A comparative study of canine epididymal sperm collection techniques and cryopreservation.

Introduction: An optimized collection method and freezing protocol for preservation of epididymal spermatozoa remains a topic of interest to many scientists. The current study focused on the collection and preservation of canine epididymal spermatozoa. During the process of collection of canine epididymal spermatozoa, blood content can occur, which may affect sperm cryopreservation in a negative way. Here, we compared first two epididymal sperm collection techniques [epididymal mincing (EM) and single incision epididymal sperm aspiration (SESA)]; and next we tried to solve the issue of blood content using an erythrocyte lysis buffer (ELB). Methods: Hence spermatozoa were collected after weighing the epididymides, either by EM or SESA, and sperm quality assessed prior to and post freezing (concentration, total sperm output (TSO), motility, viability and morphology). Next, new sperm samples were collected from eight epididymides by EM and subjected either to a standard freezing protocol or to an ELB treatment freezing protocol. Post-thaw sperm parameters (concentration, TSO, motility, viability and morphology), including intracellular reactive oxygen species (ROS) and lipid peroxidation were assessed. The correlation between the weight of the epididymis and the TSO was evaluated based on the collection technique, and differences in sperm parameters were detected both within different collection techniques and between different pre-freezing treatment protocols. Results: There was a very strong correlation between the weight of the epididymis and the TSO for the EM technique (p = 0.002, R2 = 0.6), along with an increased sperm motility with EM compared to SESA (median 80%, inter-quartile range (IQR) 88-65 and median 67.5%, IQR 72.5-52.5, respectively; (p = 0.002). Post-thaw samples subjected to ELB treatment freezing protocol had lower motility and higher intracellular ROS compared to the standard freezing protocol (motility: median 56.25%, IQR 60-48.75 and median 70%, IQR 72.5-63, respectively; p = 0.01; ROS: median 78.5%, IQR 81.25-75.5 and median 70%, IQR 70.5-68.75, respectively; (p = 0.04). Discussion: The results indicated that EM is a better technique to harvest epididymal spermatozoa despite the presence of some blood content. Furthermore, the ELB treatment should not be implemented to remove those red blood cells prior to cryopreservation of epididymal spermatozoa in dogs.

Canine and Feline Epididymal Semen-A Plentiful Source of Gametes.

Canine and feline epididymal semen provide an additional source of gametes to preserve the genetics of valuable breeding dogs and tomcats, especially for those that fail to ejaculate, need castration as a therapy or die unexpectedly. Moreover, since it is quite common to perform castration of non-breeding dogs and cats, the development of a gene bank of epididymal semen collected after castration would greatly contribute to increase the genetic diversity in dogs and cats. Collection and cryopreservation of epididymal semen necessitates a full understanding of the function of the epididymis and of the characteristics of epididymal spermatozoa as opposed to ejaculated semen. During collection of epididymal semen, specific factors may have a negative effect on epididymal semen quality and freezability. Accordingly, the elimination of these triggers could enhance epididymal semen freezability and consequently positively influence post-thaw semen quality and outcome for different ARTs.

Performance of the Parasympathetic Tone Activity (PTA) index to predict changes in mean arterial pressure in anaesthetized horses with different health conditions.

The parasympathetic tone activity (PTA) index is based on heart rate variability and has been developed recently in animals to assess their relative parasympathetic tone. This study aimed to evaluate PTA index in anaesthetized horses with different health conditions and the performance of PTA variations (∆PTA) to predict changes in mean arterial pressure (MAP). Thirty-nine client-horses were anaesthetized for elective or colic surgery and divided into "Elective" and "Colic" groups. During anaesthesia, dobutamine was administered as treatment of hypotension (MAP <60 mmHg). In both groups, no significant variation of PTA and MAP were detected immediately before and after cutaneous incision. The PTA index increased 5 min before each hypotension, whereas it decreased 1 min after dobutamine administration. Horses of the Colic group had lower PTA values than those of the Elective group, whereas MAP did not differ between groups. To predict a 10% decrease in MAP, ΔPTA performance was associated with: AUC ROC [95% CI] =0.80 [0.73 to 0.85] (p < 0.0001), with a sensitivity of 62.5% and a specificity of 94.6% for a threshold value of 25%. The PTA index in anaesthetized horses appears to be influenced by the health condition. The shift toward lower PTA values in colic horses may reflect a sympathetic predominance. An increase in PTA of >25% in 1 min showed an acceptable performance to predict MAP decrease of >10% within 5 min. Even though these results require further evaluation, this index may thus help to predict potential autonomic dysfunctions in sick animals.

Evaluation of the sublingual microcirculation with sidestream dark field video microscopy in horses anesthetized for an elective procedure or intestinal surgery.

