The Complete Mitochondrial Genome and Phylogenetic Analyses of To Chicken in Vietnam.

Indigenous chicken breeds have both cultural significance and economic value since they possess unique genetic characteristics that enable them to adapt to the local environment and contribute to biodiversity, food security, and sustainable agriculture in Vietnam. To (Tò in Vietnamese) chicken, a Vietnamese indigenous chicken breed, is popularly raised in Thai Binh province; however, little known is about the genetic diversity of this breed. In this study, we sequenced the complete mitochondrial genome of To chicken for a better understanding of the diversity and origin of the breed. The results of sequencing showed that the mitochondrial genome of To chicken spans a total length of 16,784 base pairs and comprises one non-coding control region (known as the displacement-loop (D-loop) region), two ribosomal RNA genes, 13 protein-coding genes, and 22 transfer RNA genes. The phylogenetic tree analyses and estimated genetic distances based on 31 complete mitochondrial genome sequences indicated that To chicken has a close genetic distance with the Laotian native chicken breed, Lv'erwu breed in China, and Nicobari black and Kadaknath breeds in India. The result of the current study might be important for conservation, breeding, and further genetic studies of To chicken.

Association of Lactoferrin and Toll-like Receptor 2 Genotypes with Mastitis and Milk Components in Vietnamese Holstein Cattle.

Mastitis is one of the most widespread diseases in dairy cows and causes huge losses for the dairy industry. Molecular markers can be used for the quick diagnosis of mastitis infection, consequently reducing the loss caused by this disease. Lactoferrin (LTF) and Toll-like receptor 2 (TLR2) have been suggested as candidate genes for mastitis; however, their associations with the mastitis incidence and milk components have not been reported in Vietnamese Holstein cows. This study examined the association of TLR2 and LTF polymorphisms with subclinical mastitis and milk components in the Holstein breed raised in Vietnam. Among 192 samples, we identified 44 mastitis-positive samples (22.92%). The mastitis significantly reduced the fat and lactose components in milk (p < 0.001) but increased the protein concentration in milk. A total of 94 (49%) and 98 (51%) cows had AA and AB genotypes for the LTF gene, respectively. No significant association was found between the LTF genotypes and the milk component traits or mastitis incidence (p > 0.05). The interaction between LTF and mastitis incidence was significantly associated with the protein percentage (p = 0.01). A total of 78, 76, and 38 cows had genotypes GG, GT, and TT for the TLR2 gene, respectively. TLR2 genotypes were not significantly associated with mastitis incidence (p > 0.05) but were significantly associated with pH value (p = 0.03). The interaction between TLR2 and mastitis incidence was significantly associated with the fat (p = 0.02) and protein percentage (p = 0.04). Further studies are required to confirm the roles of LTF and TFL2 in mastitis in the Holstein breed in Vietnam.

Detection of non-reference porcine endogenous retrovirus loci in the Vietnamese native pig genome.

The Vietnamese native pig (VnP)-a porcine breed with a small body-has proven suitable as a biomedical animal model. Here, we demonstrate that, compared to other breeds, VnPs have fewer copies of porcine endogenous retroviruses (PERVs), which pose a risk for xenotransplantation of pig organs to humans. More specifically, we sought to characterize non-reference PERVs (nrPERVs) that were previously unidentified in the reference genome. To this end, we used whole-genome sequencing data to identify nrPERV loci with long terminal repeat (LTR) sequences in VnPs. RetroSeq was used to estimate nrPERV loci based on the most current porcine reference genome (Sscrofa11.1). LTRs were detected using de novo sequencing read assembly near the loci containing the target site duplication sequences in the inferred regions. A total of 21 non-reference LTR loci were identified and separated into two subtypes based on phylogenetic analysis. Moreover, PERVs within the detected LTR loci were identified, the presence of which was confirmed using conventional PCR and Sanger sequencing. These novel loci represent previously unknown PERVs as they have not been identified in the porcine reference genome. Thus, our RetroSeq method accurately detects novel PERV loci, and can be applied for development of a useful biomedical model.

Evaluation of genetic diversity and population structure in four indigenous duck breeds in Vietnam.

This study characterized genetic diversity and population structure of four indigenous Vietnamese duck breeds and an exotic breed for setting the conservation priority. A total of 200 samples from four duck breeds (Sincheng, Minhhuong, Muongchieng and Bauben) and an exotic breed (Supermeat) were genotyped for fifteen microsatellite markers. The average number of alleles per locus was 14.07. A moderate genetic diversity was observed for indigenous breeds as mean of observed and expected heterozygosity as Ho = 0.50 and He = 0.57, respectively. The Bauben had the lowest values of Ho (0.41) and He (0.48) while Sincheng had the highest values of Ho (0.6) and He (0.69), respectively. The inbreeding coefficients (FIS) ranged from 0.12 to 0.16, and all breeds were significantly under heterozygote deficit. Nei's genetic distance was the shortest between Minhhuong and Muongkhieng. The discriminant analysis of principal components of studied breeds resulted in four genetic clusters. The Minhhuong and Muongkhieng breeds joined the same genetic cluster while other breeds had their own clusters. These results indicated that the possibility to combine Minhhuong and Muongkhieng for reducing the cost of conservation and suggested that conservation of the Bauben should be prioritized to avoid inbreeding depression and genetic drift.

Optimization of donor cell cycle synchrony, maturation media and embryo culture system for somatic cell nuclear transfer in the critically endangered Vietnamese I pig.

