Assignment of six genes to bovine chromosome 13

Six previously unassigned genes have been assigned to the BTA13 by means of somatic cell hybrid analysis. Polymerase chain reaction primer design for the amplification of AHCY , PLTP , PPGB and PCK1 was based on nucleic acids sequence information of homologous genes on HSA20. Primers for PDYN and ASIP were designed from porcine and bovine sequence information, respectively. Homology was established by sequence analysis. These assignments support the previous finding of a conserved syntenic relationship between HSA20 and BTA13 and contribute to the understanding of the chromosomal evolution of BTA13. This is significant since the prion protein gene, thought to play a key rule in the development and course of bovine spongiforme encephalopathy is also located on BTA13.     

Assignment of new loci to river buffalo chromosomes confirms the nature of chromosomes 4 and 5

Introduction Rapid development of the river buffalo physical map can be achieved by coupling its development to that of the cattle gene map. Syntenic conservation between cattle and buffalo has been demonstrated, mainly using somatic cell hybrids (de Hondt et al. 1991; El Nahas et al. 1993, 1996, 1998; de Hondt et al. 1997; El Nahta 1996; Oraby et al. 1977), and by using in situ hybridization as reviewed by Iannuzzi (1997). G- and R-banding comparisons between cattle (2n = 60) and river buffalo (2n = 50) chromosomes have revealed a large number of banding homologies between the two species, both at early-metaphase (Gupta and Ray -Chaudhury 1978; Di Berardino et al. 1981) and prometaphase stages (Iannuzzi et al. 1990). Banding homology indicates that the five river buffalo biarmed pairs originate from centric fusion translocation between two of ten homologous cattle autosomes, which is very supportive of the hypothesis that both species have a common ancestor (Wurster and Benirschke 1968). Based on cytological analysis and banding homology between cattle and buffalo chromosomes, the five biarmed chromosomes of the river buffalo BBU1, BBU2, BBU3, BBU4, BBU5 were thought to originate from fusion of cattle chromosome (BTA) 1/25; 2/23; 8/19; 5/28; and 16/29 respectively (Iannuzzi et al. 1990; Report of the Committee for the Standardization of Banded Karyotopes of the River Buffalo 1994). However, the analysis of synteny between molecular markers assigned to different cattle syntenic groups demonstrated that BBU1 results from fusion of BTA 1 and 27 rather than 1 and 25 (El Nahas et al. 1977). This called for expanding the analysis of syntenic relationships between marker loci to confirm the nature of the other biarmed buffalo chromosomes. The purpose of this study is to test synteny between markers in buffalo and to confirm the nature of the biarmed buffalo chromosomes 4 and 5, using marker loci and somatic cell hybrids.     

水牛生精上皮细胞的冷冻保存

为探讨水牛睾丸生精上皮细胞冷冻保存的适宜冷冻保护剂及其浓度,在DMEM中分别添加不同浓度的冷冻保护剂二甲基亚砜(DMSO)、甘油(G)、丙二醇(PG)和乙二醇(EG),以及10%的胎牛血清(FBS),对3～5月龄水牛生精上皮细胞进行冷冻保存,解冻后台盼蓝染色测定细胞活率.结果显示,当以0%,5%.10%,15%,20%DMSO添加时,10%DMSO组的细胞解冻后活率均显著高于其他各组(P<0.05);当以0%,20%,25%,30%,35%,40%G添加时,35%G组的细胞解冻后活率均显著高于其他各组(P<0.05);当以0%,5%,10%,15%,20%,25%PG添加时,15%～25%PG各组的细胞解冻后活率均显著高于其他各组(P<0.05),但以20%PG组为最高;当以0%,5%,10%,15%,20%EG添加时,5%～20%EG各组的细胞解冻后活率均显著高于0%EG组,但以10%EG组为最高;而对4种冷冻保护剂各最优浓度组的细胞解冻后活率比较,10%DMSO、35%G组的细胞活率均显著高于20%PG、10%EG组(P<0.05).结果表明,以添加10%DMSO或35%G于含10%FBS的DMEM冷冻液中,采用两步慢速冷冻,液氯保存,37℃水浴1 min解冻,是一种具有较高复苏率的冷冻保存水牛生精上皮细胞的方法.     

