A dose-escalation ex vivo study on the effects of intracameral benzalkonium chloride in rabbits.

BACKGROUND: Rabbits are currently not a good model for studying diseases of the corneal endothelium because their corneal endothelial cells (CECs) maintain a high proliferative capacity throughout almost all their life. Addressing this particular feature might allow the use of this species for such a purpose. The aim of this study was to evaluate the corneal endothelial injury after intracameral benzalkonium chloride (BAC) injection into rabbit eyes ex vivo, and to establish the most suitable starting dose for an in vivo study aimed at developing an animal model of corneal endothelial disease. RESULTS: Forty rabbit eyes obtained postmortem by transconjunctival enucleation were divided into 8 groups according to the injected compound: Control (no injection), BSS, and increasing BAC concentrations (0.005%, 0.01%, 0.025%, 0.05%, 0.1% and 0.2%). At 0, 6, 24 and 48 h, ophthalmologic examination of the anterior segment, pachymetry and specular microscopy were performed, and corneas were finally vital-stained and observed under the light microscope to assess the CECs morphology and mortality rate. When compared to BSS, CECs density started to decrease significantly at 0.025% BAC concentration, while mean cell area, corneal edema and corneal thickness began to increase significantly at 0.05%, 0.005% and 0.1% BAC concentrations, respectively. Concentrations of 0.05% BAC and above caused significant increases in CECs pleomorphism (decreased hexagonality) and mortality, compared to control and BSS. CONCLUSIONS: Ex vivo intracameral BAC injection induces corneal endothelial toxicity in rabbits. However, confirmatory in vivo studies are required to develop the desired model, with 0.05% BAC being a suggested starting point.

Rapid Prototyping of a Cyclic Olefin Copolymer Microfluidic Device for Automated Oocyte Culturing.

Assisted reproductive technology (ART) can benefit from the features of microfluidic technologies, such as the automation of time-consuming labor-intensive procedures, the possibility to mimic in vivo environments, and the miniaturization of the required equipment. To date, most of the proposed approaches are based on polydimethylsiloxane (PDMS) as platform substrate material due to its widespread use in academia, despite certain disadvantages, such as the elevated cost of mass production. Herein, we present a rapid fabrication process for a cyclic olefin copolymer (COC) monolithic microfluidic device combining hot embossing-using a low-temperature cofired ceramic (LTCC) master-and micromilling. The microfluidic device was suitable for trapping and maturation of bovine oocytes, which were further studied to determine their ability to be fertilized. Furthermore, another COC microfluidic device was fabricated to store sperm and assess its quality parameters over time. The study herein presented demonstrates a good biocompatibility of the COC when working with gametes, and it exhibits certain advantages, such as the nonabsorption of small molecules, gas impermeability, and low fabrication costs, all at the prototyping and mass production scale, thus taking a step further toward fully automated microfluidic devices in ART.

Rapid Prototyping of a Cyclic Olefin Copolymer Microfluidic Device for Automated Oocyte Culturing.

Assisted reproductive technology (ART) can benefit from the features of microfluidic technologies, such as the automation of time-consuming labor-intensive procedures, the possibility to mimic in vivo environments, and the miniaturization of the required equipment. To date, most of the proposed approaches are based on polydimethylsiloxane (PDMS) as platform substrate material due to its widespread use in academia, despite certain disadvantages, such as the elevated cost of mass production. Herein, we present a rapid fabrication process for a cyclic olefin copolymer (COC) monolithic microfluidic device combining hot embossing-using a low-temperature cofired ceramic (LTCC) master-and micromilling. The microfluidic device was suitable for trapping and maturation of bovine oocytes, which were further studied to determine their ability to be fertilized. Furthermore, another COC microfluidic device was fabricated to store sperm and assess its quality parameters over time. The study herein presented demonstrates a good biocompatibility of the COC when working with gametes, and it exhibits certain advantages, such as the nonabsorption of small molecules, gas impermeability, and low fabrication costs, all at the prototyping and mass production scale, thus taking a step further toward fully automated microfluidic devices in ART.

Aggressive behavior and hair cortisol levels in captive Dorcas gazelles (Gazella dorcas) as animal-based welfare indicators.

