Identification of crossbred buffalo genotypes and their chromosome segregation patterns

Chromosome analysis on different breed types of water buffaloes (Bubalus bubalis) was undertaken to identify their karyotypes and to determine the pattern of chromosome segregation in crossbred water buffaloes. Altogether, 75 purebred and 198 crossbred buffaloes including 118 from Malaysia and 80 from the Philippines, were analyzed in this study. The diploid chromosome number of the swamp buffalo from both countries was 48 and that of the river buffalo was 50, while all F1 hybrids exhibited 49 chromosomes. The F2 hybrids consisted of three different karyotype categories (2n = 48, 2n = 49, and 2n = 50), whereas the backcrosses included two different karyotype categories each, with 2n = 48 and 2n = 49 in the three quarters swamp types and 2n = 49 and 2n = 50 in the three quarters river types. Chi-square tests on pooled data from Malaysia and the Philippines indicated that the distribution of different karyotype categories of F2 animals did not deviate significantly from the 1:2:1 ratio expected if only balanced gametes with 24 and 25 chromosomes were produced by the F1 hybrids. In the three quarters swamp and three quarters river types, the respective karyotypic categories were in ratios approximating 1:1. The distribution of chromosome categories among the F2 hybrids and backcrosses suggests that only genetically balanced gametes of the F1 hybrids are capable of producing viable F2 and backcross generations.     

Polymorphism and gene organization of water buffalo MHC-DQB genes show homology to the BoLA DQB region

In cattle (Bos taurus), there is evidence of more than 50 alleles of BoLA-DQB (bovine lymphocyte antigen DQB) that are distributed across at least five DQB loci, making this region one of the most complex in the BoLA gene family. In this study, DQB alleles were analysed for the water buffalo (Bubalus bubalis), another economically important bovine species. Twelve alleles for Bubu-DQB (Bubalis bubalis DQB) were determined by nucleotide sequence analysis. A phylogenetic analysis revealed numerous trans-species polymorphisms, with alleles from water buffalo assigned to at least three different loci (BoLA-DQB1, BoLA-DQB3 and BoLA-DQB4) that are also found in cattle. These presumptive loci were analysed for patterns of synonymous (d(S)) and non-synonymous (d(N)) substitution. Like BoLA-DQB1, Bubu-DQB1 was observed to be under strong positive selection for polymorphism. We conclude that water buffalo and cattle share the current arrangement of their DQB region because of their common ancestry.     

Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome

The cytogenetic study of 182 river buffalo (Bubalus bubalis L., 2n = 50) of Murrah, Mediterranean and Jaffarabadi breeds, from the State of S?o Paulo, was carried out to characterize their chromosomes and to detect possible chromosomal abnormalities. The karyotypes were indistinguishable with conventional staining as well as with C and replication R banding techniques. In about 44% of the sample (8 males and 72 females), an X marker chromosome due to a fragile site was shown. The frequency of metaphases expressing the fragility site on the X was highly variable, from 2.86 to 41.03%. In females, the fragile site, rarely appeared on both X chromosomes. Most of the metaphases showed only 1 marker chromosome. In R-banded metaphases using 5-bromodeoxyuridine (BrdU) treatment, it corresponded in general to the late replicating X chromosome. No correlation between the X fragile site and altered phenotype was found. Structural and numerical chromosome rearrangements were ruled out in the present sample of buffalo.     