荧光定量PCR分析水牛胚胎致密化相关基因表达方法的建立

胚胎致密化是哺乳动物早期胚胎发育过程中一个非常重要的环节,有研究发现致密化相关基因的表达水平与体外发育胚胎的质量之间存在着相互关联作用。为了分析水牛胚胎早期发育中致密化相关基因在mRNA水平上的表达并确定其定量分析方法,本研究根据黄牛有关基因序列设计了Cx43、Cx31、E-cad以及组蛋白基因(Histone H2A)引物序列,分析了黄牛和水牛致密化基因的同源性,并确定各目的基因在水牛胚胎的SYBR Green I实时定量PCR反应条件。     

Sequence analysis of the NRAMP1 genes from different bovine and buffalo breeds

The natural resistance associated macrophage protein 1 (Nramp1) has been reported to confer resistance or susceptibility to Mycobacterium bovis, Salmonella typhimurium, and Leishmania donovani in the mouse, Mus musculus. A Gly and Asp substitution at position 169 of the mouse Nramp protein is invariably associated with the resistant and susceptible phenotypes, respectively. The present study aimed to detect polymorphisms in the NRAMP1 gene from different cattle and buffalo breeds. Genomic DNAs from five breeds of cattle and four breeds of buffalo were used in the study. Sequencing showed two nucleotide substitutions found in intron 4, three in exon V, and ten in intron 5. An amino acid substitution was observed at nucleotide position 1202 in exon V of the Japanese black, Angus, Philippine and Bangladesh swamp-type buffaloes which coded for Thr, while the Korean cattle, Holstein, African N'dama, Indonesian swamp-type buffalo and the Bangladesh river-type buffalo had Ile. All the breeds of cattle and buffaloes tested in this study coded for Gly at the position in exon VI which corresponds to the same amino acid of the murine Nramp1-resistant phenotype at position 169. The phylogenetic relationship among the different breeds showed a cluster comprised mainly of cattle and another one mainly of buffaloes.     

SNP identification, linkage and radiation hybrid mapping of the porcine lamin A/C (LMNA) gene to chromosome 4q

The lamins are components of nuclear lamina and they have a profound influence on nuclear structure and functions. They are encoded by three genes, LMNA, LMNB1 and LMNB2. A genomic fragment of the porcine LMNA gene (822 bp; from exons 7 to 9) was amplified by polymerase chain reaction and comparatively sequenced. Four single nucleotide polymorphisms (SNPs) were identified in intronic sequences: G162A, G208A, T367G and C618T. The SNPs are within the restriction sites for enzymes Bsh1236I, HpaII, AluI and Bsh1236I respectively. Allele frequencies at SNPs G208A, T367G and C618T were determined by using eight pig breeds. Linkage analysis in the Hohenheim Meishan × Piétrain family placed the LMNA gene in the chromosome 4q linkage group, between MEF2D and GBA (MEF2D– 3.0 cM –LMNA– 0.2 cM –GBA). In radiation hybrid mapping LMNA was most significantly linked to SW270 on chromosome 4 (39 cR; LOD = 7.86). The LMNA gene is located in the quantitative trait loci region for some carcass traits on chromosome 4q.     

不同乳脂率德宏奶水牛乳腺组织中FATP1基因表达研究

为研究不同乳脂率德宏奶水牛乳腺组织中FATP1基因的表达情况,试验选取216头处于泌乳盛期的健康德宏奶水牛,分为高乳脂率组(H组)、中乳脂率组(M组)和低乳脂率组(L组),采集乳样,用乳品分析仪测定乳脂率,利用气相色谱法测定脂肪酸含量.采集乳腺组织,利用荧光定量PCR法检测FATP1基因的mRNA表达水平,并与乳脂率和...     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

A quantitative study of seminiferous tubular cells in the developing Murrah buffalo testis

We report here a systematic quantitative study of the seminiferous tubular cells of Murrah buffaloes. The most advanced germ cell types in the different age groups (months) were A(0) spermatogonia (SG) (1 and 3), early pachytene (6 and 9), late pachytene (12), secondary spermatocytes (15 and 18), elongating spermatids (21 and 24), elongated spermatids attached to Sertoli cells (30), elongated spermatids detached from Sertoli cells (36) and spermatozoa (42 and 48). Central primitive Sertoli cells (CPSC) and basal primitive Sertoli cells (BPSC) were present in the sex cord of one-month-old calves, while Sertoli cells (SC) were first seen in nine-month-old calves. The number of gonocytes were maximal at six months but they were not seen after this time. Prespermatogonia (PSG) and SG were at a maximum at nine months of age but PSG were not seen after 36 months. The number of SG decreased significantly after nine months up to 36 months of age.Although spermatocytes and spermatids appeared in earlier developmental stages, a rapid increase in their number was recorded after 36 months. The number of SC was maximal in 18-month-old animals. BPSC predominated in the sex cord of animals aged one to six months, SG at 9-12 months of age, primary spermatocytes from 15-30 months and spermatids from 36 to 72 months and in older animals. We concluded that a decrease in the number of SG in buffalo calves after nine months of age might be responsible for a delay in sexual maturity. Moreover, the small number of spermatocytes and spermatids present before 36 months of age may be associated with the low yield of different germ cell divisions and with the cellular degeneration. A rapid increase in the number of spermatocytes and spermatids after 36 months resulted in sexual maturity between 42 and 48 months.