Clinical and laboratorial description of the differential diagnoses of hemostatic disorders in the horse.

The process of fibrin clot formation is a series of complex and well-regulated reactions involving blood vessels, platelets, procoagulant plasma proteins, natural inhibitors, and fibrinolytic enzymes. Vasculitis can be caused by a variety of different agents as bacteria, viruses, protozoal, rickettsial organisms, toxic, drugs, medications, and neoplasms. The most common cause of vasculitis is the purpura hemorrhagica, which is associated with exposure to Streptococcus equi ssp. equi or less commonly, equine influenza. Deficiencies or defects of the hemostatic components may result in bleeding and/or thrombosis. Inherited alterations of primary hemostasis (von Willebrand disease: vWD and Glanzmann's thrombasthenia: GT) and of secondary hemostasis (hemophilia A and prekallikrein: PK deficiency) are scarcely reported in equine clinic. On the contrary, acquired alterations of primary and secondary hemostasis are commonly found. They include thrombocytopenia, platelet dysfunction due to the administration of some drugs and targeted antiplatelet agents, decreased factor synthesis (liver disease or deficiency of vitamin K), release of inactive factors, inhibition of factor activity, or excessive consumption and depletion of factors (platelets, coagulation factors, and anticoagulants factors as antithrombin (AT) and protein C). Disseminated intravascular coagulation (DIC) is the most common and complex hemostatic disorder in horses and appears to be associated with sepsis, inflammatory and ischemic gastrointestinal tract disorders and other systemic severe diseases. These alterations are commonly found in patients in intensive care units.

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders.

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47±0.46nmol/10(8) cells. Following semen cryopreservation, GSH decreased to 1.62±0.13nmol/10(8) cells, a 64% reduction (p<0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p<0.01). Addition of 1mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze-thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.

Effect of open pulled straw (OPS) vitrification on the fertilisation rate and developmental competence of porcine oocytes.

Freezing technologies are very important to preserve gametes and embryos of animals with a good pedigree or those having high genetic value. The aim of this work was to compare immature and in vitro matured porcine oocytes regarding their morphology and ability to be fertilised after vitrification by the open pulled straw (OPS) method. In four experiments 830 oocytes were examined. To investigate the effect of cumulus cells on oocyte survival after OPS vitrification, both denuded and cumulus-enclosed oocytes were vitrified at the germinal vesicle (GV) stage, then after vitrification they were matured in vitro. Besides, in vitro matured oocytes surrounded with a cumulus and those without a cumulus were also vitrified. The survival of oocytes was evaluated by their morphology. After in vitro fertilisation the rates of oocytes penetrated by spermatozoa were compared. Our results suggest that the vitrification/warming procedure is the most effective in cumulus-enclosed oocytes (22.35 +/- 1.75%). There was no difference between the order of maturation and vitrification in cumulus-enclosed oocytes, which suggests the importance of cumulus cells in protecting the viability of oocytes during cryopreservation.

The effect of nerve growth factor on nuclear progression of porcine oocytes during in vitro maturation and embryo development.

The present study examined the effect of nerve growth factor (NGF) on in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development of porcine oocytes. Cumulus-oocyte complexes were cultured with or without 1.0 ng/ml NGF for 40 h. After IVF, they were cultured in vitro for 6 days. After 10 and 20 h of IVM, there was no difference in nuclear status between the NGF-treated and control oocytes. Significant differences were detected in nuclear progression of oocytes matured in the presence or absence of NGF at 30 h of culture. A higher proportion of NGF-treated oocytes were at M-II stage compared to the control. Nevertheless, at the end of the 40-h IVM period, there was no difference in the proportion of M-II stage oocytes between the NGF-treated and control groups. NGF in IVM medium did not influence the developmental competence of putative embryos. Most embryos remained at the 2- to 4-cell stage; however, a significant amount of embryos reached the morula stage both in the NGF and the control groups. These results suggest that NGF during IVM accelerates nuclear progression of porcine oocytes by enhancing the post-diakinetic events of meiosis.

Sexing in vitro produced bovine embryos, at different stages of development, using rat H-Y antiserum.