OBJECTIVE: To compare the sublingual microcirculation between healthy horses anesthetized for elective procedures and horses with colic anesthetized for abdominal surgery and to determine the effect of mean arterial blood pressure (MAP) on the microcirculation. ANIMALS: 9 horses in the elective group and 8 horses in the colic group. PROCEDURES: Sublingual microcirculation was assessed with sidestream dark field video microscopy. Videos were captured at 3 time points during anesthesia. Recorded microvasculature parameters were De Backer score (DBS), total density of perfused vessels (PVD) and small vessels (PVD-S), total proportion of perfused vessels (PPV) and small vessels (PPV-S), vascular flow index (MFI), and heterogeneity index (HI). Blood pressure during hypotensive (MAP < 60 mm Hg) and normotensive (MAP ≥ 60 mm Hg) episodes was also recorded. RESULTS: During normotensive episodes, the elective group had significantly better PPV and PPV-S versus the colic group (median PPV, 76% vs 50%; median PPV-S, 73% vs 51%). In both groups, PPV decreased during anesthesia (elective group, -29%; colic group, -16%) but significantly improved in the elective group 15 minutes before the end of anesthesia (59%). During hypotensive episodes, PVD-S was better preserved in the colic group (11.1 vs 3.8 mm/mm2). No differences were identified for the microcirculatory parameters between normo- and hypotensive episodes in the colic group. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual microcirculation was better preserved in healthy horses anesthetized for elective procedures than in horses with colic anesthetized for abdominal surgery despite resuscitation maneuvers. Results indicated that the macrocirculation and microcirculation in critically ill horses may be independent.

Performance of the Parasympathetic Tone Activity (PTA) index to assess the intraoperative nociception using different premedication drugs in anaesthetised dogs.

The dynamic variations of the Parasympathetic Tone Activity (PTA) index were evaluated to assess nociception in dogs undergoing anaesthetic protocols with different premedication drugs. Sixty-six dogs, divided into three groups of 22 dogs each, were given different premedication drugs "morphine" (0.2 mg/kg), "morphine + medetomidine (5 µg/kg)", "morphine + acepromazine (0.03 mg/kg)", then similarly induced and maintained under general anaesthesia. The PTA, HR (heart rate) and MAP (mean arterial pressure) were assessed at S (steady-state), Cut (cutaneous incision), PTAE (PTA Event, assessed 1 min before a significant PTA decrease), HDR (Haemodynamic reaction, assessed 5 min before an increase of ≥ 20% in HR and/or MAP). For each group, the dynamic variation of PTA (ΔPTA) was calculated and a Receiver Operating Characteristics (ROC) curve was performed to detect if any of the premedication drugs may alter the performance of PTA index to predict intraoperative haemodynamic reactions. In all groups, a haemodynamic reaction was detected after Cut, PTAE and HDR and was preceded with a significant decrease of PTA, except for "morphine + medetomidine" group which showed a significant drop of PTA only at PTAE and HDR. The ΔPTA showed a fair performance in all groups (a mean [95% CI] AUC of 0.73 [0.62-0.82], 0.70 [0.59-0.79] and 0.71 [0.59-0.80] respectively in morphine, morphine + ACP and morphine + medetomidine). Although ΔPTA was rather altered by the premedication drug, it was able to predict, to a certain extent, haemodynamic reactions in all groups.

Evaluation of the Parasympathetic Tone Activity (PTA) index to assess the analgesia/nociception balance in anaesthetised dogs.

The Parasympathetic Tone Activity (PTA) is an index based on the analysis of heart rate variability that has been recently developed to assess the analgesia/nociception balance in anaesthetised animals. The present study aimed to evaluate its performance in dogs undergoing surgery. Thirty dogs admitted for elective surgeries, were anaesthetised with a standardised protocol. PTA, heart rate (HR), systolic blood pressure (SBP) and HDR (defined as an increase by >20% in HR and/or SBP within 5min) were assessed at the following predefined time-points: TStSt (steady-state, after induction of anaesthesia and before start of surgery), TClamp (clamping of surgical drapes on the skin), TCut (cutaneous incision), TPrePTA (retrospectively assessed 1min before a PTA decline of at least 20%) and TEndIso (isoflurane discontinuation). The dynamic variation of PTA over 1min (∆PTA) was calculated at each predefined time-points and its performance to predict HDR was assessed by building Receiver Operating Characteristics (ROC) curves. A significant decrease of PTA (p<0.002) was detected 1min after TClamp, TCut and TPrePTA followed by a significant increase in HR and/or SBP within 5min after the time points (p<0.01). The ΔPTA was associated with the following performance in predicting HDR: AUC ROC [95% CI]=0.80 [0.71 to 0.88] (p<0.05), with a sensitivity of 77% and a specificity of 72% for a threshold value of -18%. Although encouraging, the performance of the PTA index and its dynamic variation needs to be further evaluated, particularly in different clinical contexts.

