Short-term tolerance of equine spermatozoa to various abiotic factors.

The aim of this study was to determine the effects of various abiotic factors, such as light, physical stress (pipetting) and thermal shock, on the quality of fresh and cooled equine sperm. In experiment I, four sperm aliquots were subjected to different light exposures: (i) protected control samples (CTRL), (ii) exposed to UV light at 10 cm (UV10), (iii) exposed to UV light at 20 cm (UV20) and (iv) exposed to laboratory lighting (LAB). In experiment II, four semen aliquots were subjected to repeated pipetting for 0, 10, 20 and 30 times (CTRL, P10, P20 and P30, respectively). In experiment III, four semen aliquots at 15°C were subjected to thermal oscillations: (i) cooled control sperm at 15°C (CTRL), (ii) oscillations of 1.9°C/min to a temperature of 30°C (T30), (iii) oscillations of 1.4°C/min, with the temperature rapidly falling until reaching 1.3°C (T0R) and (iv) oscillations of 1.1°C/min, with the temperature slowly falling until reaching 4.2°C (T0S). The results revealed that after 30 min, UV10 and UV20 sperm samples showed significantly (p < .05) lower total and progressive motility values, sperm kinematic parameters and mitochondrial potential. After 45 min of exposure, differences were highly significant (p < .001). No significant differences (p > .05) were found for pipetting or thermal oscillations. The results suggest that, even if equine sperm samples are not handled in the laboratory under optimal conditions, fresh and cooled equine spermatozoa are able to resist the impact of various abiotic stimuli without any reduction in their quality. This study analyses the effect on normospermic samples, but future research could look at the tolerance that asthenozoospermic equine samples have to these abiotic influences.

Effect of trehalose- and sucrose-based extenders on equine sperm quality after vitrification: Preliminary results.

There has been a lack of research into equine sperm vitrification to date, but studies of other species suggest it may have significant potential. To evaluate the impact of various cryoprotectant agents (CPA) and vitrification on equine sperm quality, a controlled study was carried out. A total of 12 ejaculates were subjected to exposure to CPA and vitrification. Sperm was diluted in a range of CPA: fresh, control (BSA), sucrose (0.15M, 0.3M and 0.5M), trehalose (0.15M, 0.3M and 0.5M) and the combination of sucrose and trehalose (M1: 0.15M sucrose+0.5M trehalose; M2: 0.5M sucrose+0.15M trehalose). Sperm motility, viability, acrosome integrity and DNA fragmentation were assessed at the time of CPA exposure and after vitrification. The exposure of spermatozoa to various concentrations of sucrose and/or trehalose significantly reduced sperm motility, with lower concentrations resulting in higher sperm motility. Sperm viability and DNA fragmentation did not vary after exposure to CPA, but acrosome integrity fell significantly when spermatozoa were exposed to CPA with high osmolality. When spermatozoa were vitrified, motility values were significantly higher than those obtained during the exposure. Low concentrations of sucrose (0.15M and 0.3M) and trehalose (0.15M) showed the best progressive sperm motility. The vitrification-warmed procedure significantly reduced sperm viability and acrosome integrity, but DNA did not vary with any of CPA used. Equine sperm vitrification demonstrates a low capacity for preserving sperm motility, and extenders containing trehalose or sucrose at lower concentrations are associated with a better protective effect on sperm motility. After vitrification, acrosome and plasma membranes were severely impaired, while the DNA structure was maintained. Equine spermatozoa partially recover the motility after vitrification, but there is a need for further studies into the preservation of sperm membranes.

Students integrate knowledge acquisition and practical work in the laboratory.

The aim of the present work was to transfer a wider concept of teamwork and self-learning to the laboratory, encouraging students' capabilities when seeking, acquiring, and processing knowledge. This educational innovation was carried out with a total of 38 students (fourth year of degree in Biology) in the area of physiology (Advances in Reproduction course) at University of Córdoba in Córdoba, Spain. The design of the project's application methodology consisted of establishing a way in which problems would be tackled in the practical classes. For this purpose, the different tasks were set up so that students could relate them to the concepts learned in the theory classes. On the first day of class, the project was presented to the students. Groups of two to three students worked in the laboratory and set up an outline of the protocol of the practical work that they had done. This outline was performed individually and sent to the lecturers through a learning management system (Moodle). The teachers gave feedback and assessed student submissions. Upon finishing the course, students completed a survey. The project-based learning method promotes practical self-learning on the part of students. This methodology demonstrated to us that it stimulates a critical and self-critical capacity in students, both individually and in groups, and that writing didactic practical material helped students to enhance their theory knowledge. The experiment was a success in view of the scores obtained upon finishing the subject.