Testicular development and its relationship to semen production in Murrah buffalo bulls

The objectives of this study were to determine the relationship of age and body weight to testicular development and to establish norms for breeding soundness evaluations of Murrah buffalo bulls. Testicular measurements of 133 Murrah buffalo bulls of various ages were recorded with a caliper and a tape. Semen was collected twice a week for 5 weeks from groups of bulls which were 25-36 (n=17), 37-48 (n=16), 49-60 (n=14), of >60 (n=10) months of age. After examining volume, sperm concentration, and progressive motility semen was diluted in Tris-citric acid-egg yolk-fructose extender and frozen in 0.5 ml French straws. Testicular measurements of buffalo bulls were lower than those recorded for European breeds of cattle bulls. Nevertheless, like cattle bulls, scrotal circumference was highly correlated with other testicular measurements. Also, it had a significant positive relationship with semen volume and sperm concentration per ejaculate. Average sperm output per week in order of increasing age group was 15.3, 18.2, 19.8 and 23.6 x 10(9). Corresponding values for sperm output per week per gram of testis were 59.1, 45.8, 41.1, 36.2 x 10(6) indicating a reduction in spermatogenesis per unit of testis with advancing age. Compared to European breeds, daily sperm output in Murrah bulls was nearly 45% lower, presumably due to their nearly 40% lower scrotal circumference than Holstein bulls of the same age. These results indicate that in buffalo, as in cattle, scrotal circumference is a useful indicator of potential sperm output and may serve as an important criterion for selecting young bulls as AI sires.     

Draft genome of the river water buffalo

Water buffalo (Bubalus bubalis), a large‐sized member of the Bovidae family, is considered as an important livestock species throughout Southeast Asia. In order to better understand the molecular basis of buffalo improvement and breeding, we sequenced and assembled the genome (2n=50) of a river buffalo species Bubalus bubalis from Bangladesh. Its genome size is 2.77 Gb, with a contig N50 of 25 kb and the scaffold N50 of 6.9 Mbp. Based on the assembled genome, we annotated 24,613 genes for future functional genomics studies. Phylogenetic tree analysis of cattle and water buffalo lineages showed that they diverged about 5.8–9.8 million years ago. Our findings provide an insight into the water buffalo genome which will contribute in further research on buffalo such as molecular breeding, understanding complex traits, conservation, and biodiversity.     

Cytogenetic analysis of the tamaraw (Bubalus mindorensis): a comparison of R-banded karyotype and chromosomal distribution of centromeric satellite DNAs, telomeric sequence, and 18S-28S rRNA genes with domestic water buffaloes

The karyotype of the tamaraw (Bubalus mindorensis, 2n = 46) was investigated by RBG-banding technique and compared with those of the river and the swamp cytotypes of domestic water buffalo (B. bubalis). The tamaraw karyotype consisted of 6 submetacentric and 16 acrocentric autosome pairs (NAA = 56), and X and Y chromosomes. The RBG-banded karyotype of the three taxa had a high degree of homology, and the tamaraw karyotype could be explained by a Robertsonian translocation between chromosomes 7 and 15 and by a telomere-centromere tandem fusion between chromosomes 4p and 12 of the standardized river buffalo cytotype (2n = 50, NAA = 58). The buffalo satellite I and II DNAs were localized to the centromeric regions of all the tamaraw chromosomes. The biarmed chromosome 2 of the tamaraw resulting from the fusion between chromosomes 7 and 15 of the standard contained much larger amounts of the satellite I DNA than the other biarmed chromosomes, suggesting that this chromosome was formed by a relatively recent Robertsonian fusion. The (TTAGGG)n telomeric sequence was specifically localized to the telomeric region of all the buffalo chromosomes. The 18S + 28S rDNA was localized to the telomeric regions of the chromosomes 5p, 7, 19, 21, and 22 of the tamaraw and of their homologous chromosomes in the river and swamp buffalo cytotypes.     

Bull-specific effect on fertilization rate and viable embryo recovery in the superovulated buffalo (Bubalus bubalis)

This study was conducted to compare fertilization rate and viable embryo recovery rate in superovulated buffalo (n = 64) following insemination with semen from buffalo bulls (n = 5) having different fertility rates as determined by AI. Frozen-thawed semen from fertile bulls with similar post-thaw progressive motility and sperm morphology was used to inseminate buffalo at superovulatory estrus. Fertilization and viable embryo recovery rates differed among bulls, but this bull-specific effect was not related to the overall herd fertility rate as determined by AI in normal cyclic animals. These results indicate that individual bulls differ in their contribution to fertilization of superovulated donors and also to embryonic development, as determined by viable embryo recovery. Moreover, the results also suggest that buffalo bulls can be screened for optimal fertility and embryo recovery rates in superovulated donors. Further studies are warranted to ascertain the factors which contribute to such bull-specific effects.     

