Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n=9) and from explanted uteri post mortem at different gestational ages (n=91). Amniotic non-allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow-derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT-3. Isolated ovine amniotic fluid-derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small- and large-diameter tissue-engineered vascular grafts (n=10) and cardiovascular patches (n=34), laying the foundation for the use of this relevant pre-clinical in vivo assessment model for future amniotic fluid cell-based therapeutic applications. Copyright (c) 2013 John Wiley & Sons, Ltd.

Chromosome studies and other investigations on equid crossings Crossings between horses and donkeys are in nature possible and are used as mules or hinnies in agriculture. Other types of crossings are rare and scarcely investigated. Systematic experiments are only allowed with special agreements and are very time consuming and costly and therefore rare. In this report crossings between horse, Przewalski and donkey are cytogenetically and histologically investigated and their behaviour in special reproductive traits described. All animals have shown a normal chromosome set following the segregation of possible combinations. The cytogenetic results have been confirmed by a genomescan for the sex chromosomes and the EPR 23 and 24. The consequences in the evolutionary development are discussed.

Summary Associations between production traits and the genes for halothane sensitivity (HAL), S, A and H blood group systems and phosphohexose isomerase (PHI) and 6-phosphogluconate de- hydrogenase (6-PGD) enzyme systems were investigated in two lines of pigs selected for an index. The phenotypic variance-covariance matrix of the index included backfat thickness and daily gain, whereas the genetic variance- covariance matrix included daily gain, feed conversion and percentage of lean meat. The experiment was conducted at the experimental station of the Institute of Animal Produc- tion and has been underway since 1973. The same index was applied but in two opposite directions to give a superior and inferior line in relation to the production traits. One hundred twenty-nine animals of the superior line in the seventh generation and 88 animals of the inferior line in the sixth generation were studied. Forty-two percent (54/129) of the animals of the superior line were halothane- positive. No animals in the inferior line were halothane reactors. Of the halothane-positive pigs, 70.4% (38/54) in the superior line had the Hall a and 94.4% (51/54) had the SsSS geno- type, whereas only 4% (_3/75) of the Hall a and 12% (9/75) of the Sss s pigs were halothane- negative. By practicing selection at the H and S loci, it seems possible to efficiently reduce halothane sensitivity in Swiss Landrace pigs. In pigs of the superior line, there were significant differences in percentage of lean meat, carcass length, pill (pH value at 45 min to 1 h postmortem, M. longissimus) and reflec- tance values among genotypes of the HAL, S and H systems and among some genotypes of the 6-PGD system. Poorest meat quality, highest percentage of lean meat and shortest carcass length were observed in pigs homozygous for the alleles HAL n, S -~, Ha, PHIB and 6-PGDA. In the inferior line, these associations were absent. As the HAL locus is associated with the above mentioned production traits, linkage disequilibria may explain the observed asso- ciations among the H, S, PHI and 6-PGD types and production traits.

Background: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. Objectives: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. Patients/methods: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. Results and conclusions: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII: C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Table S1 RH mapping and real-time primers, annealing temperatures (Ta) and GenBank accession numbers of porcine INPP4A and SLC5A7. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Arachnomelia in Brown Swiss cattle is a monogenic autosomal recessive inherited congenital disorder of the skeletal system giving affected calves a spidery look (OMIA ID 000059). Over a period of 20 years 15 cases were sampled in the Swiss and Italian Brown cattle population. Pedigree data revealed that all affected individuals trace back to a single acknowledged carrier founder sire. A genome scan using 240 microsatellites spanning the 29 bovine autosomes showed homozygosity at three adjacent microsatellite markers on bovine Chr 5 in all cases. Linkage analysis confirmed the localization of the arachnomelia mutation in the region of the marker ETH10. Fine-mapping and haplotype analysis using a total of 34 markers in this region refined the critical region of the arachnomelia locus to a 7.19-Mb interval on bovine Chr 5. The disease-associated IBD haplotype was shared by 36 proven carrier animals and allows marker-assisted selection. As the corresponding human and mouse chromosome segments do not contain any clear functional candidate genes for this disorder, the mutation causing arachnomelia in the Brown Swiss cattle might help to identify an unknown gene in bone development.

