Strategies to improve the flow of admissions and hospital stays: a Delphi study of an adaptation of the Appropriateness Evaluation Protocol (AEP).

INTRODUCTION AND AIM: The Appropriateness Evaluation Protocol (AEP) tool analyzes inappropriate hospital stays and admissions. This study aimed to adapt the AEP questionnaire in order to analyse the appropriateness of hospital admissions and stays in our healthcare reality. METHODS: A study was conducted using the Delphi method in which 15 experts in clinical management and hospital care participated. The initial questionnaire items were taken from the first version of the AEP. In the first round, the participants contributed new items that they considered relevant in our current reality. In rounds 2 and 3, they evaluated 80 items according to their relevance using a Likert scale from 1 to 4 (maximum usefulness). Pursuant to the study's design, AEP items were considered adequate if the mean score according to the experts' evaluation was greater than or equal to 3. RESULTS: The participants defined a total of 19 new items. In the end, 47 items earned a mean score greater than or equal to 3. The resulting modified questionnaire include 17 items in "Reasons for Appropriate Admissions," 5 in "Reasons for Inappropriate Admissions," 15 in "Reasons for Appropriate Hospital Stays," and 10 in "Reasons for Inappropriate Hospital Stays." CONCLUSIONS: The identification according to expert opinion of priority items to determine the appropriateness of admissions and extended stays could be used in the future to help create an instrument to be used in our setting.

Identification of apoptotic bodies in equine semen.

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37 °C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin-V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer-assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.

Sex sorting increases the permeability of the membrane of stallion spermatozoa.

At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity was assessed using the YoPro-1 assay, while plasmalemmal stability and lipid architecture were assessed using Merocyanine 540/SYTOX green and Annexin-V, respectively. Lipid peroxidation was also investigated with the probe Bodipy(581/591)-C11. All assays were performed shortly after collection, after incubation and after sex sorting using FC. In order to characterize potential molecular mechanisms implicated in sperm damage, an apoptosis protein antibody dot plot array analysis was performed before and after sorting. While the percentage of total motile sperm remained unchanged, sex sorting reduced the percentages of progressive motile spermatozoa and of rapid spermatozoa as well as curvilinear velocity (VCL). Sperm membranes responded to sorting with an increase in the percentage of YoPro-1 positive cells, suggesting the sorted spermatozoa had a reduced energy status that was confirmed by measuring intracellular ATP content.

Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage.

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer-assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro-1) and mitochondrial membrane potential (JC-1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL-2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL-2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL-2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro-1 negative) sperm post-thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.

Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential.

Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations ≥ 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.

Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96.

The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 µM of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and membrane integrity (flow cytometry after YoPro-1/Eth staining) were evaluated. In KMT extender sperm motility significantly decreased after 45 min of incubation when sperm were incubated in the presence of concentrations of Hoechst of 45 µM or greater (P<0.05). When incubated in modified INRA96, stallion spermatozoa tolerated greater concentrations of Hoechst, because sperm motility only decreased when incubated in presence of 90 µM (P<0.05) and membrane integrity was not affected. After 90 min of incubation the same effect was observed, but in this case at concentrations over 45 µM the percentage of total motile sperm was also reduced although only in samples incubated in KMT. To produce this effect in samples incubated in Tyrodes modified INRA 96, Hoechst had to be present at concentrations over 67.5 µM. Apparently, the detrimental effect of Hoechst to stallion spermatozoa varies depending on the media, and INRA modified extender may be an alternative to KMT.

Consequences of butylated hydroxytoluene in the freezing extender on post-thaw characteristics of stallion spermatozoa in vitro.

Ejaculates from six pure Spanish stallions were split, and one subsample frozen in a commercial extender supplemented with the lipid soluble antioxidant butylated hydroxytoluene (BHT), while the other subsample served as control. After at least 4 weeks of storage, samples were thawed and post-thaw sperm quality analysed: sperm motility and kinematics using a CASA system, membrane and acrosome integrity and mitochondrial membrane potential using flow cytometry. The outcome of cryopreservation varied significantly among stallions. However, the supplementation with 1 mm BHT had no significant effect on any of the sperm parameters evaluated post-thaw.

Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa.

Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parameters studied the ratio of percentage of C16 plasmalogens related to total phospholipids was the one with the better diagnostic value. For potentially bad freezers, the significant area under the ROC-curve was 0.74, with 75% sensitivity and 79.9% specificity for a cut off value of 26.9. Also the percentage of plasmalogens respect to total phospholipids gave good diagnostic value for bad freezers. On the other hand, the percentage of C18 fatty aldehydes related to total phospholipids of the sperm membrane properly forecasted freezeability with an area under the ROC curve of 0.70 with 70% sensitivity and 62.5% specificity for a cut off value of 0.32.

Effect of cryopreservation on nitric oxide production by stallion spermatozoa.

The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an apparent molecular mass of approximately 83 kDa. On the other hand, the Ab developed against the NOS3 showed a band of approximately 96 kDa in fresh and FT sperm lysates. NO production was positively correlated with sperm motility and velocity after thaw, suggesting an NO role for the functionality of cryopreserved stallion spermatozoa; but the production of NO is compromised in egg yolk-containing extenders.