Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes

The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.     

Chromosome abnormalities in secondary pig oocytes matured in vitro

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30-48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8 %). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0 %) than in the groups matured for 30 and 36 h (9.0 %). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy (n=19-1) was identified in 22 cells (3.59 %) and a hyperhaploid (n=19+1) set of chromosomes was identified in 15 cells (2.45 %). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9 %. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.     

Embryo collection from miniature pigs and production of the piglets after transfer to common pig recipients

Embryo collection from G?ttingen miniature pigs and transfer to common pig recipients were performed. Embryos were collected 2 to 6 days after the onset of estrus. Two to 7 eggs including unfertilized ova were collected from a naturally ovulated donor and more eggs (7 to 15) were obtained from a superovulated donor. A total of 21 piglets were born after transfer of 33 miniature pig embryos to two common pig recipients. Among them 12 were stillbirths which seemed to have resulted from suffocation during the parturition. Such an embryo transfer technique will be utilized as an inventive method for the purposes such as international exchange of the particular strains of miniature pigs, establishment of a SPF miniature pig herd and so on.     

Analysis of the “Poso” wheat translocation

The two translocation chromosomes in the Poso 5B/7B translocation have been isolated in separate heterozygous aneuploid stocks (19^II+5B+T). The translocation breakpoints are in the long arm of chromosome 7B and the short arm of chromosome 5B. The translocation chromosome bearing the 5B^L pairing inhibitor was obtained as a homozygous aneuploid (19^II+T ₁ ^II ). The heterozygous aneuploid hemizygous for the pairing inhibitor (19^II+5B+T₂) was used to produce intergeneric hybrids. Only a small percentage were of the high-pairing type (17%), the majority having received chromosome 5B through the egg. This indicates a strong selection against eggs containing the translocation chromosome deficient for the pairing inhibitor.     

Do farming conditions influence brominated flame retardant levels in pig and poultry products?

Brominated flame retardants (BFR) are primarily used as flame retardant additives in insulating materials. These lipophilic compounds can bioaccumulate in animal tissues, leading to human exposure via food ingestion. Although their concentration in food is not yet regulated, several of these products are recognised as persistent organic pollutants; they are thought to act as endocrine disruptors. The present study aimed to characterise the occurrence of two families of BFRs (hexabromocyclododecane (HBCDD) and polybrominated diphenyl ethers (PBDE)) in hen eggs and broiler or pig meat in relation to their rearing environments. Epidemiological studies were carried out on 60 hen egg farms (34 without an open-air range, 26 free-range), 57 broiler farms (27 without an open-air range, 30 free-range) and 42 pig farms without an open-air range in France from 2013 to 2015. For each farm, composite samples from either 12 eggs, five broiler pectoral muscles or three pig tenderloins were obtained. Eight PBDE congeners and three HBCDD stereoisomers were quantified in product fat using gas chromatography–high-resolution mass spectrometry, or high-performance liquid chromatography–tandem mass spectrometry, respectively. The frequencies of PBDE detection were 28% for eggs (median concentration 0.278 ng/g fat), 72% for broiler muscle (0.392 ng/g fat) and 49% for pig muscle (0.403 ng/g fat). At least one HBCDD stereoisomer was detected in 17% of eggs (0.526 ng/g fat), 46% of broiler muscle (0.799 ng/g fat) and 36% of pig muscle (0.616 ng/g fat). Results were similar in concentration to those obtained in French surveillance surveys from 2012 to 2016. Nevertheless, the contamination of free-range eggs and broilers was found to be more frequent than that of conventional ones, suggesting that access to an open-air range could be an additional source of exposure to BFRs for animals. However, the concentration of BFRs in all products remained generally very low. No direct relationship could be established between the occurrence of BFRs in eggs and meat and the characteristics of farm buildings (age, building materials). The potential presence of BFRs in insulating materials is not likely to constitute a significant source of animal exposure as long as the animals do not have direct access to these materials.     

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.     

Analysis of allele segregation distortion in a swine resource family

Segregation distortion of alleles was found in several regions of the genome in allele type analyses of genetic markers for a swine resource family that had been constructed by crossing G?ttingen miniature pig and Meishan breeds. From these regions, a region on chromosome 6 presented a distortion over several consecutive markers. This chromosome 6 region was subsequently further investigated to reveal that alleles of a chromosome 6 homologue of the Gottingen miniature pig were not found in a homozygous family member. The litter size of F1-crosses which were able to produce homozygotes in this region were 24% smaller, on average, than F1-crosses which were unable to produce homozygotes. This indicated that this region of the chromosome 6 homologue contained a recessive gene or genes which could terminate fetal development. An additional 10 markers were subsequently used to investigate the region more precisely. These studies revealed that this region spans 7 cM and is located between markers Sw855 and Sw122. Since current comparative maps show that this region corresponds to the human chromosome 19 q-arm, genes positioned on the human chromosome 19 q-arm were screened to select 20 candidate genes. These included the pregnancy-specific beta-1-glycoprotein gene.     

Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos.

Polyspermy occurs frequently in the fertilization of mammalian eggs, but little is known about whether polyspermic eggs have developmental ability in vitro or in vivo. We previously reported that poly-pronuclear (PPN; 3 or more pronuclei) pig eggs developed normally to the blastocyst stage despite having fewer inner cell mass cell numbers as compared to blastocysts derived from two-pronuclear (2PN) eggs. Here it is shown that most PPN pig eggs have abnormal cleavage patterns (having 3 or more cells) in the first cell division and retarded development of pronuclei prior to syngamy as compared to 2PN eggs. Most blastocysts (14 of 18) that developed from PPN eggs showed abnormal ploidy (were haploid, triploid, and tetraploid) whereas 20 of 22 blastocysts derived from 2PN embryos were diploid. The size and morphology of most Day 40 fetuses that developed from PPN eggs appeared to be normal. Of 8 Day 40 fetuses analyzed, 1 was triploid (XXY) and another was a mosaic with both diploid (XX) and tetraploid cells (frequency of less than 10%, XXXX), and the others were diploid. Anomalies of chromosomal composition were not detected in these fetuses. Five live piglets and one dead piglet were born from two recipients of PPN eggs. It is proposed that not all pronuclei of PPN pig eggs participate in syngamy, resulting in diploid cells in the conceptus. Our data suggest that there are two types of pronuclei location in polyspermic pig eggs and that the resulting ploidy is determined at the zygote stage before the first cell division according to pronuclear location.     

Embryonation and Infectivity of Ascaris suum Eggs. A Comparison of Eggs Collected from Worm Uteri with Eggs Isolated from Pig Faeces

Ascaris suum eggs were collected from pig faeces or dissected from worms obtained from the same pigs. Eggs from the two sources were allowed to embryonate in 0.1 N H2SO4, in 1 % buffered formalin or in tap water. The embryonation of the sulphuric acid and water cultures occurred at the same speed, while the formalin cultures developed slightly more slowly. By experimental inoculation of helminthfree pigs and subsequent counting of white spots in the livers and larvae in the lungs day 7 p. i., the infectivity of eggs dissected from worm uteri and embryonated in sulphuric acid (a normal laboratory procedure) was compared with that of eggs collected from faeces and embryonated in water (i.e. more naturally developed eggs). The results suggest that the two types of eggs were equally infective. For this reason the common practice of using Ascaris eggs dissected from worms for experimental infections might be acceptable.     

Assignment of the gene for porcine insulin-like growth factor 1 (IGF1) to chromosome 5 by linkage mapping

Investigation of published sequence data from the porcine insulin-like growth factor 1 (IGF1) gene, resulted in the detection of a microsatellite in the first intron of the gene. Polymerase chain reaction (PCR) primers flanking the (CA)₁₉ repeat were constructed. Polymorphism and Mendelian segregation were documented in a three-generation pedigree and allele frequencies were determined in 74 unrelated animals from four different breeds. Seven alleles were encountered. Linkage analysis was performed in a large pedigree established for gene mapping. Linkage between the IGF1 microsatellite and an anonymous microsatellite marker, S0005, was detected. Furthermore, IGF1 and S0005 was found to be linked to the porcine submaxillary gland mucin (MUC) gene, previously assigned to chromosome 5. The results presented here extend the linkage group on pig chromosome 5 and are in accordance with conserved synteny between human chromosome 12, cattle chromosome 5, mouse chromosome 10 and pig chromosome 5.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

UV irradiation of pig metaphase chromosomes: maturation-promoting factor degradation, nuclear cytology and cell cycle progression