The objective of this study was to reestablish an extinct strain of sheep that exhibits spontaneous X-linked factor VIII deficiency closely mimicking human hemophilia A. Twenty female carriers of the trait, produced in a previous study (Bormann et al. 2006 Reprod. Fertil. Dev. 18, 201–202), were backcrossed using 3 straws of semen from their affected sire using either IVF or multiple ovulation embryo transfer (MOET). Eleven oocyte donors were synchronized with CIDRs (15 days) and superovulated with a declining dose of FSH (204 mg) twice daily for 3.5 days. Nine MOET donors were synchronized using CIDRs (14 days), superovulated with a declining dose of FSH (184 mg) BID for 3 days with pregnant mare serum gonadotropin (PMSG; 200 IU) given with the final dose of FSH, and given 1000 IU of hCG 12 h post-CIDR removal. Recipient ewes were synchronized using sponges (Ovakron, HeriotAgvet, Rowville, Victoria, Australia) containing 30 mg of flugestone acetate (14 days) and given PMSG (400 IU) at sponge removal, followed by 1000 IU of hCG 12 h post-sponge removal. Oocytes were collected via follicular aspiration during midventral laparotomy and matured as previously reported. Semen for IVF was prepared by centrifugation on a Percoll gradient. Oocytes and sperm were incubated in mTALP with 20% estrus sheep serum (modified from Bavister et al. 1977 Bio. Reprod. 16, 228–237) for 20 h, then vortexed to remove cumulus cells, and cultured in G1.3 medium (Vitrolife, Englewood, CO) with BSA until transfer. Embryos were surgically transferred into oviducts of recipients 24 to 48 h following IVF. The 9 MOET donors were surgically inseminated at the uterotubal junction with approximately 1–2.0 106 spermatazoa. Oviducts of eight of these ewes were flushed 48 h post-insemination with warm M199 containing Hanks salts, 25 mm HEPES, 10% FBS, and 0.5 µg mL–1 gentamicin. MOET embryos were surgically transferred to synchronized recipients within 5 h. One MOET donor was not flushed due to poor response and did not produce an offspring. Utilizing 140 ova, IVF produced 54 embryos for an embryo/oocyte rate of 38.6%. All IVF embryos were transferred into 15 recipients resulting in 3 lambs for a lamb/embryo rate of 5.5%. The MOET donors produced 38 embryos and 13 apparently unfertilized ova, generating an embryo/oocyte rate of 74.5%. MOET embryos were transferred into 21 synchronized recipients. MOET produced 16 lambs for a lamb/embryo rate of 42.1%. Co-transfer of 1 IVF and 1 MOET embryo into a single recipient produced one offspring. Utilizing multiple reproductive technologies over a two-year period, 8 hemophilic offspring (7 females and 1 male), 6 carrier females, and 6 unaffected males were produced. This strain of sheep will be used to produce affected offspring for stem cell-based therapies.

The Saola (Pseudoryx nghetinhensis), a new large mammal species, was discovered to science in 1993 and listed as critical endangered. Its systematic position within the family Bovidae, however, remains confused since previous studies have led to several conflicting hypotheses. In the present study, an analysis of microsatellite data was performed to determinate the taxonomic status of this enigmatic species. Cross species amplification of 19 cattle microsatellite loci was tested on a panel of 56 individuals representing 9 different species of the Bovidae. Phylogenetic analyses were carried out on matrices from the Dps and Dc genetic distances using the neighbour-joining method. The results obtained from separate and combined analyses strongly support a grouping of Pseudoryx with subtribe Bubalina, which contains all species of Bubalus and Syncerus. (Resume d'auteur)

