Retrograde Flush Research Articles

Direct open aortic cannulation under temporary retrograde cerebral flush perfusion in acute aortic dissection Stanford type A—a case report

Direct open aortic cannulation under temporary retrograde cerebral flush perfusion in acute aortic dissection Stanford type A—a case report

Effect of sperm and glycerol concentration on epididymal sperm motility parameters after thawing

The aim of this study was to evaluate the effect of different glycerol concentrations and different sperm concentrations on stallion epididymal sperm motility indicators after thawing. For statistical analysis, 25 stallions were used. Collection of the epidydimal spermatozoa was performed as a retrograde flush of cauda epididymis and ductus deferens within 12 h post castration. After evaluation, the resuspended spermatozoa were centrifuged, the supernatant removed and the spermatozoa resuspended in Gent semen extender to get three different groups with different concentrations of sperm (250 × 106 in ml, 500 × 106 in ml, 1000 × 106 in ml) and different final glycerol concentrations (2%, 4%, and 6%). Therefore, 9 different samples were finally obtained from each stallion. The spermatozoa were packed and placed in a fridge (4 °C) for 2 h, then placed in liquid nitrogen vapour (−80 to −100 °C) and after 25 min plunged into the liquid nitrogen and stored at −196 °C for at least 5 days. The selected straws were individually thawed in a 38 °C water bath for 30 s prior to post-freezing analysis. Motility indicators were assessed at 0, 60, 120, and 180 min after thawing. Parametric test was used for analysis; the measured indicators were total motility, progressive motility, curvilinear velocity, straightness, and average-path velocity. In this study, the best results were reached in samples diluted into a concentration of 1,000 × 106 in ml, regardless of the concentrations of glycerol.

Histological Analysis of Donor Lung Derived Thrombi

<h3>Purpose</h3> Donor lungs often contain clots. Retrograde flush removes a portion of these clots and improves graft quality. It's very likely that some clots remain after retrograde flush and case studies of thrombolytic treatment of donor lungs has shown a beneficial effect. We studied donor lung clots to determine their composition and age. <h3>Methods</h3> At procurement of donor lungs, in situ antegrade flush was followed by a back table retrograde flush. Retrograde flush fluid was collected and filtered using a 0,2mm filter. The material left in the filter was instantly fixed in 4% formalin for at least 24 hours and studied by a haematoxylin-Eosin(HE) stain and with Martius scarlet blue(MSB) stain. The presence and maturity of fibrin and the presence of lines of Zahn were analyzed. Zahn lines are altering bands of light and dark colors indicating the clot was formed in an area of high blood flow. Clots where then classified based on the composition of fibrin. Clot classification was based on the oldest (most dense fibrin) part of the clot. <h3>Results</h3> In the retrograde flush of all included lungs (n=15) clots were found. A total of 129 clots were found in 15 donor lungs. 12 clots were found at a 2^nd retrograde flush at EVLP. Up to 20 clots were seen in a lung varying in size from 0.3 to 70 mm. Composition varied from young, red blood cell-containing clots to more mature dense fibrin meshes (fig 1).A vast majority of clots showed either an outer layer, lines of Zahn or absence of RBC's that we consider characteristics suggestive of formation before death. <h3>Conclusion</h3> All donor lungs contain clots, emphasizing the importance of retrograde flush at retrieval. Of the clots, 39/129 were more mature and had absence of red blood cells and a more mature dense fibrin mesh. These characteristics may render these clots less susceptible to thrombolysis. However, 90/129 clots were more recently formed and might therefore be more accessible to thrombolysis. Further studies are needed to evaluate the effect of thrombolysis on donor lung clots and primary graft function.

Clinical Outcomes of Lung Transplants From Donors With Unexpected Pulmonary Embolism