The male-specific H-Y antigen is present on mammalian cell membranes and has been identified by various methods, including antiserum cytotoxicity. The objective of the present study was to determine the sex of in vitro produced (IVP) bovine embryos, at varying stages of development, by culturing in the presence of rat monoclonal H-Y antibodies. Embryos derived from IVM/IVF were classified according to the interval after IVF (48, 96 or 120 h) as Category 1, 2 or 3 if they had 4 to 8, <32, and >32 cells, respectively. Embryos of each category were cultured for 24h in TCM-199 supplemented with bovine oviductal epithelial cells, fetal calf serum (FCS), and antibiotics (Control group), to which the following had been added: guinea pig serum (GPS; C' group); H-Y antiserum (HY group); or GPS and H-Y antiserum (C' + HY group). After culture, embryos were designated as "affected" when development was arrested or one or more blastomeres was degenerate; embryos lacking these changes were designated "unaffected." The sex of each embryo was subsequently determined by chromosome analysis. After 48h of IVF (Category 1), within each of the four treatments, the proportion of unaffected embryos was higher than the proportion of unaffected embryos (81% versus 19%, P < 0.05). Similarly, the Control, C' and HY groups of Categories 2 and 3 embryos had different proportions of unaffected versus affected embryos (75% versus 25%, P < 0.05). In all these groups, the male:female ratio did not significantly differ from 1:1. In contrast, in the C' + HY group of Categories 2 and 3 embryos, the ratio of unaffected versus affected embryos was 41% versus 59% (P < 0.05) and the male:female ratio differed (P < 0.05) from the expected 1:1 ratio (approximately 0.3:1 and 4.5:1 for unaffected versus affected, respectively). In conclusion, when bovine embryos were cultured in the presence of rat monoclonal H-Y antibodies and compliment, alterations occurred in embryos that were beyond the 8-cell stage; we inferred that the antibodies cross-reacted with H-Y antigens.

Efecto del protocolo de preparación de los espermatozoides bovinos sobre el patrón de reacción acrosómica / Effect of sperm preparation protocol on the bovine acrosome reaction pattern

En este trabajo se ha analizado el proceso de reacción acrosómica mediante la tinción con lectinas de muestras de semen bovino congelado que han sido sometidos a diferentes tratamientos de preparación. La adición de heparina y cafeína supone un aumento significativo del número de espermatozoides con los acrosomas reaccionados. Al seleccionar los espermatozoides en un columna de Percoll se acelera el proceso de reacción acrosómica. Estos datos confirman que la preparación de los espermatozoides para la fecundación in vitro afecta al patrón de capacitación y reacción acrosómica, de manera que pueden ser determinantes para el proceso de fecundación (AU)

Níveis residuais em esponjas impregnadas com acetato de medroxiprogesterona, após tratamento de sincronizaçäo do estro e sua relaçäo com a fertilidade nas cabras em período reprodutivo / Residual levels on medroxyprogesterone acetate-impregnated sponges after estrus synchronization treatment and their relationship with fertility in cyclic goats

Preparou-se um conjunto de esponjas de poliuretano embebidas com acetato de medroxiprogesterona (MAP). O nível real do progestágeno nas esponjas foi checado com anterioridade à inserçäo das esponjas do tratamento. Treze cabras em período reprodutivo foram tratadas com esponjas intravaginais embebidas em MAP para sincronizaçäo do estro. As esponjas foram retiradas após 14 dias de tratamento. As cabras que apresentaram cio foram inseminadas artificialmente 48 horas depois de retiradas as esponjas. Os níveis residuais de MAP nas esponjas retiradas foram medidos por espectrofotometria a 241 nM e examinados em relaçäo à fertilidade. A dose real de MAP teve como média 62 mg mais ou menos 2 mg. Níveis elevados (x = 32,46 mg mais ou menos 9,84 mg) de MAP residual foram encontrados em esponjas após o tratamento. A porcentagem de sincronizaçäo dos cios foi 92,31 por cento e a taxa de prenhez, 69,23 por cento. As cabras emprenhadas tiveram um nível residual do hormônio nas esponjas significativamente maior (P < 0,01) (x = 35,56 mg mais ou menos 6,06) que as cabras näo emprenhadas (x = 20,00 mg mais ou menos 10,58 mg). Encontrou-se uma alta correlaçäo (r = + 0,7158) entre MAP residual e fertilidade. Conclui-se que há uma relaçäo entre o nível residual de MAP e a fertilidade dos cios sincronizados nas cabras em período reprodutivo

Successful intercontinental transport of porcine embryos from Europe to South America.

A total of 393 porcine embryos was transported in two trials each lasting more than 30h from Hannover, Federal Republic of Germany to Buenos Aires, Argentina. The embryos were shipped in phosphate buffered saline (PBS) medium supplemented with 10% lamb serum and packaged in 0.25ml straws placed in a modified thermos bottle at 36.5 degrees C. Upon arrival, 359 embryos (90.8%) were evaluated as morphologically intact and were transferred to 19 recipients. Twelve recipients remained pregnant. Three recipients aborted and nine recipients (47.4%) farrowed a mean number of 5.6 +/- 2.6 (x +/- SD) piglets after 115.1 +/- 1.8 d of gestation. The average birthweight was 1.1 +/- 0.2 kg. The percentage of embryos resulting in live piglets was 28.6% in farrowing recipients. These births represented the first piglets from embryos that had been stored for more than 30h in vitro and shipped from Europe to South America.