Experiments were designed to test two hypotheses. The first was that irradiation of pig metaphase chromosomes would block the normal sequence of cytological and molecular events associated with activation; the second postulated that damaged DNA would prevent eggs from progressing through the first mitotic cleavage cycle. The experimental protocol involved selectively irradiating the metaphase II plate of pig oocytes with highly focused 254 nm ultraviolet (UV) light, followed by activation using standard electroactivation procedures. The following assessments were made of different groups of eggs: (i) nuclear membrane reassembly; (ii) chromosomal cytology; (iii) changes in maturation-promoting factor kinase (MPF kinase) activity at 1 h intervals after activation; and (iv) mitotic progression of eggs containing damaged chromosomal fragments. UV irradiation neither prevented the reassembly of nuclear membranes required for pronuclear formation nor interfered with the normal pattern of MPF kinase degradation after egg activation. UV irradiation did induce a wide range of chromatin defects, including condensation and dispersal of DNA fragments which, in turn, resulted in the formation of micronuclei in the treated eggs and embryos. The presence of damaged DNA retarded, but did not inhibit, progression through the first mitotic cycle. No evidence was obtained that the subsequent mitotic cycle was adversely affected by the presence of UV-damaged DNA. Overall, these results indicate that early cleavage divisions in pig eggs are not blocked by the presence of damaged, hypercondensed chromatin. In this respect, pig eggs are similar to Xenopus eggs, but are different from bovine eggs. On the basis of these findings it is suggested that focused UV irradiation offers a simple and rapid technique for the non-invasive enucleation of pig oocytes provided that the residual hypercondensed chromatin does not affect later developmental stages.     

Localization on pig chromosome 6 of markers GPI, APOE, and ENO1, carried by human chromosomes 1 and 19, using in situ hybridization

In the pig, the linkage group around the halothane gene (HAL), composed of S-GPI-HAL-H-A1BG-PGD, has been assigned to bands p1.2----q2.2 of chromosome 6. In man, ENO1-PGD and APOE-GPI constitute two syntenic groups situated on different chromosomes (1 and 19, respectively). Since GPI and PGD are linked in the pig, we have hybridized the human cDNA probes for ENO1 and APOE to pig chromosomes. These markers were assigned to pig chromosome 6, in the q2.2----q2.4 and cen----q2.1 regions, respectively, using in situ hybridization. Since GPI and APOE are situated in the same region, we combined the use of high resolution chromosome analysis and in situ hybridization to give a more precise localization in the q1.2 and q1.2----q2.1.2 regions of chromosome 6. A possible linear order of these genes is proposed.     

Production of transgenic miniature pigs by pronuclear microinjection

Miniature pig is an attractive animal for a wide range of research fields, such as medicine and pharmacology, because of its small size, the possibility of breeding it under minimum environmental controls and the physiology that is potentially similar to that of human. Although transgenic technology is useful for the analysis of gene function and for the development of model animals for various diseases, there have not yet been any reports on producing transgenic miniature pig. This study is the first successful report concerning the production of transgenic miniature pig by pronuclear microinjection. The huntingtin gene cloned from miniature pig, which is a homologue of candidate gene for Huntington's disease, connected with rat neuron-specific enolase promoter region, was injected into a pronucleus of fertilized eggs with micromanipulator. The eggs were transferred into the oviduct of recipient miniature pigs, whose estrus cycles were previously synchronized with a progesterone analogue. A total of 402 injected eggs from 171 donors were transferred to 23 synchronized recipients. Sixteen of them maintained pregnancy and delivered 65 young, and one resulted in abortion. Five of the 68 offspring (three of which were aborted) were determined to have transgene by PCR and Southern analysis. The overall rate of transgenic production was 1.24% (transgenic/injected eggs). This study provides the first success and useful information regarding production of transgenic miniature pig for biomedical research.     

Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13

We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine-human-mouse comparative map of this region was constructed.     

The occurrence of intestinal parasites in swine slurry and their removal in activated sludge plants

Thirteen intensive pig farms and two activated sludge treatment plants for pig slurry in north-western Spain were studied from April 2005 to June 2006 in order to evaluate the presence of enteric pathogens (Cryptosporidium, Giardia and helminths) and the efficiency with which they were removed. These parasites were present on 53%, 7% and 38% of the farms studied, respectively, with concentrations of 10(4)-10(5) oocysts per litre (/L) for Cryptosporidium, 10(3)cysts/L for Giardia and 10(2)-10(3) eggs/L for helminths. The overall removal of parasites in the pig slurry treatment plants ranged from 86.7% to over 99.99%. The results revealed a constant reduction at each stage of the treatment system, with activated sludge processes being the most effective treatment in reducing pathogens in pig slurry, 78-81% for Cryptosporidium oocysts and over 99.9% for helminth eggs. A heat drying procedure for sludge removed 4.3 log units of Cryptosporidium oocysts, demonstrating the excellent effectiveness of this treatment for reducing pathogens in sludge intended to be applied to land.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

Conserved synteny and gene order difference between human chromosome 12 and pig chromosome 5.

A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint.     

A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica).

Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS. Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y. DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18. After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA. The average insert size of the library was 405 bp. Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA. Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences. A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1. However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.