Pulmonary embolism (PE) is unexpectedly detected in some donor lungs during organ procurement for lung transplantation. Anecdotally, such lungs are usually implanted; however, the impact of this finding on recipient outcomes remains unclear. We hypothesized that incidentally detected donor PE is associated with adverse short-term and long-term outcomes among lung transplant recipients. We analyzed a prospectively maintained database of all lung donors procured by a single surgeon and transplanted at our institution between 2009 and 2018. A standardized approach was used for all procurements and included antegrade and retrograde flush. Pulmonary embolism was defined as macroscopic thrombus seen in the pulmonary artery during the donor procurement operation. A total of 501 consecutive lung procurements were performed during the study period. The incidence of donor PE was 4.4% (22 of 501). No organs were discarded owing to PE. Donors with PE were similar to donors without PE in baseline characteristics and Pao2. Recipients in the two groups were also similar. Pulmonary embolism was associated with a higher likelihood of acute cellular rejection grade 2 or more (10 of 22 [45.5%] vs 120 of 479 [25.1%], P= .03). Multivariable Cox modeling demonstrated an association between PE and the development of chronic lung allograft dysfunction (hazard ratio 2.02; 95% confidence interval, 1.23 to 3.30; P= .005). Lungs from donors with incidentally detected PE may be associated with a higher incidence of recipient acute cellular rejection as well as reduced chronic lung allograft dysfunction-free survival. Surgeons must use caution when transplanting lungs with incidentally discovered PE. These preliminary findings warrant corroboration in larger data sets.

Warm retrograde perfusion can remove more fat from lung grafts with fat embolism in a porcine model.

In lung transplantation, unexpected pulmonary emboli, including thrombi and fat, have been observed with high probability and are associated with potential primary graft dysfunction. We evaluated a new perfusion method using warm retrograde flushing that removes more fat than conventional cold retrograde flushing. We developed a novel porcine donor model for pulmonary fat embolism by administering autologous fat in the left pulmonary artery. The left pulmonary artery and the left superior and inferior pulmonary veins were cannulated for flushing and collecting these solutions. After flushing, the left lung was reperfused under observation for 3h. Two groups underwent warm and cold additional retrograde flush (WS; warm solution group, CS; cold solution group). The fat removal rate in the antegrade flush was equal in both groups (3.0 ± 0.6% vs 3.0 ± 0.4%, p = 0.46); however, the rate was significantly greater in the WS group in retrograde flush (25.2 ± 3.2% vs 8.0 ± 1.4%, p = 0.01). Histology with Oil Red O staining and its software analysis showed more residual fat in the CS group (0.12 ± 0.01% vs 0.38 ± 0.07%, p = 0.01). There was no significant difference in the pulmonary function and hemodynamics during the 3-h period after reperfusion. Warm retrograde perfusion can remove more fat from lung grafts with fat embolism in a porcine donor model.

Comparison of the Effects of Five Semen Extenders on the Quality of Frozen-Thawed Equine Epididymal Sperm

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1–4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

The Effect of Pre- and Post-injection Procedural Factors on Blastocyst Rate After ICSI

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1–4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

The Effect of Four Different Commercial Semen Extenders on the Motility of Stallion Epididymal Sperm

To preserve epididymal sperm, only a limited number of sperm is available, and an optimum processing method that is applicable for the majority of stallions is therefore crucial for successful preservation. The aim of the present study was to evaluate the effect of four different extenders that are commercially available for chilled semen on the motion characteristics of stallion epididymal sperm. Sperm were harvested by retrograde flush from 20 epididymides after the routine castration of 10 stallions. Aliquots of sperm samples were diluted with each of the four extenders ([E1] skim milk-based, [E2] containing defined milk protein, [E3] containing egg yolk, and [E4] containing caseinate). Total motility (TMOT %) and progressive motility (PMOT %) assessed immediately after sperm harvesting and during prolonged storage were highest in extenders with selected milk proteins (E2: 54, 24–79 and 50, 19–78; E4: 57, 23–82 and 52, 14–80) compared to the skim milk-based extender (E1: 40, 1–66 and 37, 0–62) and the egg yolk-containing extender (E3: 21, 6–48 and 13, 2–40; median, min–max for TMOT and PMOT, stored for 48 hours at 4°C, respectively). Motility values were similar for extenders E2 and E4 during the entire storage period (P > .05), while extenders E1 and E3 yielded significantly lower values (P < .05). Using E1, motility increased during the storage period but did not reach values similar to those of E2 or E4. Curvilinear velocity (VCL), amplitude lateral head displacement (ALH), and beat-cross frequency (BCF) differed among all extenders after sperm harvesting (P < .05), but not after 24 (VCL, ALH) and 48 hours (VCL, ALH, BCF; P > .05). Based on the motility results, we recommend extenders containing defined milk protein to process epididymal sperm.

