Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (Sus scrofa) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.

Porcine in vitro production (IVP) systems, including in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes and their subsequent in vitro culture (IVC), have been modified by many researchers, but are still at a low level because of a low developmental rate of embryos to the blastocyst stage and their poor qualities. Our objectives were to establish reliable IVP procedures for porcine blastocysts and to examine the ability of the blastocysts to develop to term after transfer to recipients. Porcine cumulus-oocyte complexes were matured in vitro under 5% O(2) or 20% O(2), fertilized in vitro under 5% O(2), and subsequently cultured under 5% O(2) in 1) IVC medium supplemented with glucose (IVC-Glu) from Day 0 (the day of IVF) to Day 6; 2) IVC-Glu from Days 0 to 2, then IVC medium supplemented with pyruvate and lactate (IVC-PyrLac) from Days 2 to 6; 3) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6; and 4) IVC-PyrLac from Days 0 to 6. There were no significant differences in blastocyst formation rates on Day 6 between the 5% O(2) and 20% O(2) conditions (19.9% and 14.0%, respectively). However, the quality of blastocysts, as evaluated by the total cell number, was better after IVM under 5% O(2) than under 20% O(2) (mean cell number, 43.5 and 37.8, respectively). When IVP embryos were cultured in IVC-PyrLac from Days 0 to 2 and subsequently in IVC-Glu from Days 2 to 6, the rate of blastocyst formation (25.3%) and cell number (48.7) were higher than the rates (5.8% to 18.1%) and numbers (35.4 to 37.1) with the IVC-Glu then IVC-Glu, the IVC-Glu then IVC-PyrLac, and the IVC-PyrLac then IVC-PyrLac regimens, respectively. We then prepared conditioned medium (CM) from culture of porcine oviductal epithelial cells for 2 days in IVC-PyrLac and evaluated its effect on development to the blastocyst stage. Cultivation in CM for the first 2 days, followed by IVC-Glu for a further 4 days, had a significantly greater effect in increasing the number of cells in the blastocyst (58.3) than did in IVC-PyrLac (48.4). Finally, we evaluated the ability of blastocysts, generated by IVM under 5% O(2) and IVC in CM, to develop to term. When Day 5 expanding blastocysts (mean cell number, 49.7) were transferred to an estrus-synchronized recipient (50 blastocysts per recipient), the recipient remained pregnant and farrowed eight normal piglets. Furthermore, when Day 6 expanded blastocysts (mean cell number, 80.2) were transferred to two estrus-synchronized recipients, both gilts remained pregnant and farrowed a total of 11 piglets. These results suggest that an excellent piglet production system can be established by using this modified IVP system, which produces high-quality porcine blastocysts. This system has advantages for the generation of cloned and transgenic pigs.

In order to locate the genetic regions in the swine genome that are responsible for economically important traits, a resource population has been constructed by mating two female Meishan pigs with a male Göttingen miniature pig. In subsequent generations, 265 F2 offspring were produced from two F1 males and 19 F1 females. The F2 offspring were scored for eight traits including growth rate, teat number, vertebra number and backfat thickness, and genotyped for 318 genetic markers spanning the swine genome. Least-square analysis revealed quantitative trait loci (QTL) effects for vertebra number on chromosomes 1 and 2; for teat number on chromosomes 1 and 7; for birth weight on chromosome 1; for average daily gain between 4 and 13 weeks of age on chromosomes 9 and 10; for backfat thickness on chromosome 7; and for backskin thickness on chromosome 3.

