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Individual variation in buffalo somatic cell cloning efficiency is related to glycolytic metabolism

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Science China(Life Sciences) 2022年第10期65卷 2076-2092页

作者： Chan Luo Zhiqiang Wang Jinling Wang Feng Yun Fenghua Lu Jiayuan Fu Qingyou Liu Deshun Shi State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources Guangxi UniversityNanning 530005China College of Animal Science and Technology Guangxi UniversityNanning 530005China

Mammalian individuals differ in their somatic cell cloning efficiency,but the mechanisms leading to this variation is poorly understood.Here we found that high cloning efficiency buffalo fetal fibroblasts(BFFs)displayed robust energy metabolism,looser chromatin structure,high H3 K9 acetylation and low heterochromatin protein 1α(HP1α)expression.High cloning efficiency BFFs had more H3 K9 ac regions near to the upstream of glycolysis genes by Ch IP-seq,and involved more openness loci related to glycolysis genes through ATAC-seq.The expression of these glycolysis genes was also found to be higher in high cloning efficiency BFFs by q RT-PCR.Two key enzymes of glycolysis,PDKs and LDH,were confirmed to be associated with histone acetylation and chromatin openness of BFFs.Treatment of low cloning efficiency BFFs with PS48(activator of PDK1)resulted in an increase in the intracellular lactate production and H3 K9 acetylation,decrease in histone deacetylase activity and HP1αexpression,less condensed chromatin structure and more cloning embryos developing to blastocysts.These results indicate that the cloning efficiency of buffalo somatic cells is associated with their glycolytic metabolism and chromatin structure,and can be improved by increasing glycolytic metabolism.

关键词： buffalo cloning efficiency chromatin openness histone acetylation glycolytic metabolism

buffalo anti-PDC-109 antibodies improve the semen quality profiles and in-vitro zona binding index and minimize the cryoinjury of sperm in cryopreserved buffalo semen

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Asian pacific Journal of Reproduction 2023年第3期12卷 131-143页

作者： S.S.Ramteke J.S.Rajoriya A.M.Shende S.K.Ghosh J.K.Prasad P.Perumal MAFSU-College of Veterinary and Animal Sciences UdgirMaharashtraIndia NDVSU-College of Veterinary Science and Animal Husbandry RewaMadhya PradeshIndia MAFSU-Nagpur Veterinary College NagpurMaharashtraIndia ICAR-Indian Veterinary Research Institute IzatnagarUttar PradeshIndia ICAR-Central Island Agricultural Research Institute Port BlairAndaman and Nicobar IslandsIndia

Objective:To optimize the concentration of PDC-109 protein in semen for higher cryopreservability and fertility by sequestration or neutralization of PDC-109 by its antibodies(anti-PDC-109 antibodies)in bubaline species.Methods:PDC-109 protein was purified by applying two-step chromatography procedures.Purified protein was injected in rabbits to raise antibodies.These raised anti-PDC-109 antibodies were used in neutralization or sequestration of PDC-109 in in-vitro model.Ejaculates were collected from buffaloes and splited for four groups.Group 1 received egg yolk Tris glycerol extender,without anti-PDC-109 antibodies,while group 2 to 4 received anti-PDC-109 antibodies 266μg/mL,80μg/mL,and 26μg/mL in Tris-fructose-citrate buffer,respectively.Semen quality parameters viz.,forward progressive motility,viability,total morphological abnormality,acrosomal integrity,plasma membrane integrity,cryoinjury and in-vitro zona binding index were evaluated.Results:Semen quality parameters of neat semen were within the normal range of bubaline species.Sperm motility,livability,acrosomal integrity,plasma membrane integrity,and cholesterol content of sperm were decreased and total sperm abnormality was increased significantly in post-thaw semen compared to those in pre-freeze and fresh semen(P<0.05).Semen in group 2 had higher sperm motility,livability,acrosomal integrity,plasma membrane integrity,and cholesterol content of sperm and lower total sperm abnormality significantly compared to those in group 1,3 and 4 at pre-freeze and post-thaw stages(P<0.05).Conclusions:Sequestration or neutralization of PDC-109 by its antibodies significantly improves pre-freeze,and post-thaw semen quality parameters and in-vitro zona binding index with simultaneously reducing cryoinjury or cryodamage in the sperm of bubaline species.

