Equine seminal plasma and sperm membrane: Functional proteomic assessment.

During ejaculation, a large amount of seminal plasma proteins interact with the sperm membrane, leading to a series of biochemical and structural changes implicated in sperm function and gamete interaction. However, the roles of the majority of these proteins remain unknown. This study aimed to investigate the proteome and functionality of the major equine proteins of seminal plasma and the sperm membrane. Seminal plasma and enriched-membrane proteins (150 µg) were separated by two-dimensional gel electrophoresis, and the respective maps were analyzed. Protein identification was performed by in-gel digestion and tandem mass spectrometry (GeLC-MS/MS). Samples were also submitted to in-solution digestion (complex protein mixture) and identified by shotgun analysis by LC-MS/MS; bioinformatic tools were used to investigate protein functions. Seminal plasma and sperm membrane extract maps contained 91.0 ± 8.2 spots and 245.3 ± 11.3 spots, respectively, within the 3-10 pH range. In total, the most abundant proteins identified in 2D maps and in complex protein mixtures included 24 proteins for seminal plasma and 33 for sperm membrane extract, with a high degree of confidence (P < 0.05). Of these, HSP1, CRISP3 and KLK1E2 were the most abundant in seminal plasma; HSP1 was highly abundant in sperm membrane extract, in many isoforms, which is related to membrane destabilization and may compromise sperm preservation. HSP1-polybromo-1 interactions suggested a role in DNA stabilization. Prosaposin was identified in seminal plasma and may play a role in the fertilization process. IZUMO4, a member of the IgSF family involved in the prefertilization stages, was identified in 2D gel and MS/MS analysis of sperm membrane extract. Ten proteins of seminal plasma were found to interact with the sperm membrane and were related to binding and catalytic activities (clusterin, CRISP3, epididymal sperm-binding protein 1, kallikrein1E2, seminal plasma protein A3, and HSP1). Additionally, other identified proteins were associated with DNA integrity, capacitation and recognition of pregnancy. These findings indicate that the binding of specific proteins to the plasma membrane during ejaculation may influence sperm survival after cryopreservation and may play a role in decreasing the quality in stallions with toxic seminal plasma. Elucidation of these interactions is an important step in understanding the biological processes related to equine fertility and facilitates future investigations on the selection and application of low freezability semen strategies.

Influence of spermatozoal lipidomic profile on the cryoresistance of frozen spermatozoa from stallions.

The membrane of spermatozoa, which contributes to cellular cryoresistance, contains numerous lipids with a composition that directly affects membrane fluidity and the fertilization process. In light of variations in the degree of sensitivity in equine seminal freezing, this study aimed to correlate equine semen lipids with post-thawing characteristics of spermatozoa. We used ejaculates from 34 stallions, which were evaluated (total motility ≥ 60%), frozen and thawed and reevaluated for motility of spermatozoa, membrane integrity and lipid peroxidation. Lipid extraction of the fresh semen samples was performed by liquid-liquid extraction, and fingerprinting lipid analysis was conducted by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Based on the characteristics of spermatozoa after thawing, the animals could be separated into two groups: resistant (Good Freezers, n = 5) and sensitive (Bad Freezers, n = 6) to freezing, and their MALDI-MS data were then compared. The Good Freezers group showed a higher abundance of phosphatidylcholines (m/z 796.6, 846.6, 810.6, 854.6 and 732.6). The ions of m/z 812.6, 832.6, 836.6 and 838.6 belonging to the phosphatidylcholine lipid class were also positively correlated with motility of spermatozoa, whereas that of m/z 794.6 was negatively correlated with lipid peroxidation in thawed semen. The Bad Freezer group, displayed higher abundance of one phosphatidylcholines (m/z 806.6), as well as a sphingomyelins (m/z 703.5), which were negatively correlated (univariate analysis) with kinetics of spermatozoa after thawing (m/z 703.5) and with membrane integrity (m/z 792.6). The ion of m/z 717.5, assigned to phosphatidic acid, was negatively correlated with lipid peroxidation. In general therefore, the phosphatidylcholines are associated with higher quality of spermatozoa after thawing, especially in functional capacity, and that lipid semen composition was found to influence the resistance of spermatozoa to cryopreservation and may interfere with motility, membrane integrity and lipid peroxidation in stallions.

