M. Sabés-Alsina

Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents.

The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 μM) and retinyl (RI; 0.282 or 2.82 μg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin-nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

Hair cortisol detection in dairy cattle by using EIA: protocol validation and correlation with faecal cortisol metabolites

Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein-Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R 2=0.98 and mean percentage error of -10.8 ± 1.55%. The extraction efficiency was 89.0 ± 23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.

Daily exposure to summer temperatures affects the motile subpopulation structure of epididymal sperm cells but not male fertility in an in vivo rabbit model

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction.

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.

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.

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.

Feather corticosterone evaluated by ELISA in broilers: A potential tool to evaluate broiler welfare

The measure of corticosterone (CORT) in feathers has been recently recognized as a valid and easily obtainable measure of chronic glucocorticoids secretion in avian species. This measure provides meaningful interpretations of how individuals respond to environmental perturbations. The growing interest of the public toward animal-food production welfare shows the need for improving and expanding objective tools to evaluate this issue. The present study evaluates whether it is possible to detect CORT in broiler feathers, and thus, assess if it would be a useful measure to study broiler welfare. Twenty-two broilers were randomly selected from an intensive farm. Four to 6 dorsal feathers were collected from each bird, and sex, weight, and morphological aspects of feather status were recorded. We tested the feasibility for detecting CORT in broiler feathers by ELISA, which had never been done before, and an assay validation test was performed. No significant relationships were found between feather CORT concentrations and physiological variables such as sex, weight, and fault bars in broilers. To our knowledge, this is the first study that uses broiler feathers as a matrix that provides a retrospective record of their hypothalamic-pituitary-adrenal activity. Results indicate that ELISA is a valid tool to detect feather CORT levels in broilers.

Retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The study tried to confirm the harmful effects of high temperatures on epididymal sperm cells in comparison with other temperatures (scrotal, environmental, and refrigeration temperatures), the main objective was the assessment of the addition of retinol as an antioxidant agent to improve sperm quality parameters. Testes from 10 bulls were collected from a slaughterhouse. Sperm cells were flushed from the cauda epididymis and deferent duct and assessed for sperm quality parameters at recovery. Afterward, sperm cell samples were exposed to one of four different temperatures (4 °C, 22 °C, 32 °C, and 41.5 °C for 3 hours) in presence or absence of retinol in the storage extender. Percentages of viability and morphologic abnormalities were determined using eosin-nigrosin staining. Acrosome integrity and sperm plasma membrane integrity were assessed by fluorescence Pisum sativum agglutinin lectin (FITC-PSA) staining and the hypo-osmotic swelling test, respectively. Total and progressive motility were analyzed by computer-assisted sperm analysis. Sperm quality parameters were mainly affected by high temperatures (41.5 °C). The addition of all-trans-retinol to the storage extender did not show any effect on sperm quality parameters. However, the percentage of sperm cells with altered acrosome was significantly reduced when retinol was present in the extender under heat stress conditions (41.5 °C). In conclusion, retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

Relationship between feather corticosterone and subsequent health status and survival in wild Eurasian Sparrowhawk

AbstractIn birds, integrated levels of corticosterone (CORT) measured in feathers (CORTf) allow us to make inferences on past levels of stress demands. It has been suggested that levels of CORTf track carry-over effects across seasons. Nevertheless, our understanding of how this measure can be used to assess future health status is far from complete. In this study, we aimed to investigate whether CORT deposited in feathers over the moulting period was related to subsequent mortality and health status in wild raptors admitted to rehabilitation centres. Thirty-four Eurasian Sparrowhawks (Accipiter nisus) admitted during the non-moulting period were sampled. Body condition (BC) was used as an indicator of health status to classify individuals’ health as good, poor or cachexia depending on their pectoral muscle score. Mortality was recorded over the non-moulting period. Other potential sources of CORTf variation were assessed, such as sex, age and feather type, primary or body covert feathers. While CORTf did not vary with age or sex, significant differences were found between primary and body feathers, highlighting the importance of sampling the same feather type. Our results also revealed that birds in poor BC showed higher CORTf levels than individuals in good condition; however, CORTf levels in cachectic birds did not differ from those in good condition. This finding suggests caution when assuming that only high CORTf levels represent individuals in poor condition, and limits the utility of CORTf for the prediction of BC. The present study also showed that individuals which died following admission had higher CORTf levels than individuals which survived, suggesting the potential utility of CORTf as a metric for the study of subsequent mortality.ZusammenfassungZusammenhang zwischen Feder-Kortikosteron und späterem Gesundheitszustand und Überleben bei Europäischen Sperbern Der Messung von Kortikosteron in Federn (CORTf) erlaubt Rückschlüsse auf vergangene Stresssituationen. Es werde angenommen, dass über CORTf-Spiegel Carry-over Effekte über die Zeit hinweg verfolgt werden können. Gleichwohl ist unser Verständnis, wie solche Werte zur Abschätzung des zukünftigen Gesundheitszustandes genutzt werden können bei Weitem nicht vollständig. Ziel dieser Studie war zu untersuchen, ob in Federn zur Mauserzeit eingelagertes Kortikosteron (CORT) mit späterer Mortalität und dem Gesundheitszustand von Sperbern, die in Greifvogelauffangstationen eingeliefert wurden, zusammenhängen. Dazu beprobten wir 34 außerhalb der Mauserperiode eingelieferte Europäische Sperber (Accipiter nisus). In Abhängigkeit vom Brustmuskelwert wurde die individuelle Gesundheit als gut, schwach oder abgemagert klassifizieren. Zudem wurde die Sterblichkeit über die Nichtmauserzeit ermittelt. Andere potentielle Quellen für Unterschiede im CORTf wie bspw. Geschlecht, Altern und Federtypen, Großgefieder oder Körperfedern wurden ebenfalls berücksichtigt. Während CORTf nicht mit Alter und Geschlecht variierte, wurden signifikante Unterschiede zwischen Groß- und Körpergefieder gefunden, was die Bedeutung der Beprobung desselben Federtyps hervorhebt. Unsere Ergebnisse zeigen auch, dass Vögel mit schlechter Kondition höhere CORTf Werte zeigten als Individuen in guter Kondition, wobei aber die CORTf Werte abgemagerter Vögel sich nicht von denen in guter Kondition unterschieden. Dieses Ergebnis unterstreicht die nötige Vorsicht, wenn angenommen wird, dass nur hohe CORtf Werte für Individuen mit schwacher Kondition stehen und reduziert gleichzeitig die Nützlichkeit von CORTf zur Vorhersage von Körperkonditionen. Die vorliegende Studie zeigt auch, dass Individuen, die später starben, höhere CORTf Werte hatten als Individuen, die überlebten, was zeigt, dass Federkortikosteron als ein Maß für spätere Mortalität verwendet werden kann.

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.