关键词： Anti-PDC-109 antibodies buffalo In-vitro capacitation In-vitro fertilization Semen

Effect of cholesterol-loaded cyclodextrin enriched extenders on the quality of pre- frozen and frozen buffalo semen

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Asian pacific Journal of Reproduction 2022年第3期11卷 146-152页

作者： Asmaa A.Mostafa Mohamed S.El-Belely Sayed T.Ismail Reda I.El-Sheshtawy Mohamed I.Shahba Abassia Frozen Semen Center General Organization for Veterinary ServicesCairoEgypt Department of Theriogenology Faculty of Veterinary MedicineCairo UniversityGizaEgypt Department of Animal Reproduction and Artificial Insemination National Research CentreDokkiGizaEgypt

Objective:To investigate the effects of non-permeable cryoprotectant,cholesterol-loaded cyclodextrin,when added at different concentrations into cooled and frozen-thawed semen extended with Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders.Methods:A total of 40 ejaculates from four buffalo bulls were collected using artificial vagina.Ejaculates were extended with one of Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders which contained different concentrations[0(control),0.75,1.50,2.25 and 3.00 mg/mL]of cholesterol-loaded cyclodextrin.The extended semen samples were cooled to 5曟and then frozen slowly to-196曟in 0.25 mL ministraws before being stored in liquid nitrogen pending its evaluation.Sperm motility,live sperm,normal sperm morphology,sperm membrane integrity and acrosome morphology were measured.Results:Supplementation of cholesterol-loaded cyclodextrin improved progressive motility,viability,morphology and acrosome as well as plasma membrane integrities at 1.50-2.25 mg/mL depending upon types of used extenders and stages of pre-and post-freezing process(Pbuffalo sperm quality.The most suitable concentration is 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.

关键词： buffalo Semen Tris Cooling Cryopreservation Cholesterol-loaded cyclodextrin Semen evaluation

L-carnitine improves developmental competence of buffalo oocytes in vitro

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Asian pacific Journal of Reproduction 2022年第5期11卷 236-242页

作者： Avijit Kumar Modak Md Nuronnabi Islam Asma Khatun Md Hasanur Alam Ireen Akter AKM Ahsan Kabir Md Abul Hashem Mohammad Moniruzzaman Department of Animal Science Bangladesh Agricultural UniversityMymensingh-2202Bangladesh

Objective:To investigate the effect of L-carnitine on in vitro maturation and subsequent in vitro embryo production of buffalo oocytes.Methods:Cumulus oocyte complexes(COCs)were aspirated from ovaries of slaughtered buffaloes.COCs were classified into good and fair qualities based on morphological observation of numbers and integrity of cumulus cells surrounding the oocyte.Both categories of COCs were placed in in vitro maturation medium with supplementation of different concentrations(0,0.250,0.375 or 0.500 mg/mL)of L-carnitine.Oocytes from both qualities were in vitro fertilized and in vitro cultured for 7 days,to examine the developmental competence.Results:Supplementation of L-carnitine to in vitro maturation medium increased the cumulus cell expansion rate of COCs to grade A,and reduced the cumulus cell expansion of COCs to grade B and grade C in both good and fair quality oocytes.Similarly,L-carnitine induced the in vitro meiotic progression of buffalo oocytes to metaphaseⅡin both good and fair quality oocytes.Additionally,L-carnitine reduced the rate of oocyte degeneration in both good and fair quality oocytes.L-carnitine increased the rate of cleaved formation at day 2 and blastocyst formation at day 7 during in vitro culture in both qualities of oocytes.Moreover,a higher rate of blastocyst production was observed in L-carnitine-treated fair quality oocytes,which was higher than the results in the untreated good quality oocytes.Conclusions:L-carnitine enhances meiotic maturation and subsequent embryo development from both good and fair quality buffalo oocytes.

关键词： buffalo Cumulus oocyte complex Embryo In vitro maturation In vitro fertilization L-carnitine

Understanding divergent domestication traits from the whole-genome sequencing of swamp-and river-buffalo populations

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National Science Review 2020年第3期7卷 686-701页

