Development of single blastomeres derived from two-cell embryos produced in vitro in pigs.

The objective was to investigate development of single blastomeres derived from IVP two-cell porcine embryos. There was no difference (P > 0.05) in blastocyst rates among intact two-cell embryos (IN), zona-free two-cell embryos (ZF), and single blastomere (SB) groups (50.0 ± 9.7, 57.4 ± 5.7, and 45.1 ± 7.2%, respectively; mean ± SEM). However, blastocyst yield for the SB group (90.2 ± 14.4%, based on the original number of two-cell embryos before blastomere separation) was higher (P < 0.05) than those of IN and ZF groups. Although the number of inner cell mass (ICM) and trophectoderm (TE) cells in SB blastocysts (6.2 ± 0.8 and 15.5 ± 1.1, respectively) was lower (P < 0.05) than those in IN (12.4 ± 1.3 and 26.0 ± 3.8) and ZF blastocysts (10.7 ± 1.6 and 26.4 ± 3.4), ICM:TE ratios did not differ significantly among groups. Expressions of transcripts associated with cellular organization (TUBA1 and TUBB) were reduced (P < 0.05) in SB versus IN blastocysts. However, there was no significant difference among groups for expression of transcripts associated with responses to stress (HSPE1, HSPD1, and HSPCA) or glucose catabolism (ENO1, COX6C, COX7B, NDUFA4, NDUFA13, UCRC, and UQCRFS1) in blastocysts. The percentage of the sister blastomere pairs in which both cells developed to blastocysts (36.6 ± 5.3%) or both degenerated (46.3 ± 10.3%) were higher (P < 0.05) than that of the pairs in which one developed to blastocyst while the other degenerated (17.1 ± 7.8%). When both pairs developed to blastocysts, one blastocyst had more (P < 0.05) ICM and TE cells (8.2 ± 1.2 and 20.2 ± 2.1, respectively) than the other (5.2 ± 0.9 and 13.5 ± 1.1), although ICM:TE cell ratios were not significantly different. In conclusion, blastomere separation at the two-cell stage significantly increased blastocyst yield from IVP porcine embryos. This might be a useful approach for conservation of rare pig breeds, in which low numbers of embryos limited the success of embryo transfer.

Phylogenetic position of the saola (Pseudoryx nghetinhensis) inferred from cytogenetic analysis of eleven species of Bovidae.

Previous morphological and molecular analyses failed to resolve the phylogenetic position of the critically endangered saola (Pseudoryx nghetinhensis) with respect to its placement in Bovina (cattle, bison, and yak) or Bubalina (Asian and African buffaloes). In the present study, G- and C-banding, Ag-staining and FISH with 28S and telomeric probes was undertaken for 17 bovid species. An analysis of these data allowed us to identify 49 structural rearrangements that included autosomes, gonosomes and 17 different NOR sites. The combined data set was subjected to a cladistic analysis aimed at: (i) providing new insights on phylogenetic relationships of the saola and other species within the subfamily Bovinae, and (ii) testing the suitability of different classes of chromosomal characters for phylogenetic reconstruction of the family Bovidae. The study revealed that nucleolar organizing regions (NORs) are phylogenetically informative. It was shown that at least one, or sometimes two of these characters punctuate divergences that include nodes that are the most basal in the tree, to those that are the most recent. In this context, the shared presence of three NORs in saola and species of Syncerus and Bubalus strongly suggests the saola's placement within the subtribe Bubalina. This contrasts with Robertsonian rearrangements which are informative only at the generic level. These findings suggest that NORs are an important and frequently overlooked source of additional phylogenetic information within the Bovidae that may also have applicability at higher taxonomic levels, possibly even for Pecora.

Application of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis).

The aim of the present study was to assess the applicability of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis). A total of 127 microsatellite markers were tested on a male and a young female Saola. An efficient amplification was observed for 123 markers (96.8%), 73 markers (59.3%) were polymorphic. Four loci (BM2304, BMS1928, BMS779 and ILSTS006) on cattle chromosomes 1, 4, 7 and 8, respectively, failed to amplify in Saola. Two cattle Y-chromosome-specific microsatellite markers (INRA126 and BM861) were successfully amplified from both sexes in Saola. However, two additional markers (INRA124 and INRA189) on Y-chromosome failed to amplify in the female animal. These results show that most of the bovine microsatellite markers are applicable in Saola and therefore they can be used to study the phylogenetic relationships and the genetic diversity of the Saola population.