Early and late analysis of postpartum stress in newborn foals / Análise do estresse no pós-parto imediato e tardio em potros neonatos

The aim of this study was to evaluate newborn foals stress in the first 48 hours of life. Heart rate (HR), heart rate variability (HRV) and serum cortisol concentrations, blood glucose, and lactate were determined in foals (n = 20, Paint Horse). Cortisol concentrations decreased significantly between four and 48 hours, with the highest concentration at 4 hours after birth. Positive correlations between cortisol and lactate occurred between 12 and 16 hours. And there was a negative correlation between cortisol and lactate 4 hours after birth. Among the values obtained for HR, there was significantly difference between the the value obtained at twelve hours of life moment and all the other moments measured, the highest values were at birth, 24 and 48 hours of life. Among HRV indexes, there were no statistical differences between the variables analysed. The cortisol, HR and HRV alterations were consistent to neonatal stress, but might be related to extrauterine environment adaptation associated to sympathoadrenal response, since those foals were healthy.(AU)

O objetivo deste estudo foi avaliar o estresse em potros neonatos nas primeiras 48 horas de vida. FC, VFC, concentrações de cortisol sérico, glicemia e lactatemia foram determinadas em potros (n = 20; Paint Horse). As concentrações de cortisol reduziram significativamente, entre quatro e 48 horas, sendo o maior valor observado às quatro horas após o nascimento. Correlações positivas entre cortisol e lactato ocorreram entre 12 e 16 horas de vida. E ocorreu correlação negativa entre cortisol e lactato às quatro horas de vida. Os valores de FC divergiram significativamente entre os momentos ao nascimento, quatro, oito, 16, 20, 24, 36 e 48 horas quanto comparados com as 12 horas de vida, sendo os maiores valores ao nascimento, às 24 e às 48 horas. Entre os índices de VFC, não foram observadas diferenças estatísticas para as variáveis analisadas. As alterações encontradas para cortisol, FC e VFC são condizentes com um quadro de estresse neonatal, mas podem estar relacionadas com o período de adaptação à vida extrauterina, associada a uma resposta simpatoadrenal, visto que os potros apresentavam-se saudáveis.(AU)

The 14bp-deletion allele in the HLA-G gene confers susceptibility to the development of hepatocellular carcinoma in the Brazilian population.

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class I molecule involved in tumor escape mechanisms. Considering that the HLA-G 14bp insertion/deletion polymorphism is located at the 3' untranslated region (3'UTR) in exon 8, and since it has been associated with the magnitude of HLA-G production, we studied the association of 14bp insertion/deletion polymorphism with the risk of developing hepatocellular carcinoma (HCC). A total of 109 HCC patients followed at the University Hospital, Faculty of Medicine of Ribeirão Preto, São Paulo, Brazil, and 202 healthy controls from the same geographic area were genotyped for the 14bp insertion/deletion polymorphism using polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis. Compared to controls, the frequency of the 14bp deletion allele was overrepresented in HCC patients (65% versus 56%, respectively, P = 0.0326). The 14bp deletion conferred an odds ratio (OR) of 1.46 [95% confidence interval (CI): 1.04-2.05]. Similarly, the deletion/deletion genotype was marginally overrepresented in HCC patients (45% versus 35% in controls, P = 0.0871), conferring an OR of 1.54 (95% CI: 0.96-2.48). The frequencies of the deletion/insertion or insertion/insertion genotypes observed in patients were not statistically different from those observed in controls (P > 0.05). Our results suggest that the 14bp-deletion allele in HLA-G gene is associated with HCC susceptibility in a Brazilian population.

Seasonal aspects of reproductive physiology in captive male maned wolves (Chrysocyon brachyurus, Illiger 1815).