Karyotype analysis of mithun (Bos frontalis) and mithun bull x Brahman cow hybrids

We examined the cytogenetics of mithun (Bos frontalis), a domesticated version of the Asian gaur, and hybrids (F(1) generation) produced by artificial insemination of Brahman cows (Bos indicus) with mithun semen. Reproductive potential was also examined in the F(1) generation and a backcrossed heifer for utilization of heterosis. Metaphase chromosome spreads were examined by conventional staining and fluorescence in situ hybridization hybridized with the entire chromosome 1 of mithun as a specific probe. Chromosome 1 of mithun was found to be equivalent to Bos taurus chromosomes 2 and 28. The karyotype of the female mithun (N = 4) comprised 58 chromosomes, including 54 acrocentric and four large submetacentric chromosomes, without the four acrocentric chromosomes found in the domesticated species B. indicus. However, one of the four female mithuns with a normal mithun phenotype had an abnormal karyotype (2n = 59), indicating introgression from B. taurus or B. indicus. The F(1) karyotypes (N = 6, 3♂3♀) of the mithun bull × Brahman cow cross had 2n = 59, intermediate between their parents; they were consistent heterozygous carriers with a centric fusion involving rob(2;28), as expected. Two pronounced red signals were seen in the mithun karyotypes, three red signals in the mithun × Brahman hybrids, and four red signals in the Brahman cattle, in good agreement with centric fusion of bovine rob(2;28). The female backcross hybrid (N = 1) with 2n = 59 had a similar chromosome configuration to the F(1) karyotypes and had rob(2;28). Such female backcross hybrids normally reproduce; however, the F(1) bulls (N = 3) had not yet generated normal sperm at 24 months.     

Analysis of conserved microsatellite sequences suggests closer relationship between water buffalo Bubalus bubalis and sheep Ovis aries.

The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.     

河流型水牛与沼泽型水牛杂交后代(2n=49) 染色体遗传与繁殖力的研究

通过2n=50和2n=49两种核型三品种杂交水牛繁殖记录分析和其中2n=49三品种杂交水牛联会复合体及其精子染色体研究,结果表明,后者虽然公母都是可育的,由于它产生两种正常配子（n=24,n=25）和两种异常配子（n=24+1,n=25-1）,自群繁殖导致其子代染色体多态性（2n=50,2n=49和2n=48）;其异常配子,与正常配子结合,则产生非整倍性,致其繁殖力降低,表现为情期配种受胎率降低12.3%;年受胎率降低6.4%;产仔间隔长97.6天;终生（11岁）产仔数减少1.33～1.54头。 Abstract:After analysis of reproduction records of two types of karyotypes (2n=50 & 2n=49) of triple crossbreed buffaloes (TCB) and studies of synaptinemal complex and sperm chromosome of 2n=49 TCB,the results showed that 2 sorts of normal gametes (n=24 and n=25) and 2 sorts of abnormal gametes (n=24+1 and n=25-1) were produced in 2n=49 TCB.Thus,both male and female of 2n=49 TCB are reproducible,and chromosomal polymorphyism (2n=50,2n=49 & 2n=48) occurred in the progenies after intermating.But its fertility decreased because of aneuploidy combined between normal and abnormal gametes.Compared with 2n=50 TCB,the conception rates for individual inseminations and for whole year reduced 12.3% and 6.4%,calving interval were prolonged by 97.6 days,and calf numbers in its lifetime (up to 11-year old) were lower by approximate 1.5 calves,respectively.     