Our objective was to develop a method of endowing oocytes from porcine primordial follicles with full maturation and fertilizing ability as a model for ovarian xenografting of large mammals. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of both kidneys of ovariectomized athymic mice. The host mice were treated with 5 IU of equine chorionic gonadotropin (eCG) for 10 days (eCG-10), 30 days (eCG-30), or 60 days (eCG-60) after detection of cornified epithelial cells in their vaginal smears. Cumulus-oocyte complexes, ovarian grafts, and blood samples were obtained 48 h after eCG treatment. Forty-five to 70 days after grafting, the host mice in all groups for the first time showed vaginal cornification, accompanied by the formation of a small number of antral follicles in the grafts. However, we recovered large numbers of full-sized oocytes only from mice in the eCG-60 group; the numbers of full-sized oocytes in the other groups were low. Peripheral levels of total inhibin were highest in the eCG-60 group; this supports our finding that the most enhanced growth of antral follicles occurred in this eCG-60 group. Of 573 oocytes obtained from the eCG-60 group, 98 (17%) were at the metaphase II stage after in vitro culture for maturation. Moreover, 55% of matured oocytes with the first polar body (n = 20) were fertilized in vitro. These results clearly demonstrate that fertilization of oocytes from porcine primordial follicles is achievable by a combination of xenografting and in vitro culture.

A resource family of pigs has been constructed by using a boar of Göttingen miniature pig and two sows of Meishan pig as parents. In the construction of the family, two F1 males and 18 F1 females were intercrossed to generate 143 F2 offspring. The members of the family were genotyped using 243 genetic markers including 26 markers developed in our laboratory in order to generate a linkage map of markers for use in detecting quantitative trait loci (QTLs) in the family. The markers consisted of 237 microsatellites, five PRE-1 markers, and one RFLP marker. The linkage map was revealed to cover all 18 autosomes and the X chromosome; and the total length of the sex-averaged linkage map was calculated to be 2561.9 CM. Four out of the 26 markers developed in our laboratory exended the current linkage map at the termini of chromosomes 1p, 5p, 11p, and Xq. The linkage maps of all the chromosomes except for chromosome 1 were found to be longer in females than in males. Concerning chromosome 1, the length of the linkage map showed no difference between females and males, which was attributed to low recombination rates between markers localized in the centromeric region in females. The average ratio of female-to-male recombination was calculated to be 1.55.

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.

Two injection methods were examined for making chimeras between Chinese pigs (Meishan) and European pigs (Landrace or Landrace x Large White). Furthermore, mitochondrial DNA (mtDNA) polymorphism was detected as a cell marker for the analysis of chimerism. In the first experiment, blastomeres were transplanted into embryos at the 4-16-cell stage. Of 41 transplanted embryos transferred into 3 females, 12 were single-colored, but no overt chimeras were obtained. Judging from coat color and mtDNA in white blood cells, 2 piglets in 2 litters were derived from injected blastomeres, and 10 piglets in 3 litters were derived from recipient blastomeres. In the second experiment, inner cell mass cells of Day 6 Landrace embryos were injected into blastocoels of Day 6 Meishan embryos. Of 35 injected embryos transferred into 3 females, 2 overt chimeras of each sex were obtained in a single litter. In the overt male chimera, mtDNA clearly showed chimerism in spleen, pancreas, brain, kidney, lung, liver, heart, testis, and small intestine. The overt female chimera showed chimerism not only in blood but also in germ line according to a progeny test. No chimerism was detected in any of the 21 single-colored piglets in the second experiment.

The objective of this study was to investigate the presence of tumor necrosis factor-alpha (TNF-alpha) in the central nervous system and the effects of lipopolysaccharide on central and peripheral concentrations of TNF-alpha, behavioral conditions (standing or lying), elimination scores (defecation or urination), rectal temperature, and food intake (as-fed basis) in Chinese Meishan pigs. Intravenous injections of lipopolysaccharide resulted in increased (P < 0.05) plasma concentrations of TNF-alpha and cortisol. Although urination was not affected by the administration of lipopolysaccharide, defecation was stimulated (P < 0.05). Lipopolysaccharide increased (P < 0.05) rectal temperature and standing rate, and inhibited (P < 0.05) food intake in pigs. To determine whether TNF-alpha is present in the porcine central nervous system as well as in peripheral blood, TNF-alpha and its specific transcripts in brain tissues (hypothalamus, amygdala, or hippocampus) and the pituitary were determined. The abundance of TNF-alpha messenger RNA and immunoreactive TNF-alpha were observed in all tissues, and the concentrations of TNF-alpha were increased (P < 0.05) by the intramuscular injection of lipopolysaccharide. These results suggest that TNF-alpha is present in the central nervous system, and plays some roles in its biological regulation in Chinese Meishan pigs.