Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification     

K Sirisha  NL Selokar  M Saini  P Palta  RS Manik  MS Chauhan  SK Singla 《Reproduction in domestic animals》2013,48(4):538-544

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.  相似文献   

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro     

A Yadav  KP Singh  MK Singh  N Saini  P Palta  RS Manik  SK Singla  RC Upadhyay  MS Chauhan 《Reproduction in domestic animals》2013,48(5):858-865

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.  相似文献   

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation     

MK Singh  KP Singh  D Kumar  RA Shah  T Anand  MS Chauhan  RS Manik  SK Singla  P Palta 《Reproduction in domestic animals》2013,48(2):284-291

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.  相似文献   

Quantitative risk assessment for the annual risk of exposure to Trichinella spiralis in imported chilled pork meat from New Zealand to Singapore     

EKB Richardson  N Cogger  WE Pomroy  MA Potter  RS Morris 《New Zealand veterinary journal》2013,61(5):269-277

Abstract

AIMS: To determine the annual likelihood of exposure to an infectious dose of Trichinella spiralis from consuming imported pork meat from New Zealand to Singapore.

METHODS: Input values specific for chilled pork meat imported into Singapore from New Zealand were used in a quantitative risk-assessment model. The model, designed to allow any combination of importing and exporting countries, was divided into two components, viz the release assessment, and the exposure assessment that assessed the annual risk of exposure to the consumer (ARC). The former estimated the likelihood that a contaminated fresh meat product from New Zealand would arrive at Singapore's border, and took into consideration the prevalence of disease on different types of farms. The latter determined the likelihood over a year that a person in Singapore would consume one or more servings of imported fresh meat from New Zealand that contained a burden of greater than or equal to one larva(e) of T. spiralis per gram after preparation for consumption.

RESULTS: The ARC for offal was 2.41 × 10^?7, which was below the pre-selected safety threshold of 1.00 × 10^?6. The ARC for lean meat was 2.39 x 10^?5, which was above the acceptable safety threshold.

CONCLUSIONS: The study demonstrated that continued routine testing at slaughter is unnecessary for pig offal produced commercially, and provided a model with which to further assess management of the risk of exposure to T. spiralis in lean meat.

CLINICAL RELEVANCE: The potential of Trichinella species to cause disease in humans is a public health concern, and has created adverse effects on the international trade of fresh lean meat without regard to the surveillance measures employed by particular pork-producing countries.  相似文献   

Relationship between the fungal complex causing Fusarium head blight of wheat and environmental conditions     

Xu XM  Nicholson P  Thomsett MA  Simpson D  Cooke BM  Doohan FM  Brennan J  Monaghan S  Moretti A  Mule G  Hornok L  Beki E  Tatnell J  Ritieni A  Edwards SG 《Phytopathology》2008,98(1):69-78

ABSTRACT Over 4 years, the environmental conditions and the causal agents of Fusarium head blight (FHB) disease of wheat were determined in field sites in four European countries: Hungary, Ireland, Italy, and the United Kingdom. Polymerase chain reaction-based methods were used to detect each species causing FHB and quantify its DNA (as a measurement of fungal abundance) in the samples. Canonical correspondence analysis (CCA) was used to determine the relationship of the incidence and abundance of each species with weather variables. CCA indicated that little variability in the species prevalence data was explained by the weather variables. In contrast, a greater proportion of variability in abundance data was accounted for by the weather variables. Most samples contained two or more species and statistical analysis suggested that these species tended to coexist at field sites. CCA also indicated that there were differences in the relationships of the prevalence and abundance of the six FHB species with environmental variables. Fusarium poae was associated with relatively drier and warmer conditions, whereas F. graminearum was associated with warmer/humid conditions. F. avenaceum and F. culmorum were both associated with niches of cooler/wet/humid conditions. Two Microdochium species were associated with regions of relatively cool/moderate temperatures and frequent rainfalls of short duration. The results also suggested that environmental conditions differentially affect the infection and colonization processes, and the comparative abundance of the six species.  相似文献   

Mining the Watkins collection of wheat landraces for novel sources of eyespot resistance     