作者： Xier Luo Yu Zhou Bing Zhang Yi Zhang Xiaobo Wang Tong Feng Zhipeng Li Kuiqing Cui Zhiqiang Wang Chan Luo Hui Li Yanfei Deng Fenghua Lu Jianlin Han Yongwang Miao Huaming Mao Xiaoyan Yi Cheng Ai Shigang Wu Alun Li Zhichao Wu Zijun Zhuo Do Da Giang Bikash Mitra Mohammad Farhad Vahidi Shahid Mansoor Sahar Ahmed Al-Bayatti Eka Meutia Sari Neena Amatya Gorkhali Sigit Prastowo Laiba Shafique Guoyou Ye Qian Qian Baoshan Chen Deshun Shi Jue Ruan Qingyou Liu Animal Biotechnology Department Agricultural Biotechnology Research Institute of Iran-North Region 11. National Institute for Biotechnology and Genetic Engineering 12. Animal Genetic Sources DepartmentDirectorate of Animal Resources Ministry of Agriculture 13. Department of Animal Science Faculty of Agriculture Syiah Kuala University 14. Animal Breeding DivisionNational Animal Science Research Institute Nepal Agriculture Research Council 15. Animal Science Department Universitas Sebelas Maret 16. International Rice Research Institute State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources Guangxi University CAS Key Laboratory of Genome Sciences and Information Beijing Institute of Genomics Chinese Academy of Sciences National Engineering Laboratory for Animal Breeding Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural AffairsCollege of Animal Science and Technology China Agricultural University Guangdong Laboratory for Lingnan Modern Agriculture Genome Analysis Laboratory of the Ministry of Agriculture Agricultural Genomics Institute at Shenzhen Chinese Academy of Agricultural Sciences CAAS-ILRI Joint Laboratory on Livestock and Forage Genetic Resources Institute of Animal ScienceChinese Academy of Agricultural Sciences International Livestock Research Institute Faculty of Animal Science and Technology Yunnan Agricultural University Bacgiang Agriculture and Forestry University Cellular Immunology Lab Department of Zoology University of North Bengal

Domesticated buffaloes have been integral to rice-paddy agro-ecosystems for millennia, yet relatively little is known about the buffalo genomics. Here, we sequenced and assembled reference genomes for both swamp and river buffaloes and we re-sequenced 230 individuals(132 swamp buffaloes and 98 river buffaloes)sampled from across Asia and Europe. Beyond the many actionable insights that our study revealed about the domestication, basic physiology and breeding of buffalo, we made the striking discovery that the divergent domestication traits between swamp and river buffaloes can be explained with recent selections of genes on social behavior, digestion metabolism, strengths and milk production.

关键词： Bubalus bubalis genome buffalo domestication re-sequencing traits

Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection

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Journal of Animal Science and Biotechnology 2015年第4期6卷 395-401页

作者： Fanli Meng Hui Li Xiaoli Wang Guangsheng Qin Bj?rn Oback Deshun Shi Animal Reproduction Institute State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresourcesGuangxi University AgResearch Ltd. Ruakura Research CentreReproductive Technologies

Background： Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i） how much linear DNA to inject and ii） when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein （EGFP） plasmid into buffalo zygotes. Results： First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/pL DNA was significantly higher than from those injected with 5 pg/mL. However, 50 ng/pL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/pL DNA. Second, zygotes injected early （7-8 h post-insemination [hpi]） developed better than those injected at mid （12-13 hpi） or late （18-19 hpi） time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections （16.4 % vs 7.7 %）. With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions： Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.

关键词： buffalo Cytoplasmic injection Plasmid Transgenic Zygote

Involvement of Quebracho tannins in diet alters productive and reproductive efficiency of postpartum buffalo cows

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Animal Nutrition 2019年第1期5卷 80-86页

作者： Sobhy M.A.Sallam Marwa F.A.Attia Adel N.M.Nour El-Din Samir Z.El-Zarkouny Adel M.Saber Hani M.El-Zaiat Moustafa M.Zeitoun Department of Animal and Fish Production Faculty of AgricultureAlexandria University Animal Production Research Institute Agricultural Research CenterDokki

This study was conducted to investigate the effects of 10 weeks supplementation of Quebracho tannins(QT; 0 [control], 100 [QT100] or 200 g/[cow$d] [QT_(200)]) to 30 multiparous postpartum buffalo cows(10 cows per group) on milk yield and composition, blood metabolites and reproductive performance.Supplementation of QT100 had no significant effect on milk yield, whereas QT_(200) decreased(P buffalo cows displayed negative consequences on their productive and reproductive performances.