大戟科野桐属4种6个居群的核型研究

采用常规压片法对大戟科野桐属4种(含变种)6个居群进行了体细胞染色体数目和核型研究。研究结果显示其染色体数目为2n=22或2n=44,核型均为1A。核型公式分别为:绒毛野桐2n=4x=44m;白背叶2n=20m 2sm或2n=2x=22m或2n=20m(2sat) 2sm;广西白背叶2n=2x=22m;毛桐2n=2x=22m。其中白背叶[Mallotus apelta(Lour.)Muell.-Arg.]、广西白背叶[M.apelta(Lour.)Muell.-Arg.var.kwangsiensisMetic.]和毛桐[M.barbatus(Wall.)Muell.-Arg.]的染色体数目和核型均为首次报道。根据野桐属细胞分类学资料推测,多倍化和异基数变异是野桐属属内物种形成的重要方式。     

百合属4种植物的核型研究

采用常规压片法对4种百合属植物野百合(L.brow n ii F.E.B row n ex M ie llez.)、兰州百合(L.d av id iiDuchartre var.un icolor(Hoog.)Co Hon.)、川百合(L.d av id ii Duchartre)、湖北百合(L.henry i B aker)进行了核型研究.结果表明,4种百合的染色体数目均为2n=24,核型除川百合为3A外,其余3种均为3B型.核型公式分别为:野百合2n(2x)=24=4m(2SAT) 2sm(2SAT) 4st 14t,兰州百合2n(2x)=24=2m(2SAT) 2sm 10st(2SAT) 8t 2T,川百合2n(2x)=24=2m(2SAT) 2sm 12st(3SAT) 8t,湖北百合2n(2x)=24=4m 18st 2t,其中湖北百合染色体核型为首次报道.通过比较发现,兰州百合与川百合的核型最为相似,亲缘关系相近;核型不对称性为兰州百合>川百合>野百合>湖北百合,以湖北百合的核型较为原始.     

In vitro production of cattle-water buffalo (Bos taurus--Bubalus bubalis) hybrid embryos

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using inter species gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.     

Selection of buffalo bulls: Sexual behavior and its relationship to semen production and fertility

Forty-four buffalo bulls, used for artificial insemination, were studied to develop libido, mating ability and sexual behavior indices for selection purposes. For each index, 5 categories (i.e., excellent, very good, good, fair and poor) were established. The sexual behavior index was found to be more reliable than the libido and mating ability indices. Buffalo bulls in good to excellent categories were considered acceptable sires.

Reaction time, sexual aggressiveness, and scores of libido, mating ability and sexual behavior differed significantly among the various categories of the 3 indices. Libido significantly correlated with mating ability (r=0.89; P<0.001). Sexual behavior expressed significant relationship with age (r=0.41; P<0.01) and body weight (r=0.48; P<0.01), but was nonsignificant with the scrotal circumference (r=0.28; P>0.05) of buffalo bulls. However, these relationships were absent (P>0.05) in the acceptable sires. Semen production was correlated with sexual behavior in only the fair and poor categories of buffalo bulls (r=0.84; P<0.005). Sexual behavior had no relationship with the fertility rate of buffalo bulls (r=0.44; P>0.05). It is concluded that the sexual behavior index can be used successfully for the selection of buffalo bulls. Excellent- to good bulls should be used in an artificial breeding program if they qualify in the other selection indices.     

紫苏属植物染色体数目和核型分析

通过对26份紫苏属材料进行染色体数目的观察,并对其1种(包括紫苏和白苏)及2变种材料各一份进行核型分析.结果显示,紫苏属植物染色体数均为2n=40,其核型公式分别为紫苏[P.frutescens(L.)Britton var.frutescens(zi-su)]2n=40=20m(2SAT) 10sm 10st;野生紫苏[P.frutescens var.purpurascens(Hayata)H.W.Li]2n=40=18m 14sm(2SAT) 8st;回回苏[P.frutescens var.crispa(Benth.)Deane ex Bailey]2n=40=10m(2SAT) 14sm 16st;白苏[P.frutescens(L.)Britton var.frutescens(bai-su)]2n=40-24m 12sm(2SAT) 4st.结果表明,紫苏属不同原(变)种植物的染色体数日相同,但在核型上存在一定的差异,其染色体核型为首次报道.     

