Both MPU plugs in most men (74%) had a regular shape. All of these men reached azoospermia by 18 months. 91% of the 11 men who had 1 regular and 1 irregular shaped MPU plug reached azoospermia by 18 months. This percentage fell to 67% for the 3 men whose MPU plugs were both irregular. The physicians had removed irregularly shaped MPU plugs in 5 men earlier and the elastomer filled the vas lumen and the surrounding vas surface. Recanalization did not occur in these 5 patients. 1 man with MPU vas occlusion developed a hematoma. 3 men experienced hemaspermia within 2 weeks after vas occlusion, but medication made it disappear within 4 weeks. Injection of 1% procaine solution may have damaged the seminal vesicles, resulting in the hemaspermia. Longterm complications did not occur.

Occlusive or non-occlusive methods of vas deferens in males.

PIP: The spermicidal effect in vitro of 3 copolymers, maleic anhydride-vinyl acetate-butyl acrylate, acrylic acid-butyl acrylate, and methacrylic acid-butyl acrylate was evaluated. The copolymers were synthesized by solution polymerization using 2-azo-bis-iso-butyronitrile as initiator. The effect of mole ratio of co-monomers, initiator concentration, and temperature on polymerization were investigated. The acidity of the copolymer increased with acidity, while solubility decreased. The polymers were insoluble at pH 5, but soluble at pH 7.35. Swelling in water, determined gravimetrically, also varied with pKa. For spermicidal assays, co-polymers were soaked in Tyrode's solution pH 7.4 at 37 degrees Celsius for 48 hours, and samples of the supernatant were tested with motile human sperm according to the WHO Laboratory Manual. All 3 co-polymers were spermicidal in vitro. These materials were judged suitable for research on reversible vas sterilization.^ieng

Developmental and environmental regulation of a phenylalanine ammonia-lyase-beta-glucuronidase gene fusion in transgenic tobacco plants.

A 1.1-kilobase promoter fragment of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (EC 4.3.1.5) gene PAL2 was translationally fused to the beta-glucuronidase reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disk transformation. The distribution of beta-glucuronidase activity in these transgenic plants is very similar to that of endogenous PAL2 transcripts in bean, with very high levels in petals; marked accumulation in anthers, stigmas, roots, and shoots; and low levels in sepals, ovaries, and leaves. Histochemical analysis of the spatial pattern of beta-glucuronidase activity showed that the PAL2 promoter is highly active in the shoot apical meristem, the zone of cell proliferation immediately adjacent to the root apical meristem, and in the early stages of vascular development at the inception of xylem differentiation. Wounding and light evoke specific changes in the spatial pattern of beta-glucuronidase activity in stems, including induction in the epidermis. These data indicate that the PAL2 promoter transduces a complex set of developmental and environmental cues into an integrated spatial and temporal program of gene expression to regulate the synthesis of a diverse array of phenylpropanoid natural products.

Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PAL1 and PAL2 are expressed in shoots and only PAL1 is expressed in leaves. Strikingly, PAL2 is expressed at very high levels in petals, where PAL1 is only very weakly expressed and PAL3 is not expressed. All three genes are induced by mechanical wounding of hypocotyls, but fungal infection only activates PAL1 and PAL3. Illumination of etiolated hypocotyls activates PAL1 and PAL2 but not PAL3. Corresponding differential patterns of synthesis of specific PAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.

Organization and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris.

Chalcone synthase (CHS) catalyzes the first and key regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. In bean (Phaseolus vulgaris L.) and other members of the Leguminoseae, chalcone synthase is also involved in the synthesis of the isoflavonoid-derived phytoalexin antibiotics characteristic of this family. We have demonstrated that the haploid genome of bean contains a family of about six to eight CHS genes, some of which are tightly clustered. Treatment of bean cells with fungal elicitor activates several of these genes leading to the accumulation of at least five and probably as many as nine distinct CHS transcripts encoding a set of CHS isopolypeptides of Mr 42-43 kDa but with differing pI in the range pH 6-7. In elicited cells specific transcripts and encoded polypeptides are differentially induced with respect to both the extent and kinetics of accumulation. Wounding or infection of hypocotyl tissue also activates several CHS genes with marked differences in the pattern of accumulation of specific transcripts and encoded polypeptides in wounded compared to infected tissue or elicited cells, indicating operation of more than one cue for defense gene activation. Illumination induces accumulation of a different set of CHS transcripts including only one of the set hitherto demonstrated to be induced by biological stress. The organization and differential regulation of the CHS gene family in bean are discussed in relation to the functions of this enzyme in adaptative and protective responses to diverse environmental stresses.

Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression.

Derivatives of plasmid pRK290 that are useful for cloning and for analyzing gene expression in a wide variety of Gram-negative bacteria are described. A smaller broad host range plasmid derived from RK2, with properties similar to that of pRK290, is also described.