Abstract Hemophilia A, or Factor VIII (FVIII) deficiency, is the most common severe hereditary coagulation disorder, affecting 1 in 5000 male live births. Animal models in dog, mouse, and rabbit have been developed and used to study FVIII function and to evaluate new methods of treatment and prevention of inhibitor formation. Unfortunately, for unknown reasons, results obtained using these models didn’t always result in successful therapies when applied to humans. For new treatments to be safely and successfully translated from surrogate models to clinical trials, it is critical to develop an animal model that simultaneously and accurately parallels normal human physiology while mimicking human hemophilia’s physiopathological process. Due to its striking physiological and anatomical similarities to humans, sheep are considered an ideal model to study a vast array of pathologies. The aim of these studies was to re-establish, study, and characterize an extinct line of sheep with a spontaneous bleeding disorder that closely recapitulated human hemophilia A (ThrombHaemost68:618,1992). Thus, we used frozen semen from an affected male to generate hemophilia A carriers. We obtained 20 females that when compared to pooled control sheep plasma, exhibited slightly increased PTT levels (38.1±1.1; N=30s), normal PT and platelet number, and slightly decreased FVIII:C (70±3%). Levels of Fibrinogen, FIX, vWF activity and vWF:ag were also normal. A second round of reproductive manipulations using the carriers’ oocytes and the affected semen produced 23 more animals, 16 of which were obligate carriers with a similar phenotype. The other 8 animals exhibited prolonged bleeding from the umbilical cord that promptly stopped upon administration of purified human FVIII concentrate using recommended dosing. Due to the unfeasibility of clamping the umbilical cord, therapy with human FVIII was continued each 12 hours until the umbilical cord dropped off. Blood collected prior to the administration of FVIII showed that these animals had almost non-existent levels of FVIIIc, and an extremely prolonged PTT (91.5±2.9, N=30.3) with normal levels of platelets, fibrinogen, FVII, FIX, and vWF. 2 of the animals died shortly after birth due to extensive hematomas related to lambing trauma. The other 6 animals, now 5 months old (maturity 6–9 months), developed clinical symptomatology closely mimicking that of human patients with severe hemophilia A. Each of these animals had between 2–6 episodes of severe bleeding including hemarthroses of the elbow, shoulder, hip, and knee, multiple muscle hematomas, including 1 hematoma of the tongue and 1 episode of mild hematuria. All of the bleeding episodes resolved upon administration of 1–2 treatments with human FVIII. Animals have thus far received between 964-4546U of human FVIII. Of interest is that low-titer inhibitors (1.3; 2.6; 3 BU) were detected in 3 of the animals showing that the nature of the mutation present in these sheep renders them prone to inhibitor development. Characterization of the mutation is currently underway. We hope that this large animal model will contribute to a better understanding of hemophilia and the development of novel treatments that can directly translate to human patients, such as stem cell transplantation and gene therapy-based approaches.

The evolutionary development of mammals involves mutations and fixations of chromosomal types. The Y chromosome polymorphism in cattle is important for the breeding strategy, since chromosomal incompatibilities in crossings result in fertility problems. In bulls of various breeds in Switzerland, data on chromosome status have been collected for over 20 years. Data from 7 years were analysed in this study through chromosome measurements and their normalization. Some highly significant differences were found between the 7 groups of breeds, especially between Holsteins and the original Swiss breeds Braunvieh and Simmental. Fleckvieh (purebred or crossbred) did not differ significantly from Black or Red Holsteins. The results were discussed with respect to fertility problems. The observed Y chromosome polymorphism should be taken into account in breeding, and research in this field should be continued.

BACKGROUND:The wild gaur (Bos gaurus) is an endangered wild cattle species. In Vietnam, the total number of wild gaurs is estimated at a maximum of 500 individuals. Inbreeding and genetic drift are current relevant threats to this small population size. Therefore, information about the genetic status of the Vietnamese wild gaur population is essential to develop strategies for conservation and effective long-term management for this species. In the present study, we performed cross-species amplification of 130 bovine microsatellite markers, in order to evaluate the applicability and conservation of cattle microsatellite loci in the wild gaur genome. The genetic diversity of Vietnamese wild gaur was also investigated, based on data collected from the 117 successfully amplified loci.RESULTS:One hundred-thirty cattle microsatellite markers were tested on a panel of 11 animals. Efficient amplifications were observed for 117 markers (90%) with a total of 264 alleles, and of these, 68 (58.1%) gave polymorphic band patterns. The number of alleles per locus among the polymorphic markers ranged from two to six. Thirteen loci (BM1314, BM2304, BM6017, BMC2228, BMS332, BMS911, CSSM023, ETH123, HAUT14, HEL11, HEL5, ILSTS005 and INRA189) distributed on nine different cattle chromosomes failed to amplify wild gaur genomic DNA. Three cattle Y-chromosome specific microsatellite markers (INRA124, INRA126 and BM861) were also highly specific in wild gaur, only displaying an amplification product in the males. Genotype data collected from the 117 successfully amplified microsatellites were used to assess the genetic diversity of this species in Vietnam. Polymorphic Information Content (PIC) values varied between 0.083 and 0.767 with a mean of 0.252 while observed heterozygosities (Ho) ranged from 0.091 to 0.909 (mean of 0.269). Nei's unbiased mean heterozygosity and the mean allele number across loci were 0.298 and 2.2, respectively.CONCLUSION:Extensive conservation of cattle microsatellite loci in the wild gaur genome, as shown by our results, indicated a high applicability of bovine microsatellites for genetic characterization and population genetic studies of this species. Moreover, the low genetic diversity observed in Vietnamese wild gaur further underlines the necessity of specific strategies and appropriate management plans to preserve this endangered species from extinction.