Impact of ultra-rapid freezing on the motility, morphology, viability and acrosome integrity of epididymal cat sperm diluted in sucrose-based extenders.

The objective of the present study was to investigate the influence of different sucrose-based extenders on the motility, morphology, viability and acrosomal integrity of epididymal cat spermatozoa cryopreserved by ultra-rapid freezing method. Nine cats were castrated, and collected semen was diluted 1 : 1 with Dulbecco`s phosphate-buffered saline-BSA1%-based extender supplemented with different sucrose concentrations (0, 0.25, 0.4 and 0.6 m). After ultra-rapid freezing, samples were thawed and sperm motility, morphology, viability and acrosome status were assessed. At thawing, the number of progressively motile (p < 0.01) and morphologically normal (p < 0.01) sperm was higher in the sucrose-supplemented groups than in the sucrose-free group. Viability of spermatozoa cryopreserved without sucrose was significantly reduced. In extender supplemented with 0.4 m sucrose, spermatozoa viability showed higher values (57.0 ± 4.7; p < 0.01). No significant differences were detected among groups for sperm acrosome integrity. Results support that cat sperm survive after ultra-rapid freezing using sucrose as a cryoprotectant, and the best results were achieved when 0.4 m of sucrose was used. This is the first report on sperm ultra-rapid freezing of cat sperm and further studies on extenders, sperm management or cryovials should be carried out to improve sperm cryosurvival.

Genetic analysis of haematological and plasma biochemical parameters in the Spanish purebred horse exercised on a treadmill.

The novel aim of this study was to describe the reference values of different haematological and biochemical parameters in the Spanish purebred horse (Andalusian, SPB) in each of the stages of a programmed exercise on a treadmill system, and to establish heritability and genetic correlations for these haematological and biochemical parameters. For this, 94 young SPB male horses (4.22 ± 2.27 years old) were used. An increasing intensity exercise test at 4, 5, 6 and 7 m/s was carried out on a treadmill (6% inclination). Total red blood cells, total white blood cells, neutrophils and lymphocytes counts; haematocrit, haemoglobin, lactate, uric acid, creatinine and total plasma proteins concentrations and aspartate transaminase, lactate dehydrogenase, creatine-quinase activities were determined. To conclude: (i) the reference values for each parameter were determined for each of the exercise test stages (ii) all the parameters analysed manifested a medium-high heritability and a high repeatability. These results will, in the near future, determine the measuring guidelines for improving the SPB horse's athletic ability on an objective treadmill system and for selecting these animals in response to those parameters.

Effect of different extenders on in vitro characteristics of feline epididymal sperm during cryopreservation.

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS-egg yolk-based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25-ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0- and 6-h post-thaw. Experiment 1 compared the effect of three non-feline commercial extenders - based on TRIS-egg yolk (Triladyl), egg-yolk-free medium (AndroMed) and skimmed milk-egg yolk (Gent) - on the quality of frozen-thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p < 0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen-thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS-egg yolk-based extenders, one non-feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing-thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing-thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS-egg yolk-based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.

Utility of the discriminant analysis to categorize untrained Spanish Pure Bred horses on the basis of biochemical and muscle variables before and after a standardized exercise test.

We hypothesized that the information obtained from a discriminant analysis could be used to objectively discriminate horses untrained from early ages, in agreement with certain physiological characteristics. In the biopsies of 24 Spanish Pure Bred horses (1.5-3 years old) before and after a standardized exercise test (SET; 4-7 m/s with a change of velocity of 1m/s every 2 min) muscle enzymes, substrate and metabolites were determined. Also, diverse plasma and blood parameters were considered. Three pre-exercise groups (A1: six horses; A2: seven horses and A3: eleven horses) and two post-exercise groups (B1: sixteen horses; B2: eight horses) were defined from a correspondence analysis. Forward stepwise discriminant analysis selected 11 variables which differentiated the groups between each other both pre- and post-exercise. The results of the present study suggested the utility of a discriminant analysis to categorize horses in agreement with certain physiological variables. It could be used for establishing different types of training in each group by expert trainers.