The aim of the study was to evaluate the seasonality of andrological characteristics and hormonal profile of captive maned wolves (Chrysocyon brachyurus, Illiger 1811). Three adult males were evaluated from the Companhia Brasileira de Metalurgia e Mineração Scientific Breeding Center in Araxá, MG, Brazil, over 13 months. Semen was collected 2-3 times weekly and analysed. Scrotal circumference, biometrics and testicular volume were measured. Stool samples were collected 2-3 times weekly to analyse corticosteroid and testosterone metabolite concentrations. A success rate of 100% was achieved in the collection attempts during the breeding season (BS) and 77.8% during the non-breeding season (NBS). The interval to achieve penile erection was 1-5 min in the BS and 6-10 in the NBS (p < 0.001). Of the ejaculates collected, 80.0% contained sperm during BS, while 28.6% did during the NBS. The ejaculate had only one fraction, was odourless, predominantly translucent (72.4%), with a watery appearance, pH 6.7 and osmolarity of 352.8 mOsmol. Seasonal influences were seen in ejaculate volume (1.3 ml vs 0.4 ml), number of spermatozoa per ejaculate (73.9 × 10(6) vs 6.1 × 10(6) ) and percentage of live sperm (82.0% vs 66.1%) between the BS and NBS (p < 0.05), respectively. A high percentage of major sperm defects were observed in both seasons (50.1% in BS; 65.7% in NBS). Testicular volume was larger (p < 0.05; right testicles 13.1 cm(3) in BS vs 4.0 cm(3) in NBS, while left testicles 12.9 cm(3) in BS vs 5.3 cm(3) in NBS) and testicular consistency increased in the BS. No difference was seen in the basal faecal metabolite concentrations of testosterone; however, the corticosteroid concentrations were higher in the BS. Based on these results, it is possible to conclude that the collection of semen is feasible in captive maned wolves without compromising libido, seminal characteristics and reproductive behaviour and that sperm production is influenced by seasonality; however, it appears that there is no seasonal influence on basal testosterone concentrations.

Comparison of two different extenders for cryopreservation of epididymal dog sperm.

The collection of epididymal sperm is an option for preservation of germplasm of genetically superior animals that need to be orchiectomized or have died. The extender type used to freeze sperm is important to avoid spermatozoal membrane damage and to preserve semen quality after cryopreservation. The objective of this study was to verify the effects of a commercial bovine extender (Bovimix(®); Nutricell, Campinas) and a traditional TRIS-citric acid-glucose-egg yolk-7% glycerol extender on cryopreservation of canine epididymal sperm. The testes of 13 adult dogs were kept at 5 °C for 24 h in saline solution, and epididymal sperm was recovered in Ringers solution without lactate and were evaluated for motility. Samples with ≥ 80% motility were pooled and then divided before dilution and packaging in 0.5 ml plastic straws, equilibration at 4 °C for 1 h, freezing in nitrogen vapour for 20 min and storing at -196 °C. The straws were thawed at 56 °C for 10 s and were evaluated for motility by computer assisted analysis (CASA). The semen parameters, sperm movement index, linearity, total motility and rapid progressive motility were statistically higher in Bovimix(®) than TRIS. In contrast, amplitude of lateral head displacement, slow sperm and static sperm were lower in Bovimix(®). Despite the high percentage of sperm defects in epididymal cells, regardless of the extender, we concluded that Bovimix(®) is a viable alternative for the freezing of canine epididymal sperm.

Treatment of hepatocellular carcinoma with liver transplantation: a single-center experience from Brazil.

INTRODUCTION: Orthotopic liver transplantation (OLT) is the treatment of choice of hepatocellular carcinoma (HCC) for patients with cirrhosis, mainly those with early HCC. Herein we have present the clinical characteristics and outcomes of cirrhotic patients with HCC who underwent OLT from cadaveric donors in our institution. METHODS: From May 2001 to May 2009, we performed 121 OLT including 24 patients (19.8%) with cirrhosis and HCC within the Milan criteria. In 4 cases, HCC was an incidental finding in the explants. RESULTS: The patients' average age was 55 +/- 10 years, including 82% men. Fifty percent of patients were Child class B or C. The average Model for End Stage Liver Disease for Child A, B, and C categories were 11, 15, and 18, respectively. The HCC diagnosis was made by 2 dynamic images in 16 cases; 1 dynamic image plus alphafetoprotein >400 ng/mL in 4; and 4 by histologic confirmation. Twenty patients received a locoregional treatment before OLT: 6 percutaneous ethanol injection, 9 transarterial chemoembolization, 1 transarterial embolization, and 4 a combination of these modalities. The median follow-up after OLT was 19.7 months (range, 1-51). A vascular invasion was observed in the explant of 1 patient, who developed an HCC recurrence and succumbed at 8 months after OLT. Two further patients, without vascular invasion or satellite tumor displayed tumor recurrences at 7 and 3 months after OLT, and death at 2 and 1 month after the diagnosis. The remaining 25 patients have not shown a tumor recurrence. CONCLUSION: In the present evaluation, OLT patients with early HCC and no vascular invasion showed satisfactory results and good disease-free survival. Strictly following the Milan criteria for liver transplantation in patients with HCC greatly reduces but does not completely avoid, the chances of tumor recurrence.