Cytogenetic comparison of saola (Pseudoryx nghetinhensis) and cattle (Bos taurus) using G- and Q-banding and FISH.

In order to cytogenetically describe the new bovid species saola (Pseudoryx nghetinhensis), comparative G- and Q-banding of saola and cattle (Bos taurus) chromosomes as well as FISH-mapping of 32 type-I markers (29 Texas markers and three additional markers) on saola chromosomes were performed. Saola was shown to have a diploid number of 2n = 50 chromosomes possessing five biarmed autosomal pairs and an acrocentric X chromosome. Homology of saola and cattle chromosomes was indicated by banding patterns and by marker hybridization suggesting that all five biarmed pairs in saola originate from centric fusions involving ten cattle autosomes. However, small intrachromosomal rearrangements cannot be excluded. In this study the first preliminary homology map of these two species is presented.

Characterization of G-banded chromosomes of a female saola (Pseudoryx nghetinhensis,2n = 50) and X chromosome i dentification by means of fluorescent in situ hybridization.

The saola (Pseudoryx nghetinhensis) is a newly discovered large mammal species, belongs to the subfamily Bovinae and is listed as being endangered. Due to the limitation of the material available, no cytogenetic studies have been carried out on this species. In the present study, preliminary cytogenetic analysis was undertaken on cultured female fibroblast cells to characterize the karyotype organization of saola. An examination of 120 Giemsa stained metaphases showed the diploid chromosome number of 2n = 50, including five bi-armed chromosome pairs. The distribution of constitutive heterochromatin in saola was studied. However, the variability in the size of C-bands was not significant on all the homologous chromosomes. The X chromosome pair, corresponding to the largest telocentric chromosomes, was identified by fluorescent in situ hybridization (FISH) using a bacterial artificial chromosome clone (BAC 0577G05, which maps to BTAXq25-->q33). In comparison to the standard karyotype of cattle (ISCNDB 2000), a G-banded ideogram of saola (about 390 band level) was presented. This work, therefore, provided a basic insight into the karyotype organization of this endangered species and will be particularly useful to improve the understanding of differences of genomes between related species.

Evidence from DNA that the mysterious 'linh duong' (Pseudonovibos spiralis) is not a new bovid.

In 1993, several horns of an unknown mammal were collected in the south of Vietnam. Due to the unusual characteristics of its horns, the 'linh duong', as named by Vietnamese hunters, was quickly described as belonging to a new monospecific genus of bovid, i.e. Pseudonovibos spiralis Peter & Feiler, 1994. The taxonomic status of Pseudonovibos was a highly controversial subject, and it has been suggested that this enigmatic species may be related to three different groups of Bovidae: Antilopini (gazelles), Bovini (cattle, bisons, buffaloes), and Caprini sensu lato (goats, sheep and allies). To assess the phylogenetic relationships of the linh duong within the family Bovidae, two different DNA markers, the nuclear lactoferrin and the mitochondrial cytochrome b genes, were sequenced from bone samples of four trophies collected during 1925 in Indochina. Results show that the mysterious horns of linh duong belong to domestic cattle (Bos taurus). Thus, the linh duong is not a new mammal and the scientific name Pseudonovibos spiralis should be abandoned.

Efficient cryopreservation of bovine blastocysts derived from nuclear transfer with somatic cells using partial dehydration and vitrification.

Preservation by vitrification of Day 7 and Day 8 bovine blastocysts derived from nuclear transfer with cumulus cells was compared with preservation of in vitro fertilized blastocysts. In Experiment 1, embryos were vitrified in PBS containing 60% ethylene glycol. In Experiment 2, they were vitrified in combination with partial dehydration using a solution of 39% ethylene glycol + 0.7 M sucrose and 8.6% Ficoll. In Experiment 1, survival and hatching rates were 44 and 95% for nuclear transferred embryos, and 78 and 55% for in vitro fertilized embryos, respectively. In Experiment 2, survival and hatching rates were 93 and 95% for nuclear transfer embryos, and 77 and 85% for in vitro fertilized embryos, respectively. It is concluded that Day 7 and Day 8 bovine blastocysts derived from cumulus cells could be cryopreserved without the loss of viability by a simple and efficient method using a combination of partial dehydration and vitrification.