Estudio del hemograma en caballos de pura raza española ejercitados en cinta rodante / No disponible

No disponible

Estudio de la evolución del perfil enzimático en el músculo del toro de lidia con el entrenamiento mediante la utilización de la biopsia muscular / No disponible

No disponible

Estudio de la evolución del perfil enzimático en el músculo del toro de lidia con el entrenamiento mediante la utilización de la biopsia muscular / No disponible

No disponible

Effects of training on phagocytic and oxidative metabolism of peripheral neutrophils in horses exercised in the aerobic-anaerobic transition area.

Using simple techniques, the neutrophil function, in its phagocytosis and oxidative metabolism stages, was evaluated in horses. This was done before and after moderate exercise at the aerobic-anaerobic threshold (standardized heart rate 150 beats/min and lactate level of 3.07 +/- 0.21 mmol/L). The objective was to determine whether regular training and moderate exercise improved the neutrophil function. A group of 19 horses was used; 11 of these were untrained and the remainder trained for national jumping events. The exercise test consisted of a 5 min trot followed by a 3 min gallop on a long lunge. Blood samples were taken for analysis before, immediately after and 15 min after exercise. The results showed that (a) there is a difference in the internalization of particles (PI, PP and PE) by neutrophils from trained and untrained horses at a single time point during active recovery, and PP is higher in trained horses immediately after exercise; and (b) oxidative metabolism is significantly lower in untrained animals before and 1 min after exercise. The moderate exercise at the aerobic-anaerobic threshold did not have any influence on the peripheral blood neutrophil function of the phagocytosis and oxidative metabolism of particles.

Nonspecific immune response of peripheral blood neutrophils in two horse breeds (Anglo-Arabian and Spanish-Arabian): response to exercise.

The aim of the present paper was: (1) to find out if there were any differences in the nonspecific immunological pattern of peripheral blood neutrophil between two breeds of horses (AA and SA); (2) to evaluate the effects of an exercise in the aerobic-anaerobic threshold. This has been observed in a group of 11 untrained horses (6 SA and 5 AA) of 2.5 years old. No statistically significant differences were found in the different stages of immune response between the rest and immediately after physical exercise to two breeds. However, the chemotaxis was significant higher at rest in the AA than SA breed. A positive correlation was found at rest between the phagocytic and oxidative metabolism activity for AA breed and a negative correlation too between the adherence and chemotaxis with phagocytic capacity, immediately after exercise test, for the same breed.

Lead and lead toxicity in domestic and free living birds.

At present, domestic and wild fauna are being exposed to aspects and factors which are foreign to the habitat in which they live. One that stands out is the enormous amount and variety of chemical compounds which, in many cases, are highly complex and which are constantly being released into the atmosphere, mainly from agricultural and industrial activity. All these substances affect some species more than others, whether they be plants or animals, from the most insignificant micro-organism to the most evolved species, among them birds. Finally, another cause of mortality in many birds is plumbism, namely death caused by the ingestion of lead. Lead has been one of the main causes of poisoning in man since ancient times due to its use in many activities although it is only recently that this toxicity has been recognized. Moreover, the use of lead pellets for shooting has resulted in the release into the environment of millions of these over many years, with serious repercussions for many bird species populations, which have ingested them either directly or indirectly. Added to this use of lead in cynegetic activities is the fate of the lead weights (sinkers or ballast) used by rod fishers, which sink to the bottom or accumulate on the banks of rivers, lakes, lagoons or reservoirs. The problem arises when these pellets or weights are ingested by birds, mainly Anatidae, which mistake them for the small stones or grit they use to triturate food in their gizzards. Small particles of lead enter the digestive tract, start dissolving in the form of lead salts, are incorporated into the bloodstream and the rest of the body, accumulate in organs like the liver or kidneys, and cause physiological or behavioural changes. When certain concentrations of lead are reached, the birds then die. If lead-poisoned birds are consumed by carrions or predators, the latter also ingest the lead so that they may also be affected or die from plumbism since, being a heavy metal, its degradation and/or elimination is very difficult. There is, therefore, no doubt that millions of birds die annually worldwide from lead poisoning (in the U.S.A., around 3,000,000), this problem being most acute in marshland. The solutions could include the introduction of legislation regulating or banning shooting, in the use of non-toxic ammunition in marshes and protected areas, the substitution of lead pellets for other non-toxic ones, such as steel, bismuth, tungsten or other suitable metals, and to go on studying other possible alternatives to end such a dramatic situation for birds all over the world.

