l-carnitine enhances the kinematics and protects the sperm membranes of chilled and frozen-thawed Peruvian Paso horse spermatozoa.

l-carnitine (LC) transports fatty acids to the mitochondria for energy production, reducing lipid availability for peroxidation through ß-oxidation. This research examines the effect of LC supplementation to two skimmed milk-based extenders on the cryosurvival of chilled (5°C) and frozen-thawed Peruvian Paso horse spermatozoa .An initial experiment determined the optimal LC concentration (0, 1, 5, 10, 25, and 50 mM) when added to INRA-96® and UHT (skimmed milk + 6% egg yolk) extenders, using nine ejaculates from three stallions chilled for up to 96 h. Subsequently, the effect of 25 mM LC supplementation (the optimal concentration) on chilling (INRA-96) and freezing (INRA-Freeze®) extenders was evaluated using eight pooled samples from sixteen ejaculates (2 ejaculates/pool) from four stallions. Results indicated that all LC concentrations produced significantly higher values (P<0.05) for kinematic variables (total [TM] and progressive motilities, curvilinear [VCL] and straight-line [VSL] velocity, and beat-cross frequency [BCF]), and the integrity of plasma/acrosome membranes (IPIA) compared to non-supplemented chilled sperm samples for up to 96 h with both extenders. Moreover, the use of 25 mM LC was more efficient (P<0.05) in preserving the post-chilled values of velocity, BCF, and IPIA for the long term than lower LC concentrations (1-10 mM). Post-thaw values of total motility, the amplitude of lateral head displacement (ALH), and IPIA were significantly improved (P<0.05) when INRA-Freeze extender was supplemented with 25 mM LC. In conclusion, supplementation of l-carnitine to skimmed milk-based extenders enhanced kinematic variables and protected the membrane integrity in chilled and frozen-thawed Peruvian Paso horse spermatozoa.

Effect of Melatonin and Caffeine Supplementation to Freezing Medium on Cryosurvival of Peruvian Paso Horse Sperm Using a Two-Step Accelerating Cooling Rate.

This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96®, and finally frozen with INRA-Freeze® with either no supplementation (as control), 1 µM ME, or 2 mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution® 2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater (p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences (p < 0.01) between stallions (1-4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.

EUropean prospective cohort study on Enterobacteriaceae showing REsistance to CArbapenems (EURECA): a protocol of a European multicentre observational study.

INTRODUCTION: The rapid worldwide spread of carbapenem-resistant Enterobacteriaceae (CRE) constitutes a major challenge. The aim of the EUropean prospective cohort study on Enterobacteriaceae showing REsistance to CArbapenems (EURECA), which is part of the Innovative Medicines Initiative Joint Undertaking (IMI JU) funded COMBACTE-CARE project, is to investigate risk factors for and outcome determinants of CRE infections to inform randomised clinical trial designs and to provide a historical cohort that could eventually be used for future comparisons with new drugs targeting CRE. METHODS: A multicentre (50 sites), multinational (11 European countries), analytical observational project was designed, comprising 3 studies. The aims of study 1 (a prospective cohort study) include characterising the features, clinical management and outcomes of hospitalised patients with intra-abdominal infection, pneumonia, complicated urinary tract infections and bloodstream infections caused by CRE (202 patients in each group). The main outcomes will be 30-day all-cause mortality and clinical response. Study 2 (a nested case-control study) will identify the risk factors for target infections caused by CRE; 248 selected patients from study 1 will be matched with patients with carbapenem-susceptible Enterobacteriaceae (1:1) and with hospitalised patients (1:3) and will provide a historical cohort of patients with CRE infections. Study 3 (a matched cohort study) will follow patients in study 2 in order to assess mortality, length of stay and hospital costs associated with CRE. All patients will be followed for 30 days. Different, up-to-date statistical methods will be applied to come to unbiased estimates for all 3 studies. ETHICS AND DISSEMINATION: Before-study sites will be initiated, approval will be sought from appropriate regulatory agencies and local Ethics Committees of Research or Institutional Review Boards (IRBs) to conduct the study in accordance with regulatory requirements. This is an observational study and therefore no intervention in the diagnosis, management or treatment of the patients will be required on behalf of the investigation. Any formal presentation or publication of data collected from this study will be considered as a joint publication by the participating physician(s) and will follow the recommendations of the International Committee of Medical Journal Editors (ICMJE) for authorship. TRIAL REGISTRATION NUMBER: NCT02709408.