Pediatric myocardial infarction after racemic epinephrine administration.

Myocardial infarction is a previously unreported complication of treatment with racemic epinephrine that is used commonly in the emergency department for severe respiratory distress in bronchiolitis or croup syndrome. We describe a pediatric patient who presented with the croup syndrome and severe respiratory distress that required multiple doses of nebulized racemic epinephrine in the emergency department. The patient developed ventricular tachycardia and mild chest discomfort during one treatment, which resolved spontaneously on discontinuation of the nebulization. Persistently abnormal electrocardiograms and elevated creatine phosphokinase MB isoenzyme (CPK-MB) levels suggested a myocardial infarction had occurred. Subsequent echocardiography, cardiac catheterization, and angiography revealed an anatomically normal heart with normal coronary circulation; however, a stress nuclear study showed a small myocardial infarct. The significance of this previously unreported complication of racemic epinephrine is discussed, along with recommendations for proper use in the emergency department.

Effects of estradiol-17 beta and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet.

Twenty-four cycling swamp buffaloes with normal reproductive histories and 2-3 months postpartum were used to investigate the effect of addition of estradiol-17 beta and human chorionic gonadotrophin (hCG) to the superovulation regime on the level of ovarian stimulation and embryo production. The estrous cycles of buffaloes were synchronized by prostaglandin injection and then divided into two groups for superovulatory treatment. Those in Group 1 (n = 12) received a implant containing 3 mg norgestomet (Syncro-Mate-B) for 9 days (insertion day is Day 0), with 4000 IU of equine chorionic gonadotrophin (eCG) and 500 micrograms cloprostenol i.m. given at Day 7. Group 2 (n = 12) received the same regime as Group 1, together with 7.5 mg estradiol-17 beta given in three intramuscular injections on Days 3, 5 and 7 in decreasing doses (4.0, 2.5 and 1.0 mg, respectively) and 5000 I.U hCG i.v. coincidentally with the first insemination. Estrus was monitored visually and by placing treated animals with bulls. Each animal was inseminated twice with frozen sperm after standing estrus. The numbers of corpora lutea (CL) and follicles greater than 8 mm in diameter were recorded via palpation per rectum at 6 days after implant removal. Two days later 11 animals from Group 2 and two from Group 1 were slaughtered for direct observation of ovarian responses and for embryo collection.

Effect of estradiol supplementation on superovulation in Swamp buffalo.

The effect of estradiol-17beta (E(2)) supplementation on superovulation with (PMSG) or (FSH) was investigated in Swamp buffalo. Sixty-eight buffalo were treated in seven groups. Group 1 served as control and was superovulated by standard PMSG or FSH treatment used in routine bovine embryo transfer protocols. Group 2 was superovulated by standard PMSG regimen plus two injections of E(2) at a 48 h interval beginning one day before the onset of gonadotropin treatment (short-term supplementation) for a total dosage of 2.5 mg E(2); Groups 3 and 4 received the same regimen as Group 2, but in doses of 5.0 and 7.5 mg E(2), respectively. Group 5 received the standard FSH regimen (40% LH). Group 6 received short-term E(2) (7.5 mg) supplementation of FSH-p. Group 7 was superovulated by standard FSH regimen (40% LH) plus three injections of E(2) at 48-72 h intervals beginning five days before the onset of gonadotropin treatment (long-term supplementation) for a total dosage of 7.5 mg E(2). The number of corpora lutea (CL) and follicles >/= 8 mm in diameter were recorded by palpation per rectum and after slaughter. The mean numbers of CL and follicles were 0.99, 5.8, 8.0, 10.6, 4.0, 3.9, 8.1 and 0.25, 6.8, 6.2, 6.2, 1.6, 0.0, 4.1 for Groups 1, 2, 3, 4, 5, 6, 7, respectively. In Group 7, the rates of nonsurgical and postmortem embryo recovery were 46 and 90.4%, respectively and 54.4% of the collected ova were fertilized. These results indicate the possibility of producing viable embryos in buffalo by using E(2) supplementation for the gonadotropin treatment.