The significantly high similarity of karyotypes were shown between Bos taurus and B. indicus except for morphology of the Y-chromosome. The B. taurus-Y (BTA-Y) is submetacentric whereas the B. indicus-Y (BIN-Y) is acrocentic. Based on the hybridization of the chromosome painting technique, the difference between Y chromosomes in B. taurus and B. indicus was proposed to be originated from the pericentric inversion between these two species. However, hybridization of SRY to the sub-centromeric end of BIN-Y indicated a double pericentric inversion found between B. taurus and B. indicus. We suggested that BTA-Y and BIN-Y were originated from a common ancestor submetacentric Y chromosome with a short p-arm. The BTA-Y was formed by pericentric inversion of a major segment of q-arm and a centromere of the ancestor chromosome whereas the BIN-Y was generated from the ancestor Y chromosome by pericentric inversion of the complete p-arm together with the centromere. A proposed model of a common ancestor sub-metacentric Y of B. taurus and B. indicus had provided a new insight into the evolution of the Y chromosome in the family of Bovidae.

The cause of porcine congenital progressive ataxia and spastic paresis (CPA) is unknown. This severe neuropathy manifests shortly after birth and is lethal. The disease is inherited as a single autosomal recessive allele, designated cpa. In a previous study, we demonstrated close linkage of cpa to microsatellite SW902 on porcine chromosome 3 (SSC3), which corresponds syntenically to human chromosome 2. This latter chromosome contains ion channel genes (Ca(2+), K(+) and Na(+)), a cholinergic receptor gene and the spastin (SPG4) gene, which cause human epilepsy and ataxia when mutated. We mapped porcine CACNB4, KCNJ3, SCN2A and CHRNA1 to SSC15 and SPG4 to SSC3 with the INRA-Minnesota porcine radiation hybrid panel (IMpRH) and we sequenced the entire open reading frames of CACNB4 and SPG4 without finding any differences between healthy and affected piglets. An anti-epileptic drug treatment with ethosuximide did not change the severity of the disease, and pigs with CPA did not exhibit the corticospinal tract axonal degeneration found in humans suffering from hereditary spastic paraplegia, which is associated with mutations in SPG4. For all these reasons, the hypothesis that CACNB4, CHRNA1, KCNJ3, SCN2A or SPG4 are identical with the CPA gene was rejected.

We report the chromosomal assignment of 18 porcine genes to human homologues using the INRA-Minnesota swine radiation hybrid panel (IMpRH). These genes (CACNA1C, COL2A1, CPNE8, C3F, C12ORF4, DDX11, GDF11, HOXC8, KCNA1, MDS028, TMEM106C, NR4A1, PHB2, PRICKLE1, Q6ZUQ4, SCN8A, TUBA8 and USP18) are located on porcine chromosome 5 (SSC5) and represent positional and functional candidates for arthrogryposis multiplex congenita (AMC), which maps to SSC5. CPNE8, PRICKLE1, Q6ZUQ4 and TUBA8 were mapped to the interval for pig AMC between microsatellites SW152 and SW904. Three SNPs in TUBA8 co-segregated with the AMC phenotype in 230 pigs of our research population without recombination and could be used as a genetic marker test for AMC. In addition, we provide evidence that a small chromosomal region of HSA22q11.2 evolutionarily corresponds to SSC5q12-q22 (and contains the human homologues of porcine SW152, Q6ZUQ4, TUBA8 and USP18), while the regions flanking HSA22q11.2 on SSC5 correspond to HSA12p13 and HSA12q12. We identified seven distinct chromosomal blocks, further supporting extensive rearrangements between genes on HSA12 and HSA22 in the AMC region on SSC5.