Re: Retrogradein situversus antegrade pulmonary preservation in clinical lung transplantation: a single-centre experience

Historically, grafts were preserved by means of autoperfusion with extracorporeal circulation, topical cooling or donor core cooling. In the 1980s, the standard lung preservation method was single pulmonary artery flush perfusion, using modified Euro-Collins solution at 4°C [1]. However, reperfusion injury remained a significant factor for morbidity and early mortality after lung transplant. Since the early years of lung preservation strategies, some advances have emerged in transplant teams, such as adding prostaglandin E2 to prevent counteract temperature and potassium-induced vasoconstriction. The use of low potassium dextran glucose solutions shows better overall lung function, superior early oxygenation, higher lung compliance, lower incidence of severe primary graft dysfunction and 30-day mortality [2]. As Gohrbandt et al. point out, the route to administer the preservation solution may also have an impact on early lung graft performance [3]. Anterograde flush initiating the infusion through the pulmonary artery with pulmonary vein drainage was the traditional approach for many years [4]. This technique, however, presents a number of limitations. Flushing the pulmonary artery leaves the bronchial tissue to be preserved by topical cooling alone, raising the complication from the bronchial suture. Also, clots and fat or brain tissue emboli, especially in donors with major trauma and multiple bone fractures, may hamper uniform flushing and cooling and, therefore, impair graft function upon reperfusion. Adding a retrograde flush, whereby the preservation solution is administered via the left atrial appendage or four pulmonary veins into the pulmonary venous system and drained through the pulmonary artery, has the potential to flush not only pulmonary vessels, but also the bronchial network, contributing to the adequate perfusion of the tracheobronchial submucosa tissue, and also limiting the effect of hypothermic pulmonary arterial vasoconstriction. On the other hand, less ventilated areas, such as the posterior segments of lower lobes, are better flushed. Also residual blood and harmful constituents and any possible microthrombi that may obstruct the pulmonary vessels are washed out. The evidence from clinical series of this technique is limited [5]; authors such as Gohrbandt et al. show us that there are no clinical benefits of retrograde lung preservation alone and that it may cause or prolong mechanical ventilation and primary graft dysfunction at 72 h and prolong ICU stay [3]. On the other hand, some evidence has been published, showing that biphasic lung preservation is not only clinically feasible [6], but also related with better early oxygenation ratio, lower mean airway pressure and chest radiography score [7, 8]. Many transplant teams have now adopted a biphasic technique with an antegrade flush through the pulmonary artery followed by a retrograde flush through each of the pulmonary veins after heart excision with the lung still ventilated inside the cadaver. However, there is lack of controlled randomized studies.

Dose-dependent effects of homologous seminal plasma on motility and kinematic characteristics of post-thaw stallion epididymal spermatozoa.

Preservation of epididymal spermatozoa is important to save genetic material of endangered species and breeds, or in case of unexpected injury, which will end the breeding career of valuable sires. Seminal plasma (SP) influences sperm quality in a dose-dependent manner and its addition to preserved semen immediately before insemination may be beneficial for sperm fertility. Increased plasma membrane stability of epididymal spermatozoa reduces freezing injury of cells, and the addition of SP after freezing and thawing might have activating and protecting effects on spermatozoa within the female genital tract. In this study, epididymal spermatozoa were harvested by retrograde flush of the epididymal cauda immediately after routine castration and frozen. Seminal plasma was collected from other six stallions. Homologous SP (SP from the same species, but from a different animal) was added to frozen-thawed epididymal spermatozoa at concentrations of 0, 5, 20, 50 and 80% SP. Addition of SP increased sperm motility and influenced kinematic values in a dose-dependent manner (p < 0.05). Motility improved at concentrations of 20 and 50% SP, but did not further increase at 80% SP. There was no difference in sperm motility among SP from six different donor stallions regardless of the concentrations of SP (p > 0.05). Total and progressive motility of ten frozen-thawed epididymal spermatozoa samples collected from different stallions after dilution with extender and 5, 20, 50 or 80% SP differed significantly (p < 0.05). In conclusion, addition of homologous SP to frozen-thawed stallion epididymal spermatozoa immediately improved motility in a dose-dependent manner regardless of semen quality of SP donor stallions. This might positively influence fertility when SP is added before insemination. Moreover, there seems to be a threshold level of SP concentration for optimal improvement of sperm motility.