Ensuring welfare in captive wild animal populations is important not only for ethical and legal reasons, but also to maintain healthy individuals and populations. An increased level of social behaviors such as aggression can reduce welfare by causing physical damage and chronic stress to animals. Recently, cortisol in hair has been advanced as a non-invasive indicator to quantify long-lasting stress in many species. The sensitivity of social behavior and hair cortisol concentration was evaluated in several groups of dorcas gazelles (Gazella dorcas). Four different groups of gazelles from three different zoos were observed and the expression of intra-specific affiliative and negative social behaviors was assessed across the different groups. Hair samples were taken from sub-groups of animals and analyzed for cortisol concentrations. Significant differences between groups of dorcas gazelles were found in frequency of negative social behavior and hair cortisol concentration. Despite the low sample size, these two parameters had a positive Spearman correlation coefficient (rs = +0.80, P = 0.20). These results suggest that hair cortisol levels are sensitive to differences in the social structure of dorcas gazelles. Zoo Biol. 35:467-473, 2016. © 2016 Wiley Periodicals, Inc.

Heat stress has an effect on motility and metabolic activity of rabbit spermatozoa.

In the warm months the function of the spermatozoa can be affected by the temperature of the reproductive tract of the female exposed to hyperthermic conditions. The aim of this study was to evaluate the impact of heat stress on sperm parameters in an in vitro model and to determine if there were seasonal effects on sperm heat tolerance. Sperm samples from 32 New Zealand White rabbits were collected in two seasons and incubated at scrotal (32.5°C), body (37°C) or hyperthermic (42°C) temperatures for 3h. Sperm viability and morphology were evaluated using nigrosin-eosin staining. Motility and metabolic activity parameters were determined using computer-assisted sperm analysis and the QBlue cell viability test, respectively. The incubation of spermatozoa at 42°C decreased (P<0.05) the mean values of total motility, curvilinear (VCL) and mean velocity (VAP) as well as the metabolic activity with respect to the incubation at 32.5°C and 37°C. No seasonal effects were observed except for the highest percentages of bent and coiled tails in the cold season, and the highest mean values of VCL, linear velocity and VAP in the warm season (P<0.01). The interaction between in vitro heat stress and season was significant for metabolic activity (P=0.02). Our results suggest that rabbit spermatozoa parameters are largely modified by a short exposure to hyperthermic conditions, in terms of metabolic activity and motility parameters. Thus, a short exposure of spermatozoa to an environment of 42°C in temperature for only 3h may compromise sperm functionality. Additionally, sperm metabolic activity is influenced by season.

Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit.

The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

Daily exposure to summer circadian cycles affects spermatogenesis, but not fertility in an in vivo rabbit model.

Heat stress (HS) in mammals is a determining factor in the deterioration of spermatogenesis and can cause infertility. The aim of this study was to evaluate the effect of continuous summer circadian cycles on semen production, sperm cell features, fertility, prolificacy, and fecal cortisol metabolites from rabbits kept under an in vivo HS model. We split randomly 60 New Zealand White rabbits into two temperature-controlled rooms: The control group was maintained at comfort temperature (18 °C-22 °C) and an HS group, where the environmental temperature was programmed to increase from 22 °C to 31 °C and be maintained for 3 hours to this temperature at the central part of the day. Fecal cortisol metabolites were assessed to evaluate the stress conditions. Seminal parameters were analyzed. Although animals exposed to HS showed higher values of fecal cortisol metabolites (P = 0.0003), no differences were detected in fertility or prolificacy. Semen samples from HS males showed a significant decrease (P < 0.05) with respect to the controls in the percentage of viable spermatozoa (80.71% vs. 74.21%), and a significant (P ≤ 0.01) increase in the percentage of acrosomic abnormalities (22.57% vs. 36.96%) and tailless spermatozoa (7.91% vs. 12.83). Among motility parameters, no differences were found. This study describes a model of HS simulating a continuous summer daily cycle that allows periods of time to recover as it occurs under natural conditions. Although negative effects have been detected in several sperm parameters, fertility and prolificacy were not affected, suggesting a recovery of the reproductive function when normal conditions are reestablished.