ABSTRACT Agouti signaling protein (ASIP) is involved in the regulation of pigmentation in mammals by downregulating melanocortin 1 receptor (MC1R) activity. In wild type mice, ASIP is expressed in skin and testes. Widespread tissue expression of ASIP has been found in humans and cattle. Reverse transcription with subsequent real‐time polymerase chain reaction was used to measure ASIP expression levels in skin and five additional tissues from four cattle breeds. The expression of each sample was represented as a percentage of the expression of the housekeeping gene b‐actin . No significant difference was found in the expression levels in skin from Red Holstein, Simmental (red), Holstein (black) and Brown Swiss (brown/gray) breeds, which were all in the range of 0.2–0.3%. Expression in other tissues varied greatly between individuals, ranging from 196% in the heart of a Red Holstein animal to 0% in the liver samples of all four breeds. Additionally, we showed that expression of bovine ASIP in cell culture reduces cyclic adenosine monophosphate production in cells expressing MC1R, suggesting that bovine agouti is able to downregulate MC1R signaling.

The objective of this study was to restore a line of sheep that exhibits spontaneous X-linked factor VIII deficiency closely mimicking human hemophilia A. Six straws of frozen semen from an affected Alpine White male were obtained from Switzerland. In the first experiment the straw of semen thawed was of poor quality. Two ewes were synchronized for use as embryo donors (MOET) by means of CIDRs for 14 days and superovulated with declining doses of FSH (184 mg) twice daily for 3 days. PMSG (200 IU) was given with the final dose of FSH and 1000 IU of hCG 12 h post-CIDR removal. The ewes were surgically inseminated 24 h later. Oviducts were flushed 48 h post-insemination producing 13 unfertilized ova (UFO). Spermatozoa were used for intracytoplasmic sperm injection (ICSI) utilizing oocytes collected from superstimulated ewes by laporatomy. These ewes were synchronized with CIDRs (15 days) and superovulated with a declining dose of FSH (204 mg) twice daily for 3.5 days. Utilizing 236 oocytes, ICSI produced 189 embryos, an 80% embryo/oocyte rate. Embryos were transferred surgically to the oviducts of 17 synchronized recipients. Recipients were synchronized using sponges (Ovakron; Heriot Agvet, Rowville, Victoria, Australia) containing 30 mg of flugestone acetate (14 days) and given PMSG (400 IU) at sponge removal, followed by 1000 IU of hCG 12 h post-sponge removal. Eleven recipients produced 17 lambs for a lamb/embryo rate of 8.9%. The straw of semen utilized for the second experiment was of higher quality. Three ewes were superstimulated for use as MOET donors, as above, with increased doses of FSH (228 mg) and PMSG (500 IU). Donors were surgically inseminated and oviductal flushes were performed 40 h post-insemination, yielding 19 UFO and 12 embryos for an embryo/oocyte rate of 38.7%. Embryos were transferred to four recipients, synchronized as above with an increased dose of PMSG (600 IU). These MOET recipients produced nine lambs for a lamb/embryo rate of 75%. Semen was used to produce embryos via in vitro fertilization (IVF) using oocytes collected from superstimulated ewes (as above with an increase of FSH to 252 mg). IVF produced 91 embryos from 247 oocytes for an embryo/oocyte rate of 36.8%. Embryos were transferred to 20 recipients 24 to 48 h post-fertilization. Seven recipients maintained pregnancy and produced 10 lambs with a lamb/embryo rate of 11%. ICSI was also utilized, producing 54 embryos from 98 oocytes, an embryo/oocyte rate of 55.1%. Embryos were transferred to eight recipients; none maintained pregnancy. Through the use of multiple reproductive technologies, 36 lambs (22 carriers) were produced from two straws of semen. Carriers will be bred back to their sire in a similar program to produce affected lambs. The authors would like to acknowledge J. Liu and M. Ridha for their contributions. This work was supported by NIH Grant HL073737-12.