Our aim was to establish an efficient culture system to produce embryos by SCNT of the endangered Vietnamese I pig. Reducing the serum concentration from 10.0% to 0.2% during culture efficiently synchronized I pig fibroblasts used as donor cells at the G0/G1 stage. Oocyte maturation in a defined porcine oocyte medium (POM) supplemented with EGF and gonadotrophins resulted in higher cleavage and blastocyst rates compared with a non-defined POM containing pig follicular fluid (but without EGF) and both the defined and non-defined variants of NCSU-37. For embryo culture PZM3 and PZM5 media were superior to NCSU-37, in terms of the percentage of cleaved embryos. Addition of serum to PZM3 medium on Day 5 of culture (Day 0 = SCNT) improved blastocyst development. When SCNT embryos were transferred at the blastocyst stage, 7 of 11 recipients became pregnant. However, live offspring were not obtained. In conclusion, we established a system for the production of I pig embryos by SCNT and achieved blastocyst production rate at 26.4% by improving culture systems for donor cells, oocytes and embryos culture. Transfer of embryos resulted in pregnancies; however, live offspring were not obtained.

An assessment of genetic diversity and population structures of fifteen Vietnamese indigenous pig breeds for supporting the decision making on conservation strategies.

The study aimed to characterize genetic diversity, genetic clusters, and phylogenetic relationships of 15 Vietnamese indigenous pig breeds across the country for supporting the decision making of the conservation strategies. For this purpose, 638 samples from the breeds together with two wild pig breeds and an exotic breed were genotyped with 19 microsatellite markers recommended from FAO/ISAG for diversity studies. The higher genetic diversity was observed for indigenous breeds (mean He = 0.67) and wild breeds (mean He = 0.74); the indigenous CoAluoi breed compared the out-breed Landrace (He = 0.59). Fifteen percent of the genetic variation came from differences among breeds. The unrooted neighbor-joining dendrogram obtained from Nei's genetic distances showed three nodes with 100% supported bootstrap values. The first node included the three indigenous breeds (Hung, LungPu, and MuongKhuong), the second node included the indigenous BaXuyen and the exotic Landrace, and the third node included the two wild Thailand and Vietnam pig breeds. The discriminant analysis of principal component (DAPC) of 18 studied breeds resulted in 12 genetic clusters. Unlike the other indigenous breeds, the BaXuyen was in the same genetic cluster with the exotic Landrace-which agreed with the 100% bootstrap value of their node-so the BaXuyen should not be conserved. The five indigenous pig breeds-Huong, VanPa, Soc, ChuProng, and CoAluoi-were assigned to their own clusters, which agreed with the low supported bootstrap values of their nodes. These five breeds should be in the high conservation priority. Finally, the 9 indigenous pig breeds (MuongKhuong, LungPu, Hung, TapNa, MongCai, HaLang, Lung, Meo, and Ban breeds) formed four genetic admixture structures. These results suggest the conservation strategies should be built based on from five to nine pig groups thus reducing the cost of conservation whereas still remaining the genetic diversity of the studied breeds.

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig.

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus-oocyte complexes were collected from antral follicles of 7-8 mo old female cyclic Ban pigs and vitrified in micro-drops. Oocyte morphology, lipid content, post-warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post-warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo-tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.

Comparison of the microdrop and minimum volume cooling methods for vitrification of porcine in vitro-produced zygotes and blastocysts after equilibration in low concentrations of cryoprotectant agents.

We compared the efficacy of the microdrop and minimum volume cooling (MVC) methods for the vitrification of in vitro-produced porcine zygotes and blastocysts after equilibration in low concentrations of cryoprotectant agents. Zygotes and blastocysts were equilibrated in 2% (v/v) ethylene glycol and 2% (v/v) propylene glycol for 13-15 min. Then, they were vitrified in a medium comprised of 17.5% ethylene glycol, 17.5% propylene glycol, 0.3 M sucrose, and 50 mg/ml polyvinylpyrrolidone either by either dropping them directly into liquid nitrogen (microdrop method) or placing them on Cryotop sheets in a minimum volume of medium and plunging into liquid nitrogen (MVC method). Both zygotes and blastocysts were successfully vitrified. For the vitrification of zygotes, the MVC and microdrop methods were equally effective; however, for blastocyst vitrification, MVC was superior. For both methods, the vitrification of zygotes produced higher-quality embryos than the vitrification of blastocysts.

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region.

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y-chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y-chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.

Uncontrolled admixture and loss of genetic diversity in a local Vietnamese pig breed.

The expansion of intensive livestock production systems in developing countries has increased the introduction of highly productive exotic breeds facilitating indiscriminate crossbreeding with local breeds. In this study, we set out to investigate the genetic status of the Vietnamese Black H'mong pig breed by evaluating (1) genetic diversity and (2) introgression from exotic breeds. Two exotic breeds, namely Landrace and Yorkshire used for crossbreeding, and the H'mong pig population from Ha Giang (HG) province were investigated using microsatellite markers. Within the province, three phenotypes were observed: a White, a Spotted and a Black phenotype. Genetic differentiation between phenotypes was low (0.5-6.1%). The White phenotypes showed intermediate admixture values between exotic breeds and the Black HG population (0.53), indicating a crossbreed status. Management practices were used to predict the rate of private diversity loss due to exotic gene introgressions. After 60 generations, 100% of Black private alleles will be lost. This loss is accelerated if the admixture rate is increased but can be slowed down if the mortality rate (e.g., recruitment rate) is decreased. Our study showed that a large number of markers are needed for accurately identifying hybrid classes for closely related populations. While our estimate of admixture still seems underestimated, genetic erosion can occur very fast even through indiscriminate crossbreeding.