Selective cerebral hypothermia induced via hypothermic retrograde jugular vein saline flush in a porcine model

Objectives:Multiple methods of selective brain cooling have been used to prevent cerebral ischemia secondary to trauma and pathological or iatrogenic cerebral blood flow restriction. In this study, we tested the efficacy of hypothermic retrograde jugular vein flush (HRJVF) in eliciting selective brain hypothermia in a porcine model.Methods:Twelve swine were divided into two groups: retrograde jugular vein infusion (RJVI) with cold saline (4°C RJVI, n = 6) and with room temperature saline (24°C RJVI, n = 6). For 90 minutes, the following parameters were measured: brain parenchymal temperature, rectal temperature, intracranial pressure (ICP), mean arterial pressure, and heart rate (HR).Results:Swine receiving 4°C RJVI experienced a drop in mean brain parenchymal temperature of 1·1 ± 0·1°C, compared to 0·1 ± 0·1°C in swine receiving 24°C RJVI. At 90 minutes, mean brain parenchymal temperature in the 4°C RJVI treatment group was 35·5 ± 0·2°C, as compared to 37·1 ± 0·2°C in the 24°C RJVI treatment group (P < 0·001). In the 4°C RJVI group, the brain-systemic temperature gradient peaked 10 minutes after initiation of cooling and remained significantly different when comparing the two experimental groups (P < 0·001) throughout the duration of the 90 minutes experiment. Of note, ICP, mean arterial pressure, and HR remained constant without any significant changes or differences between treatment groups.Discussion:These results suggest that HRJVF is an effective method for selective brain hypothermia in a large animal model. Clinical application may prove effective in delaying neural ischemia.

DNA integrity of stallion spermatozoa from different segments of the epididymis

The cryopreservation of sperm from different segments of the epididymis is the ultimate opportunity to preserve the gene pool of animals of great breeding value. Despite the fact that samples from the caput epididymis are immotile, their DNA integrity could justify their use in ICSI procedures. Thus, the objective of the present study was to evaluate the DNA integrity and sperm motility of stallion sperm collected from different regions of the epididymis. Equine epididymides were obtained from 10 stallions using retrograde flush with 40mL of Botu-Semen per caudae epididymis. Sperm from the caudae epididymis were obtained with a slicing technique in association with a float-up method. The samples were divided into 3 groups: Botu-Semen (control), Fert-Talp and Sperm-Talp, and diluted 1:1 with these media. The samples were centrifuged (1000 x g/10 min) to concentrate the sperm, the supernatant was removed, and the pellets were resuspended in Botu-Crio . Samples were loaded into 0.5mL straws and cooled at 5 C for 20 min. They were then frozen in nitrogen vapor (6 cm above the level of liquid nitrogen) for an additional 20 min, plunged into liquid nitrogen and stored. Sperm chromatin structure assay was performed using a semen aliquot diluted in 200 mL of buffer solution (0.186g disodium EDTA, 0.790 g Tris-HC1, 4.380 g NaC1 in 500 mL deionized water, pH 7.4). This was added with 400 mL of detergent/acid solution. Thirty seconds later, 1.2 mL acridine orange solution was added (Evenson, In: Sorsa M, Norppa H (ed), Monitoring of

Retrograde flush is more protective than heparin in the uncontrolled donation after circulatory death lung donor

BackgroundFormation of microthrombi after circulatory arrest is a concern for the development of reperfusion injury in lung recipients from donation after circulatory death (DCD) donors. In this isolated lung reperfusion study, we compared the effect of postmortem heparinization with preharvest retrograde pulmonary flush or both. MethodsDomestic pigs (n = 6/group) were sacrificed by ventricular fibrillation and left at room temperature for 1 h. This was followed by 2.5 h of topical cooling. In control group [C], no heparin and no pulmonary flush were administered. In group [R], lungs were flushed with Perfadex in a retrograde way before explantation. In group [H], heparin (300 IU/kg) was administered 10 min after cardiac arrest followed by closed chest massage for 2 min. In the combined group, animals were heparinized and the lungs were explanted after retrograde flush [HR]. The left lung was assessed for 60 min in an ex vivo reperfusion model. ResultsPulmonary vascular resistance at 50 and 55 min was significantly lower in [R] and [HR] groups compared with [C] and [H] groups (P < 0.01 and P < 0.001) and at 60 min in [R], [H], and [HR] groups compared with [C] group (P < 0.001). Oxygenation, compliance, and plateau airway pressure were more stable in [R] and [HR] groups. Plateau airway pressure was significantly lower in [R] group compared with the [H] group at 60 min (P < 0.05). No significant differences in wet–dry weight ratio were observed between the groups. ConclusionsThis study suggests that preharvest retrograde flush is more protective than postmortem heparinization to prevent reperfusion injury in lungs recovered from donation after circulatory death donors.