北京地区铁线莲属植物的核型研究

本文研究了产于北京地区的铁线莲属9个种和东北地区的2个种的核型,其中有6种为首次报道。10种为二倍体(2n=2x=16),1种为异源四倍体(2n=4x=32)。该属10个种的核型结构可列成以下公式:2n=2x=16=10m+2st+2-4st或2-4t(2-4SAT)。此外,对4个种中的杂合性进行了讨论。     

重楼属和延龄草核型的一致性

本文报道的重楼属3个种和变种与延龄草的核型基本一致,表明它们之间的亲缘关系很近,其核型简式为:延龄草(T. tschonoskii)2n=10=4m 2m(SAT) 2st(SAT) 2t(SAT) 1B;五指莲(Paris axialis)2n=10=6m 2t 2t(SAT) 2B;狭叶重楼(P. polyphylla var. stenophylla)2n=10=2m(SAT) 4m 2st 2t;小重楼(P.polyphylla var.minora)2n=10=4m 2sm 4t.     

几种重楼的染色体核型研究

作者对重楼属(Paris)的几个种:球药隔重楼(P.fargesii),毛重楼(P.mairei),花叶重搂(P.marmorata),黑籽重楼(P.thibetica),海南重楼(P.dunniana),巴山重搂(P.bashanensis),以及多叶重楼(P.polyphylla)的两个变种狭叶重楼(var.stenophylla)和华重楼(var.chinensis)的染色体核型进行了研究,发现种间及种内不同居群(population)间的核型都存在不同程度的差别。核型简式为:球药隔重楼K(2n)=2x=10=6m+2t(SAT)+2t+3bs,毛重楼K(2n)=2x=10=6m+4t+1bs,花叶重楼K(2n)=2x=10=6m+4t,黑籽重楼K(2n)=2x=10=2m+4m(SAT)+4t,海南重楼K(2n)=2X=10=6m+2t(SAT)+2t,巴山重楼K(2n)=2x=10=6m+4st,狭叶重楼K(2n)=2x=10=6m+1st+3t,华重楼K(2n)=2x=10=6m+4t。     

Monosomy X and gonadal dysgenesis in a buffalo heifer (Bubalus bubalis)

Investigations on a 3-yr-old river buffalo heifer presenting anestrus revealed a chromosome make-up of 2n=49 in the lymphocyte cultures, compared with the 2n=50 characteristic of riverine buffalo. The missing chromosome was identified as one of the Xs by karyotypic analysis, and monosomy X was confirmed by C and G-banding techniques. Both ovaries of the heifer were underdeveloped, although the other components of the internal genitalia were normal. The phenotypic and karyotypic features confirmed this to be a case of ovarian dysgenesis with 49,XO karyotype similar to that of the Turner's syndrome in man and other mammals.     

Preliminary radiation hybrid map for river buffalo chromosome 6 and comparison to bovine chromosome 3

We present the first radiation hybrid (RH) map of river buffalo (Bubalus bubalis) chromosome 6 (BBU6) developed with a recently constructed river buffalo whole-genome RH panel (BBURH(5000)). The preliminary map contains 33 cattle-derived markers, including 12 microsatellites, 19 coding genes and two ESTs, distributed across two linkage groups. Retention frequencies for markers ranged from 14.4% to 40.0%. Most of the marker orders within the linkage groups on BBU6 were consistent with the cattle genome sequence and RH maps. This preliminary RH map is the starting point for comparing gene order between river buffalo and cattle, presenting an opportunity for the examination of micro-rearrangements of these chromosomes. Also, resources for positional candidate cloning in river buffalo are enhanced.