Benefits of moderate training to the nonspecific immune response of colts.

The aim of this work was to assess whether progressive training caused an improvement in the nonspecific immune response of colts because several unusual infections are due to defects inherent in the neutrophilic function among which respiratory diseases are a major defect in the performance of athletes taking part in professional sports activities. A group of 7 Anglo-Arabian colts belonging to the Army was selected. These animals carry out training programmes for their participation in National Jumping Competitions. During a submaximal exercise test (heart rate 150 beats/min and lactate levels maintained at aerobic-anaerobic threshold of 3 mmol/l), they were compared with 5 colts of the same breed, just beginning training exercises. Immediately after the test, the nonspecific immune capacity of neutrophilic polymorphonuclear cells was valued by adherence, chemotaxis, ingestion and digestion of foreign substances tests. The results showed significant differences between trained and nontrained animals for the adherence and foreign particle digestion tests and, beginning with a greater adherence in untrained animals, a superior effectiveness was achieved in the immune function in trained colts, whose digestive capactiy was increased with respect to the untrained ones. It was, therefore, concluded that moderate training and exercise improves and reinforces the response of the nonspecific immune system against future infections in the organisms.

Skeletal muscle fibre characteristics in young and old bulls and metabolic response after a bullfight.

Fibre type composition, activities of enzymes such as citrate synthase (CS), 3-hydroxyacyl coenzyme A dehydrogenase (HAD), lactate dehydrogenase (LDH), as well as glycogen, lactate and pH levels were analysed in muscle biopsies (m. gluteus medius) obtained after bullfighting from 10 young and 10 old bulls. No changes were seen in fibre type composition between groups, but the older bulls had higher HAD and LDH activities. Low glycogen concentrations and low pH values were found in both groups, but the lactate concentration after bullfighting was higher in the older group of bulls. The histochemical stain for glycogen revealed that type IIB fibres in both young and old bulls contained more glycogen than seen in type IIA and type I fibres. These results show that young and old bulls have similar muscle fibre type composition, but the metabolic capacity differs, with a higher glycolytic capacity and lactate production in older bulls. Furthermore, it seems that the physical and emotional stress in connection with a bullfight causes a marked depletion of glycogen, especially of type I and IIA fibres.

Differential electrocardiographic study on Iberica and Duroc breeds of pig during physical maturation.

Sequential electrocardiograms were taken of 50 pigs (25 Iberica and 25 Duroc), from the ages of 5 days to 205 days. The records were analysed to establish the normal values of the different electrocardiographic intervals (RR, QRS, QT, TQ and ST intervals), the diastole/systole quotient and heart score in the first 7 months of life, as well as age-related electrocardiographic variations. In addition, it was intended to determine which of these two breeds showed the greater heart recovery capacity. Finally, positive or negative correlations between the RR interval and the electrocardiographic incidents studied were analysed. The mean values obtained for the electrocardiographic parameters were similar in the two breeds and increased with their physical maturity. Analysis of the correlation between the duration of the heart cycle and the different electrocardiographic incidents showed a positive and significant correlation, the r-values being higher for the RR interval-TQ interval correlation. There was very little correlation between RR interval and heart score in Duroc pigs and practically no correlation between the RR interval and the QRS interval in either breed.

Locomotor response to exercise in relation to plasma lactate accumulation and heart rate in Andalusian and Anglo-Arabian horses.