11 INFLUENCE OF SPERM STIMULANTS ON EQUINE EPIDIDYMAL SPERM APOPTOSIS

Alterations to a sperm cell that are similar to apoptosis reduce the sperm cell longevity in the female reproductive tract. The aim of the present study was to evaluate the effect of sperm motility factors [caffeine, heparin, and penicillamine, hypotaurine, and epinephrine (PHE)] on phosphatidylserine translocation in equine epididymal semen. Equine epididymal samples were obtained from 10 stallions using retrograde flush with 40 mL of BotuSemen® (BS) per epididymal cauda. The samples were divided into 3 groups: BS (control), Fert-TALP (Fert), and Sperm-TALP (SP), and diluted 1 : 1 with these media. Samples containing 100 × 106 sperm cells were loaded into straws, and then incubated at room temperature (25°C) for 15 min. The samples were centrifuged (1000g for 10 min), the supernatant was removed and the pellets were resuspended in BotuCrio®. Samples were loaded into 0.5-mL straws and cooled at 5°C for 20 min. Then, they were frozen in nitrogen vapor (6 cm above the level of LN) for an additional 20 min and finally plunged into nitrogen and stored. The straws were thawed at 46°C/20 min. Sperm motility was evaluated by computer-assisted semen analysis (CASA; HTM-IVOS 12). To evaluate the pancreatic secretory trypsin inhibitor (PSTI), the Annexin V-FITC Apoptosis Detection Kit (6710KK; Pharmingen, San Jose, CA, USA) was used according to the manufacturer’s recommendations. The samples were diluted in Annexin V buffer solution to a concentration of 2 × 106 spermatozoa mL–1 and then 5 mL of Annexin V-FITC, 5 mL of propidium iodide (PI, 50 mg mL–1), and 2 mL of Hoechst 33342 dye (H342, 40 mg mL–1) were added. The samples were then homogenized and incubated for 15 min. Then, 400 µL of Annexin V buffer solution was added to obtain a final concentration of 1 × 106 spermatozoa mL–1. The samples were evaluated. Flow cytometry was carried out at BD LSR II (Becton Dickinson, Mountain View, CA, USA). Mean and standard deviation were calculated, and then the normality test was evaluated by the Kolmogorov-Smirnov test. An analysis of variance and Tukey test with a P &lt; 0.05 significance level were used to compare the mean values. Statistical analysis was performed using ANOVA followed by the Tukey test (P &lt; 0.05). There was no significant difference in the percentage of non-translocated viable cells. Total motility (36.2 ± 18.18, 52.3 ± 18.40, and 51.4 ± 22.22) and progressive motility (13.8 ± 9.27, 27.7 ± 13.48, and 28.1 ± 15.29) were assessed for BS, Fert, and SP samples, respectively. Mean values of phosphatidylserine translocation index (±SD) of dead cells (8.40 ± 5.1, 6.12 ± 3.9, and 18.6 ± 22.3), viable cells with translocation of phospholipids (61.5 ± 21.3, 63.9 ± 20.6, and 52.2 ± 27.2), viable (28.3 ± 13.6, 28.7 ± 13.7, and 27.7 ± 15.4), and dead cells with translocation of phospholipids (1.8 ± 1.1, 1.2 ± 1.9, and 1.4 ± 2.6) were registered for BS, SP, and Fert, respectively. No difference was observed among incubation media. The results of the present experiment reinforce the idea that the incubation of equine epididymal semen with either SP or Fert medium has a beneficial effect on sperm parameters without being deleterious to sperm fertility.