Case report. PET/CT appearance of desmoid tumour of the chest wall.

Desmoid tumours are rare, poorly circumscribed tumours that have a firm consistency and, although benign, have a remarkable tendency to infiltrate into surrounding structures. Extra-abdominal desmoid tumours involve mainly the extremities or the chest wall and are usually managed by wide radical resection. Moreover, desmoid tumours involving the chest wall are locally aggressive tumours with a high recurrence rate. We report a case of a pathologically proven desmoid tumour of the chest wall in a patient with a history of bilateral breast cancer and oesophageal cancer. We discuss the imaging appearances of this tumour on positron emission tomography combined with computed tomography (PET/CT) and magnetic resonance imaging.

Osteopontin in seminal plasma and sperm membrane of dogs.

The objective of this work was to describe the presence of osteopontin (OPN) in canine seminal plasma and sperm membranes. A pool of seminal plasma and sperm membrane extract from 30 dogs was used. Polyacrylamide electrophoresis gels were performed and the bands were transferred to nitrocellulose paper and Western blot was undertaken using an antibody anti-OPN. Two and 12 bands were marked in the seminal plasma (77.2 and 15.6 kDa) and sperm membrane extracts (70.6-26.6 kDa), respectively. However, from 12 marked bands in the sperm membrane extract, only three (46.4, 37.7 and 36.5 kDa) were strongly marked. We conclude that, seminal plasma and sperm membranes from dogs contain different isoforms of OPN; yet, further studies will be necessary to determine their function in this species.

Fertilizing capacity of frozen epididymal sperm collected from dogs.

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 x g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 mum in diameter, with a dark ooplasm surrounded by three- or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70 degrees C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO(2) at 38 degrees C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.

Liver mitochondrial function in familial amyloidotic polyneuropathy.

The objective of the present study was to analyze hepatic mitochondrial function in patients with familial amyloidotic polyneuropathy (FAP) undergoing cadaveric donor orthotopic liver transplantation. From February 2005 to May 2007, eight patients with FAP, ranging in age from 34 to 41 years and with Model for End-Stage Liver Disease scores ranging from 24 to 29. Underwent orthotopic transplantation using a liver from a deceased donor by the piggyback method. Immediately before beginning the recipient hepatectomy in a patient with FAP, a biopsy was obtained for analysis of mitochondrial function (FAP group). The control group consisted of 15 patients undergoing hepatic surgery to treat small tumors of the liver. Mitochondrial respiration was determined on the basis of oxygen consumption by energized mitochondria using a polarographic method. The membrane potential of the mitochondria was determined spectrofluorometrically. Data were analyzed statistically by the Mann-Whitney test, with the level of significance set at 5%. State 3 and 4 values, respiratory control ratio, and membrane potential were 47 +/- 8 versus 28 +/- 10 natoms O/min/mg protein (P < .05); 14 +/- 3 vs 17 +/- 7 nat.O/min/mg.prot.mit. (P > .05); 3.6 +/- .5 vs 1.7 +/- 0.7 (P < .05); and 135 +/- 5.2 vs 135 +/- 6 mV (P > .05) for control versus FAP patients, respectively, demonstrating a decreased energy status of the liver in FAP.

Liver transplantation at a university hospital, faculty of the medicine of Ribeirão Preto, University of São Paulo: results for the first 60 recipients.

The purpose of the present article was to present the series operated by a Liver Transplant Group of the interior of the State of Sao Paulo, Brazil. Sixty patients were transplanted from May 2001 to May 2007. Thirty percent of the patients had alcoholic cirrhosis. 18.3% had C virus-induced cirrhosis, 10% had C virus- and alcohol-induced cirrhosis, 6% had B virus-induced cirrhosis, 13.3% had cryptogenic cirrhosis, 8.3% autoimmune cirrhosis, 13.3% had familial amyloidotic polyneuropathy (FAP), and 13.3% had hepatocellular carcinomas. The series was divided by a chronological criterion into two periods: A (n = 42) and B (n = 18) with the latter group operated based upon the Model for End-stage Liver Disease (MELD) criterion. Sixty-nine percent were men. Age ranged from 14 to 66 years. Period A included 12% Child A: 59.2%, Child B; 24%, Child C; and 4.8%, FAP. Period B comprises 22.2% Child A: 11.1%, Child B: 33.3%, Child C: and 33.3%, FAP. MELD scores ranged from 8 to 35 for period A and from 14 to 31 for period B. Intraoperative mortality was 2/42 patients for period A and 0/18 for period B, overall postoperative mortality was 40% including for period A, 35% among Child B and C patients, and 5% among FAP and Child A patients (P < .05) and 16.6% for period B among 11.1% Child B patients and 5.5% FAP patients; 3.3% of patients required retransplantation due to hepatic artery thrombosis. Real postoperative survival was 60% during period A and 83.3% during period B, with an overall survival rate of 67% for the two periods. The present results show levels of postoperative mortality, (especially during period B), and survival rates similar to those reported by several other centers in Brazil.