The KUPKB: a novel Web application to access multiomics data on kidney disease.

The information gathered from the large number of omics experiments in renal biology is underexplored, as it is scattered over many publications or held in supplemental data. To address this, we have developed an open-source Kidney and Urinary Pathway Knowledge Base (KUPKB) that facilitates simple exploration of these omics data. The KUPKB currently comprises 220 data sets (miRNA, mRNA, proteins, and metabolites) extracted from existing publications or databases. Researchers can explore the integrated data using the iKUP browser, and a simple template is provided to submit new omics data sets to the knowledge base. As an example of iKUP's use, we show how we identified, in silico, calreticulin as a protein induced in human interstitial fibrosis and tubular atrophy (IFTA) in chronic kidney transplant rejection; a link that would have been difficult to establish using existing Web-based tools. Using immunohistochemistry, we validated in vivo this in silico result in human and rat biopsies of IFTA, thus identifying calreticulin as a potential new player in chronic kidney transplant rejection. The KUPKB provides a simple tool that enables users to quickly survey a wide range of omics data sets and has been shown to facilitate rapid hypothesis generation in the context of renal pathophysiology.

Pargyline reduces renal damage associated with ischaemia-reperfusion and cyclosporin.

BACKGROUND: The slow deterioration of the kidney graft is characterized histologically by interstitial fibrosis and tubular atrophy (IFTA). Immunological and non-immunological stress is the main cause of progression towards IFTA. Our study focused on the non-immunological injuries induced by ischaemia-reperfusion (IR) and cyclosporin (CsA) toxicity, which remain the two stress factors putting a damper on the outcome of the renal graft. Endogenous reactive oxygen species (ROS) are essentially produced by mitochondria, and we have previously shown that the blockage of the mitochondrial enzymes monoamine oxidases (MAOs) prevents H2O2 production in the early reperfusion stage following IR. METHODS: We used a rat model of IFTA consisting in unilateral nephrectomy followed by IR and daily CsA administration. Four weeks after IR, we analysed renal function, histological alterations and a number of inflammatory and fibrotic genes. RESULTS: We observed, 28 days after pargyline-mediated blockade of MAO (before or after IR), improved renal function as well as a net decrease in renal inflammation associated to lower IL-1ß and TNF-α gene expression. However, significant reduction in apoptosis, necrosis and fibrosis was only observed when pargyline was administrated before IR. This protective effect was associated to decreased expression of TGF-ß1, collagen types I, III and IV and also to the normalization of antioxidant (SOD1, catalase) and inflammatory (COX2, LOX5) gene expression. CONCLUSION: It appears that the blockage of ROS produced by MAO and subsequent cell death might be an effective protective strategy against IFTA progression.

Congenital ureteropelvic junction obstruction: human disease and animal models.

Ureteropelvic junction (UPJ) obstruction is the most frequently observed cause of obstructive nephropathy in children. Neonatal and foetal animal models have been developed that mimic closely what is observed in human disease. The purpose of this review is to discuss how obstructive nephropathy alters kidney histology and function and describe the molecular mechanisms involved in the progression of the lesions, including inflammation, proliferation/apoptosis, renin-angiotensin system activation and fibrosis, based on both human and animal data. Also we propose that during obstructive nephropathy, hydrodynamic modifications are early inducers of the tubular lesions, which are potentially at the origin of the pathology. Finally, an important observation in animal models is that relief of obstruction during kidney development has important effects on renal function later in adult life. A major short-coming is the absence of data on the impact of UPJ obstruction on long-term adult renal function to elucidate whether these animal data are also valid in humans.

Delayed treatment with plasminogen activator inhibitor-1 decoys reduces tubulointerstitial fibrosis.

We examined the capacity of delayed inhibition of plasminogen activator inhibitor-1 (PAI-1) to reduce tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO) in mice. Small peptides mimicking parts of urokinase (uPA) and tissular plasminogen activator (tPA) and serving as decoy molecules for PAI-1 were administered daily during the late stages (3 to 8 days) of UUO. Treatment with PAI-1 decoys reduced interstitial deposition of fibronectin, collagen III and collagen IV without changes in macrophage and myofibroblast infiltration. Interestingly, while PAI-1 activity was reduced and the combined uPA and tPA activity was increased, the antifibrotic effect was obtained without modification of plasmin activity but with increased of hepatocyte growth factor (HGF) expression. We show for the first time that treatment with small PAI-1 decoy peptides reduces established tubulointerstitial fibrosis. This protective effect probably resulted from increased degradation of the extracellular matrix by an HGF dependent mechanism.