226 CRYOPRESERVED EJACULATED AND EPIDIDYMAL SPERM COLLECTED FROM THE SAME HOLSTEIN BULLS USED FOR IN VITRO FERTILIZATION

Exposure to seminal plasma may modify the ability of sperm to survive cryopreservation, undergo capacitation, and fertilize oocytes. The present work was designed to compare embryo development after IVF of oocytes with ejaculated and epididymal bovine sperm from bulls previously tested and showing similar responses to freezing. However, we also found that this ejaculated and epididymal sperm differed in their in vitro culture dynamics (capacitation, viability, and auto-acrosome reaction) and ability to fertilize oocytes in vitro. Ejaculated and epididymal sperm were collected from the same fertile mature Holstein bulls (n = 4) by artificial vagina and post-castration retrograde caudal epididymal flush, respectively. Collection of epididymal sperm was conducted 2 weeks after the last collection of ejaculated sperm. After collection, ejaculated and epididymal sperm were cryopreserved and stored in LN until use. Before IVF, a viable sperm population was isolated by centrifugation through a Percoll density gradient. Ejaculated and epididymal sperm were then added to fertilization drops at a final concentration of 1 × 106 mL–1, and IVF was conducted with and without the capacitation agent heparin. Oocytes were washed and randomly assigned to one of four treatment groups (ejaculated ± heparin or epididymal ± heparin). Embryo development was determined at 72 and 186 h after IVF. Differences in the mean values among treatment groups were analysed by one-way ANOVA, followed by the Holm-Sidak pairwise multiple comparisons. Embryo cleavage after IVF using ejaculated sperm without heparin (45.2%) was significantly lower (P &lt; 0.05) than in all other groups. Cleavage rates of ejaculated sperm with heparin (56.9%) and epididymal sperm with (58.1%) and without (57.5%) heparin were found to be similar. No difference was noted between ejaculated and epididymal sperm in blastocyst development, although the inclusion of heparin did significantly (P &lt; 0.01) increase blastocyst development in both ejaculated (9.3 compared with 23.6%) and epididymal sperm (2.5 compared with 23.3%). In conclusion, cryopreserved ejaculated and epididymal sperm collected from the same bulls can be successfully used for the in vitro production of bovine blastocysts without changing the existing protocols. This may increase the efficiency when using epididymal sperm in assisted reproductive techniques.

154 CRYO-ACROSOME REACTION/CAPACITATION OF EJACULATED AND EPIDIDYMAL BOVINE SPERM AND THEIR IN VITRO FERTILIZATION RATES

Seminal plasma has been shown to affect the composition and function of sperm. This exposure may alter the ability of sperm to endure cryopreservation, undergo capacitation and fertilize oocytes. Earlier studies demonstrated that the freezing response of epididymal and ejaculated sperm from the bulls utilised in these studies was similar (Alapati et al. 2009). Subsequent studies are designed to investigate the response of ejaculated and epididymal bovine sperm to cryopreservation and their ability to undergo capacitation. Ejaculated and epididymal sperm from the same bulls (n = 4) were collected by an artificial vagina and retrograde caudal epididymal flush, respectively and were cryopreserved in standard egg-yolk glycerol extender. Sperm was compared by level of cryo-acrosome reaction/capacitation and their fertilization rates in vitro with and without the capacitating agent heparin. Upon evaluation, a sample was thawed and washed and the viable sperm population was isolated through a discontinuous Percoll® gradient. Sperm viability and acrosome status were assessed by fluorescent staining with propidium iodine and fluorescein isothiocyanate-PNA followed by evaluation with flow cytometry. Capacitated sperm were then induced to undergo the acrosome reaction by exposure to lysophosphatidylcholine (10 μg mL–1). Cryo-capacitation was determined by the difference between cryo- and lysophosphatidylcholine-induced acrosome-reacted sperm levels. Differences in the mean values between groups were analysed by ANOVA. Ejaculated and epididymal sperm differed by level of cryo-acrosome reaction (15 vs 4%, respectively; P &lt; 0.001), but did not differ by level of cryo-capacitation (6.2 vs 5.9%, respectively; P = 0.92). The ability of epididymal and ejaculated sperm to fertilize oocytes in vitro with and without heparin was also investigated. Sperm were thawed and washed and the viable population was isolated through a Percoll® gradient. Oocytes were washed and randomly assigned to a treatment group, either ejaculated ± heparin or epididymal ± heparin. Oocytes and sperm were added to a capacitating medium (TALP) with and without heparin. Following 18 h of incubation, fertilization rate was determined by pronuclear fluorescent staining with Hoechst 33342 followed by confirmation with aceto-orcein staining. Differences in the mean values among treatment groups were analysed by one-way ANOVA followed by Holm-Sidak pairwise multiple comparisons. Fertilization rates of epididymal sperm with and without heparin and ejaculated sperm with heparin were similar (76, 80 and 70%, respectively). Ejaculated sperm without heparin was lower than all other groups, with a fertilization rate of 42% (P &lt; 0.001). In summary, epididymal sperm displayed lower levels of cryo-acrosome reaction but were similar by level of cryo-capacitation. In addition, epididymal and ejaculated sperm differed in the need for a capacitating agent such as heparin when used in vitro. This may be useful to increase efficiency when using epididymal sperm in assisted reproductive techniques.