Acute chagasic myocardiopathy after orthotopic liver transplantation with donor and recipient serologically negative for Trypanosoma cruzi: a case report.

Chagas disease (American trypanosomiasis) is caused by the protozoan parasite Trypanosoma cruzi. Chagas disease following solid-organ transplantation has occurred in Latin America. This report presents the occurrence of Chagas disease despite negative serological tests in both the donor and the recipient, as well as the effectiveness of treatment. A 21-year-old woman from the state of Sao Paulo (Brazil) underwent cadaveric donor liver transplantation in November 2005, due to cirrhosis of autoimmune etiology. Ten months after liver transplantation, she developed signs and symptoms of congestive heart failure (New York Heart Association functional class IV). The echocardiogram, which was normal preoperatively, showed dilated cardiac chambers, depressed left ventricular systolic function (ejection fraction = 35%) and moderate pulmonary hypertension. Clinical investigation discarded ischemic heart disease and autoimmune and other causes for heart failure. Immuno fluorescence (immunoglobulin M and immunoglobulin G) and hemagglutination tests for T cruzi were positive, and abundant T cruzi amastigotes were readily identified in myocardial biopsy specimens. Treatment with benznidazole for 2 months yielded an excellent clinical response. At the moment of submission, the patient remains in functional class I. This case highlighted that more appropriate screening for T cruzi infection is mandatory in potential donors and recipients of solid-organ transplants in regions where Chagas disease is prevalent. Moreover, it stressed that this diagnosis should always be considered in recipients who develop cardiac complications, since negative serological tests do not completely discard the possibility of disease transmission and since good results can be achieved with prompt trypanocidal therapy.

Characteristics of seminal plasma proteins and their correlation with canine semen analysis.

The objectives were to separate canine seminal plasma proteins (with SDS-PAGE) and to determine the correlation between specific proteins and semen characteristics. Three ejaculates from 20 mixed-breed dogs, of unknown fertility, were collected by digital manipulation. Ejaculate volume and color, sperm motility, sperm vigor, percentage of morphologically normal spermatozoa, and membrane integrity (hypoosmotic swelling test and fluorescent staining) were assessed. For each dog, seminal plasma was pooled from all three ejaculates and proteins were separated with SDS-PAGE, using polyacrylamide concentrations of 13% and 22% in the separation gels. After staining, gel images were digitized to estimate molecular weights (MW) and integrated optical density (IOD) of each lane and of individual bands. Total seminal plasma protein concentration was 2.19+/-1.56 g/dL (mean+/-SD; range 1.12-5.19 g/dL). A total of 37 protein bands were identified (although no dog had all 37 bands). In the 13% gel, molecular weights ranged from 100.6 to 17.1 kDa, with four bands (49.7, 33.2, 26.4, and 19.5 kDa) present in samples from all dogs. In the 22% gel, molecular weights ranged from 15.6 to 3.6 kDa, with nine bands (15.6, 13.5, 12.7, 11.7, 10.5, 8.7, 7.8, 5.6, and 4.9 kDa) present in samples from all dogs. Combined for both gels, the majority of bands (85%) had molecular weights <17 kDa, with B20 (15.6 kDa) in high concentrations in samples from all dogs. There were positive correlations (P < or = 0.01) between two bands, B4 (67 kDa) and B5 (58.6 kDa), and sperm motility (r=0.66 and r=0.46), sperm vigor (r=0.56 and r=0.66), percentage of morphologically normal spermatozoa (r=0.55 and r=0.59), the hypoosmotic swelling test (r=0.76 and r=0.68), and fluorescent staining (r=0.56 and r=0.59), respectively. In conclusion, 37 proteins were identified in seminal plasma; two were significantly correlated with semen characteristics.