To establish the effect of the locomotor pattern on heart rate (HR) and plasma lactate (LA), 28 horses, 18 Andalusian (AN) and 10 Anglo-Arabian (AA), aged 3-4 years, were studied. After a warm-up period, the horses performed a four-step test at 5, 6, 7 and 8 m/s, covering 1000 m at each velocity. HR was monitored, LA was analysed at rest and after each workload, and images were filmed. The locomotor parameters determined were stride duration (SD), frequency (SF) and length (SL), step and bipedal support durations, stance (restraint and propulsion) and swing phase durations, and stride vertical component. The HR and LA were significantly higher in the AN horses from velocities of 5 m/s. Similarly, the stride vertical component was higher in the AN horses at the trot and in the leading forelimbs at the gallop. Conversely, at all the galloping velocities, swing phase duration and stride length were longer in the AA horses. Significant correlations between HR, LA and locomotor pattern were only found in the AN horses. It was concluded that the greater stride vertical component in the AN horses limits SL and the cranial advancement of the hoof, with the result that reaching a longer SL triggers an increase in HR and LA. AA horses reach a balance between SL and SF, improving the efficiency of the gait.

Locomotor, cardiocirculatory and metabolic adaptations to training in Andalusian and Anglo-Arabian horses.

The effects of two training programmes in 20 Andalusian and 12 Anglo-Arabian horses were evaluated by an increasing intensity work test at velocities of 4, 5, 6, 7 and 8 m sec(-1). Heart rate was monitored and blood samples were drawn at rest and after each velocity to analyse packed cell volume, haemoglobin concentration, plasma lactate and potassium levels. Furthermore, the programmes were video-taped and stride length, duration and frequency, stance (restraint and propulsion), swing phase durations and stride vertical component were measured. The training protocol of the Andalusian horses produced significant decreases in the cardiovascular, haematological and metabolic responses to exercise. Locomotory training adaptation consisted of an increased stride frequency and a reduced stride length and vertical stride component. The last variable was the limiting factor of stride length both before and after training in the Andalusian horses. A different training protocol for show-jumping competition in Anglo-Arabian horses failed to show significant differences in the studied parameters to the work test, although an increase in stride length at velocities of over 6 m sec(-1) was observed. Stride vertical component did not have an effect on the physiological response to exercise, either before or after training.

Cardiovascular and metabolic adaptations in horses competing in cross-country events.

The cardiovascular and metabolic response to two cross-country events (CC*: preliminary level and CC*** advanced level) were analysed in 8 male eventing horses (4 Anglo-Hunter and 4 Anglo-Arabian). This study focused on the establishment of the main metabolic pathways involved in the muscle energy resynthesis during the competitions. Heart rate (HR) was recorded throughout the CC events. Jugular venous blood samples were withdrawn before the warm-up period, immediately after the competitions and at 5 and 10 min in the recuperation period. The following haematological parameters were studied: red blood cells (RBC), packed cell volume (PCV), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cells (WBC), and number and percentages of lymphocytes (LYM) and granulocytes and monocytes (GRAN). One fraction of blood was centrifuged and, in plasma, lactate (LA), total plasma protein (TPP) and the rate of LA disappearance were determined. The competitions induced significant increases in RBC, Hb, PCV, MCV and TPP. Plasma LA response exceeded the anaerobic threshold of 4 mmol/l, reaching a maximum level of 13.3 mmol/l. HR ranged from 140 to more than 200 bpm, peaking at 230 bpm, revealing a limitation in the oxygen supply to the working muscles. It was concluded that muscle energy resynthesis during a CC event is provided both through oxidative processes and glycolysis with LA formation. Therefore, both stamina and power exercises are required for eventing horses.

The use of functional indexes to evaluate fitness in Andalusian horses.

The fitness of 8 Andalusian horses between 3 and 4 years of age was analysed. The animals were subjected to an exercise test on a sandy track consisting of 2 stages of different intensities. The first stage was of submaximal intensity at 4 speeds which increased progressively (4.17, 5.56, 6.94 and 8.33 m/sec.) covering a distance of 1,000 m in each level. Between each of these speeds, the horses rested for 2 min. The second stage was a maximal speed test over the same distance carried out 2 min after the ending of the maximal phase. Data of heart rate, plasma lactate concentration, velocity, PCV and pH in the blood were obtained. Maximum heart rate, maximum velocity, VLA2, VLA4, peak lactate, minimum pH and maximum PCV were considered functional indexes. A principal component enabled us to segregate horses according to their fitness and in relation to the information provided by the trainers in charge of these horses. The most discriminant variables in order to segregate horses were pHmin, VLA4, HRmax, VLA2 and Vmax. Differences between horses in relation to PCVmax were not found. The influence of each one of these functional indexes on the test exercise tolerance was discussed.