C. Burt  L. L. Griffe  A. P. Ridolfini  S. Orford  S. Griffiths  P. Nicholson 《Plant pathology》2014,63(6):1241-1250

Eyespot is an economically important stem base disease of wheat caused by the soilborne fungal pathogens Oculimacula yallundae and Oculimacula acuformis. The most effective method of controlling the disease is host resistance. However, there are only three genetically characterized resistances in wheat varieties and further sources of resistance are required. Previous studies have identified resistances in wild relatives, but use of these resistances has been limited by linkage drag with deleterious traits exacerbated by low rates of recombination. Therefore, the identification of novel resistances in hexaploid wheat germplasm is desirable. The Watkins collection currently consists of 1056 hexaploid wheat landraces that represent global wheat diversity at the time of its collection in the 1920s and 1930s. As such, it may contain beneficial agronomic traits such as eyespot resistance. The Watkins collection was screened for resistance to O. yallundae based on a glasshouse test of all 1056 accessions and a polytunnel test of 44 accessions selected from a previous field trial. Resistant lines identified in these tests were retested against both O. yallundae and O. acuformis. This identified 17 accessions with resistance to one or both of the pathogen species. From these, two accessions (1190094.1 and 1190736.3) provided a high level of resistance to both pathogen species. An F₄ population derived from accession 1190736.3 indicated that the resistance to O. acuformis in this accession is conferred by a single gene and therefore would be suitable for introgression into elite wheat varieties to provide an alternative source of eyespot resistance.  相似文献   

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs     

LR Maschio  RS Cordeiro  SR Taboga  RM Góes 《Reproduction in domestic animals》2010,45(3):516-524

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.  相似文献   

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine     

A Mukherjee  D Kumar  KP Singh  MS Chauhan  SK Singla  P Palta  RS Manik 《Reproduction in domestic animals》2010,45(6):1118-1121

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.  相似文献   

Off-axis crustal thickness across and along the east pacific rise within the MELT area   总被引：1，自引：0，他引：1  

JP Canales  RS Detrick  S Bazin  AJ Harding  JA Orcutt 《Science (New York, N.Y.)》1998,280(5367):1218-1221

Wide-angle seismic data along the Mantle Electromagnetic and Tomography (MELT) arrays show that the thickness of 0.5- to 1. 5-million-year-old crust of the Nazca Plate is not resolvably different from that of the Pacific Plate, despite an asymmetry in depth and gravity across this portion of the East Pacific Rise. Crustal thickness on similarly aged crust on the Nazca plate near a magmatically robust part of the East Pacific Rise at 17 degrees15'S is slightly thinner (5.1 to 5.7 kilometers) than at the 15 degrees55'S overlapping spreading center (5.8 to 6.3 kilometers). This small north-south off-axis crustal thickness difference may reflect along-axis temporal variations in magma supply, whereas the across-axis asymmetry in depth and gravity must be caused by density variations in the underlying mantle.  相似文献   

Seroprevalence and association with abortion of leptospirosis in cattle in Ontario.   总被引：4，自引：1，他引：3       下载免费PDF全文

J F Prescott  R B Miller  V M Nicholson  S W Martin    T Lesnick 《Canadian journal of veterinary research》1988,52(2):210-215

Sera were collected using a systematic random sampling from 348 cattle herds in Ontario, in proportion to the cattle population in different areas. One cow in five from 296 dairy herds and one in three from 52 beef herds were sampled. The sera were analyzed for prevalence of antibodies to Leptospira interrogans serovar grippotyphosa, hardjo, icterohaemorhagiae and pomona using the microscopic agglutination test. Herd seroprevalence (one or more animals with titer greater than or equal to 80) in beef and dairy herds combined was grippotyphosa 2%, hardjo 13.8%, icterohaemorrhagiae 10.1% and pomona 25.8%; 39% of all herds showed evidence of leptospiral infection with one or more serovars; 44.2% of 52 beef herds had serological evidence of infection with serovar hardjo compared to 8.4% of 296 dairy herds (P less than 0.0001). Seroprevalence of other serovars was not significantly different between beef and dairy herds. The proportion of beef animals seropositive for hardjo and for pomona increased with age, particularly for hardjo; 26.5% of beef animals aged nine years or over were seropositive for hardjo. Dairy animals showed a significant rise of hardjo but not pomona titers with age. The seroprevalence of pomona infection was significantly higher in dairy cattle in eastern Ontario than in other regions. Thirty-four (6.1%) of 553 aborted bovine fetuses had leptospires detected by immunofluorescence techniques. Sixty-five percent of these fetuses were from submissions made between November and January. Leptospires were identified as serovar hardjo by specific immunofluorescence. There appeared, however, to be a paradoxical serological response in that eight aborting cows had antibody titers to pomona rather than hardjo.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献