关键词： buffalo Tannins Milk traits Blood metabolites Ovarian follicles Conception rate

The role of MC1R gene in buffalo coat color

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Science China(Life Sciences) 2010年第2期53卷 267-272页

作者： MIAO YongWang1,2,WU GuiSheng3,WANG Lei2,LI DaLin4,TANG ShouKun5,LIANG JianPing2,MAO HuaMing2,LUO HuaiRong3 & ZHANG YaPing1,6 1 Laboratory for Conservation and Utilization of Bio-resource,Yunnan University,Kunming 650091,China 2 Faculty of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China 3 Kunming Institute of Botany,Chinese Academy of Sciences,Kunming 650204,China 4 Yunnan Institute of buffalo Science and Technology,Kunming 650021,China 5 Animal Husbandry and Veterinary Station of Luxi city,Luxi 678400,China 6 State Key Laboratory of Genetic Resources and Evolution,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming 650223,China Laboratory for Conservation and Utilization of Bio-resource Yunnan University Kunming China Faculty of Animal Science and Technology Yunnan Agricultural University Kunming China Kunming Institute of Botany Chinese Academy of Sciences Kunming China Yunnan Institute of buffalo Science and Technology Kunming China Animal Husbandry and Veterinary Station of Luxi city Luxi China State Key Laboratory of Genetic Resources and Evolution Kunming Institute of Zoology Chinese Academy of Sciences Kunming China

Melanocortin-1 receptor (MC1R) plays a major role in pigmentation in many species.To investigate if the MC1R gene is associated with coat color in water buffalo,the coding region of MC1R gene of 216 buffalo samples was sequenced,which included 49 black river buffalo (Murrah and Nili-Ravi),136 swamp buffalo (Dehong,Diandongnan,Dechang,Guizhou,and Xilin) with white and gray body,and 31 hybrid offspring of river buffalo Nili-Ravi (or Murrah) and swamp buffalo.Among the three variation sites found,SNP684 was synonymous,while SNP310 and SNP384 were nonsynonymous,leading to p.S104G and p.I128M changes,respectively.Only Individuals carrying homozygote EBR/EBR were black.The genotype and phenotype analysis of the hybrid offspring of black river buffalo and gray swamp buffalo further revealed that the river buffalo type allele EBR or the allele carrying the amino acid p.104S was important for the full function of MC1R.The in silico functional analysis showed that the amino acid substitutions p.G104S and p.M128I had significant impact on the function of MC1R.Above results indicate that the allele EBR or the allele carrying the amino acid p.104S was associated with the black coat color in buffalo.

关键词： buffalo coat color MC1R

Expression of BSE-associated proteins in the CNS and lymphoreticular tissues of cattle and buffalo

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Science Bulletin 2016年第17期61卷 1377-1383页

作者： Hui Zhao Si-Qi Wang Li-Li Qing Lin-Lin Liu Ya-Ping Zhang State Key Laboratory for Conservation and Utilization of Bioresource Yunnan University Kunming 650091 China School of Life Science Yunnan University Kunming 650091 China State Key Laboratory of Genetic Resources and Evolution Kunming Institute of Zoology Chinese Academy of Sciences Kunming 650223 China

Bovine spongiform encephalopathy(BSE),which has been documented in 190,000 cases of BSE-infected cattle,to our knowledge,has not been reported in buffalo.Prion protein(PrP) is critical to susceptibility and development of BSE.Moreover,a new PrP-like protein,Shadoo(Sho),has been shown to have overlapping expression patterns and shared functions with PrP.Therefore,we hypothesize that differences in expression at the transcriptional level and/or the post-transcriptional level of the two genes may be associated with these specific differences between cattle and buffalo.We compared the relative mRNA expression of the prion protein gene(PRNP) and the Shadoo gene(SPRN) in 3% RNA samples using real-time PCR.We also analyzed PrP/Sho protein from 96 samples by Western blot.Our results demonstrated significantly lower PrP expression in the cerebellum,obex,mesenteric lymph node,and bronchial lymph node tissues,but higher relative expression of Sho in the cerebrum and spleen in buffalos compared with catties.Although these results support our primary assumption,Sho and PrP expressions did not correlate with corresponding mRNA expression,suggesting that the biological modulations of both PrP and Sho proteins are at post-translational levels.Moreover,positive correlations between PRNP and SPRN were found in cattle and buffalo cerebrum as well as in buffalo obex.Conversely,negative correlations between PrP and Sho were detected in buffalo cerebellum and obex tissues.These findings suggest that additional post-transcriptional studies are warranted to elucidate mechanisms behind prion diseases.

关键词： BSE PRNP SPRN Cattle buffalo