Collection and evaluation of epididymal sperm in captive agoutis ( Dasyprocta aguti)

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1–2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 μL, pH 6.9, sperm concentration 748 x 10 6 sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 μm long and 3.13 ± 0.35 μm wide, with an area of 13.01 ± 2.01 μm 2. Midpieces were 5.33 ±0.44 μm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 μm, and overall sperm length was 40.12 ± 2.44 μm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species.

505: Retrograde Flush Is More Effective Than Heparinization in the Uncontrolled Non-Heart-Beating Donor

505: Retrograde Flush Is More Effective Than Heparinization in the Uncontrolled Non-Heart-Beating Donor

Retrograde Arterial Flush of the Liver Graft in Living Donor Liver Transplantation

ABSTRACT Formal hepatic arterial flush to preserve the liver graft in living donor liver transplantation (LDLT) is not recommended by most transplant centers because direct cannulation may injure the intima of the hepatic artery. The authors describe a method of retrograde arterial flush of the liver graft without arterial cannulation by hepatic venous outflow occlusion (HVOO) in LDLT. First, we proved no backflow of the hepatic artery without HVOO by portal flush to pig livers. Then we used HVOO on 15 LDLT cases (Group HVOO). The results were compared with those of 24 counterpart LDLT cases (Group non-HVOO) without hepatic artery flush. The two-week posttransplantational liver functions were not different between two groups except that the day-three and day-seven serum bilirubin levels were lower in Group HVOO (day-three total bilirubin: 4.99 ± 4.04 mg/dl versus 7.65 ± 4.33 mg/dl, p =.016; day-seven total bilirubin: 5.06 ± 5.02 mg/dl versus 9.57 ± 6.09, p =.005). The rates of vascular complications, six-month graft survival, and biliary anastomotic stricture in Group HVOO were 0, 93.3, and 13.3% respectively, which were not different from those of Group non-HVOO. In summary, to avoid intima injury, the retrograde arterial flush of liver graft by HVOO is safe in LDLT. The short-term results showed the effect of decreasing early functional cholestasis but the long-term benefits need further evaluation, especially with regards to biliary anastomotic complications.

Late Retrograde Perfusion of Donor Lungs Does Not Decrease the Severity of Primary Graft Dysfunction

The ideal preservation strategy has yet to be established in lung transplantation. This clinical study compares primary graft dysfunction using antegrade and retrograde perfusion of donor lungs. Over a 6-year period, 153 consecutive patients underwent lung transplantation in our institution. Group I consists of 65 patients who received lungs preserved with an antegrade flush of modified Euro-Collins solution. Group II includes 65 patients who received lungs preserved with an antegrade flush of low-potassium dextran (LPD) solution. Group III consists of 23 patients who received lungs preserved with an antegrade and a preimplantation retrograde flush of LPD solution. Endpoints evaluated were the following: acute lung injury (ALI) score, time to achieve a fraction of inspired oxygen (Fio2) of 40% and a positive end-expiratory pressure (PEEP) of 5, length of ventilation, length of intensive care unit (ICU) stay, 90-day operative mortality, and patient survival rates. The patient demographic data, underlying diagnosis, number of single and double lung transplants, use of cardiopulmonary bypass, and mean ischemic times were similar in all 3 groups. The mean ALI score (6.2, 5.8, and 6.0) and the median length of ventilation (23.5, 24.0, and 27.0 hours) in groups I, II, and III, respectively, were not significantly different. The length of ICU stay, the 90-day operative mortality, and the survival rates were not significantly different in the 3 groups. Our results suggest that late retrograde perfusion of donor lungs does not decrease the severity of primary graft dysfunction when compared with standard antegrade techniques.