Characteristics of waitlisted patients for liver transplantation at a liver transplantation unit in the city of Ribeirão Preto, São Paulo, Brazil, especially considering Child and Model for End Stage Liver Disease (MELD) scores.

Liver transplantation represents the most effective therapy for patients suffering from chronic end-stage liver disease. Until recently, in Brazil liver allocation was based on the Child-Turcotte-Pugh score and the waiting list followed a chronological criterion. The aim of this study was to show the clinical and laboratory patterns of our patients awaiting a liver transplantation. Seventy-nine medical records were reviewed in January 2005 to classify patients according to their age, sex, cause of cirrhosis, and Child and Model for End Stage Liver Disease (MELD) scores. The mean age of patients was 47 years; 70% were men. The main diagnosis was liver cirrhosis (97%): 27% alcoholic, 26% viral hepatitis, 20% alcoholic plus viral hepatitis, 13% cryptogenic, and 11% other causes. Sixty-three patients (80%) were Child B or C. The average MELD, scores for Child A, B, and C were 10 +/- 5, 13 +/- 3.4, and 21 +/- 4.3, respectively. Nine deaths (11%) on the waiting list occurred in 2005. Among these, 1 patient was Child B with MELD 10, while the others were Child C, with mean MELD scores of 21 +/- 3.8. Twelve patients (15%) received cadaveric orthotopic liver transplantation. Thus, in this small series, the higher MELD scores corresponded to Child C class and mortality on the waiting list.

Evaluation of fertilizing potential of frozen-thawed dog spermatozoa diluted in ACP-106 using an in vitro sperm--oocyte interaction assay.

The aim of present study was to evaluate frozen canine semen with ACP-106 (Powder Coconut Water) using an in vitro sperm--oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106 containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106 containing 20% egg yolk and 12% glycerol. Samples were thawed at 38 degrees C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 +/- 14.8% and it was significant higher than the total motility estimated by CASA (23.0 +/- 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 +/- 3.1% and 94.3 +/- 3.1%, respectively, post-thaw percentage of intact plasma membrane was only 35.1 +/- 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106 was efficient for maintain the in vitro fertility potential of dog spermatozoa.

Therapeutic effect of hyperbaric oxygen in hepatic artery thrombosis and functional cholestasis after orthotopic liver transplantation.

Among the postoperative complications, hepatic artery thrombosis can occur in up to 10% of adult orthotopic liver transplants and intervention is indicated when this occurs within 30 days by retransplantation. Primary graft dysfunction, which can occur in up to 30% of the cases and is another potential complication, although reversible, has a relatively high mortality rate. Hyperbaric therapy, an efficient mode of tissue oxygenation, is being used in an increasing number of clinical situations. We report here two cases where hyperbaric oxygen therapy greatly benefited patients with complications after orthotopic liver transplantation: one with hepatic artery thrombosis and the other with primary graft dysfunction. Both patients showed rapid clinical recovery with gradual reduction of liver and canalicular enzymes soon after commencing hyperbaric oxygen therapy.

Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa.

Frozen-thawed epididymal spermatozoa have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of felid species, which are threatened with extinction. The aim of this study was to compare the effects of freezing and thawing on domestic cat semen collected by electroejaculation (EL) and from the epididymides (EP) and vasa deferentia. Ten adult cats were anesthetized, electroejaculated and immediately thereafter, orchiectomized. Epididymal spermatozoa were collected through the compression of caudae epididymidis and vasa deferentia. Spermatozoa were frozen-thawed following a single protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) were assessed on two occasions, immediately after collection and after freezing and thawing. There were no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa for any of the variables. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosomal changes are known to affect sperm fertilising capacity, further studies are needed to optimize cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa.

Prolonged interval between parturition of normal live pups in a bitch.

On day 64 after artificial insemination, a six-year-old primiparous briard bitch whelped three live pups between 05.00 and 08.00. It was presented at 11.00 on the same day with failure to complete parturition. On ultrasound examination, a normal live fetus was observed and the bitch was treated with oxytocin three times during the day (1.0, 2.0 and 2.0 iu intramuscularly), with no effect. The following day, a higher dose of oxytocin (5.0 iu) was administered intramuscularly at 11.00, after a uterine ultrasound examination confirmed viability of the fetus. At 18.00 of the same day, the bitch whelped the fourth normal live pup, 37 hours after initiation of parturition and 34 hours after expulsion of the last fetus. Effectiveness of oxytocin and normal versus prolonged parturition due to uterine inertia are discussed.