Production of Agu piglets after transfer of embryos produced in vitro.

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing piglets of Agu, an Okinawan indigenous pig breed. After collection of oocytes from surgically dissected ovaries, they were subjected to in vitro maturation. After in vitro maturation/fertilization, a total of 616 putative embryos were transferred into four commercial Western pig recipients, one of which became pregnant and farrowed a total of eight Agu piglets. These results demonstrate that in vitro embryo production using ovaries from Agu females is useful for breeding management and conservation of indigenous breeds.

Embryo production by intracytoplasmic injection of sperm retrieved from neonatal testicular tissue of Agu pigs after cryopreservation and grafting into nude mice.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

Comparison of growth performances, carcass characteristics, and meat qualities of Okinawan indigenous Agu pigs and crossbred pigs sired by Agu or Duroc boar.

Indigenous Okinawa Agu pigs are crossed with Large White × Landrace (WL) pigs to improve their meat production, but there is little information regarding the crossbreeding effects. The study aims to compare growth, carcass characteristics, and meat qualities of Agu pigs with those of WL crossbreeds with Agu sires (WLA) or Duroc sires (WLD). WLA pigs showed better growth performance and carcass characteristics and less intramuscular fat (IMF) contents than Agu ones, but they had higher fat deposition, smaller longissimus dorsi muscle area, and higher IMF contents than WLD pigs. Agu pigs showed higher water holding capacity than the other two breeds. The inner layer of Agu backfat contains higher and lower proportions of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acid (PUFA), respectively, than that in WLD animals. WLA animals had intermediate values for the fatty acid content in the inner backfat, although MUFA contents were equal to those of Agu pigs. Fatty acid profiles in IMF were similar to those in the backfat. These results indicate that crossbreeding of Agu with WL pigs improves growth performance and carcass quality. Particularly, WLA pigs have higher IMF contents and MUFA concentrations and lower PUFA concentrations than WLD pigs.

Evaluation of the genetic structure of indigenous Okinawa Agu pigs using microsatellite markers.

OBJECTIVE: Agu pigs are indigenous to the Okinawa prefecture, which is the southernmost region of Japan. Agu pigs were exposed to a genetic bottleneck during the 20th century, due to the introduction of European pig breeds. The objective of this study was to elucidate the genetic structure of Agu pigs and to determine their relationships with those of five European breeds, two Chinese breeds and Ryukyu wild boar using microsatellite markers. METHODS: A total of 203 DNA samples consist of 8 pig breeds were used in this study. Genotyping was performed using 21 microsatellite markers distributed across 17 chromosomes. RESULTS: Numbers of effective alleles in Agu pigs were fewer than in European breeds and Ryukyu wild boar. Among domestic pigs, Agu pigs had the lowest heterozygosity (0.423) and highest inbreeding coefficient (FIS = 0.202), indicating a severe loss of heterozygosity in Agu pigs possibly due to inbreeding. Neighbor-joining tree analysis was performed based on Reynolds' genetic distances, which clustered Agu pigs with Duroc pigs. However, principal component analysis revealed a unique genetic position of the Agu pig, and the second principal component separated Agu pigs from all other breeds. Structure analysis with the optimal assumption of seven groups (K = 7) indicated that Agu pigs form an independent cluster from the other breeds. In addition, high and significant FST values (0.235-0.413) were identified between Agu pigs and the other breeds. CONCLUSION: This study revealed a substantial loss of genetic diversity among Agu pigs due to inbreeding. Our data also suggest that Agu pigs have a distinctive genetic structure, although gene flows from European breeds were observed.

Maternal lineage of Okinawa indigenous Agu pig inferred from mitochondrial DNA control region.

OBJECTIVE: The Agu is the only native pig breed in Japan, which is reared in Okinawa prefecture, the southernmost region in Japan. Its origins are considered to be of Asian lineage; however, the genetic background of the Agu is still unclear. The objective of this study was to elucidate the maternal lineage of the Okinawa indigenous Agu pig with the use of the mitochondrial DNA (mtDNA) control region. METHODS: The mtDNA control regions of Agu pigs were sequenced and the phylogenetic relationship among Agu, East Asian and European pigs was investigated with the use of 78 Agu individuals. RESULTS: Twenty-seven polymorphic sites and five different haplotypes (type 1 to type 5) were identified within the Agu population. Phylogenetic analysis indicated that types 1 and 2 were included in East Asian lineages; however, the remaining types 3, 4, and 5 were of European lineages, which showed a gene flow from European pigs in the 20th century. Sixty-seven out of 78 Agu individuals (85.9%) possessed mtDNA haplotypes 1 and 2 of the East Asian lineage, which were identical to two haplotypes of ancient mtDNA (7,200 to 1,700 years before the present) excavated at archaeological sites in Okinawa. CONCLUSION: This study confirmed that the East Asian lineage is dominant in the maternal genetic background of the Agu population, supporting the hypothesis that the ancestors of the Agu pig were introduced from the Asian continent.