Significance of Non‐conventional Parameters in the Evaluation of Cooling‐induced Damage to Ram Spermatozoa Diluted in Three Different Media

The objectives of this study were two. First, to compare three base media with different sugar composition as an initial step to achieve a good chemically‐defined extender for ram sperm refrigeration. The second one, to determine which sperm quality parameters may be more useful for revealing differences between sperm samples. One medium contained 200 mm sucrose and 2.8 mm glucose (SM), another only disaccharides (D) such as sucrose, trehalose, maltose and lactose (75 mm each); and the third one (D+M) included a mix of monosaccharides (50 mm glucose, 20 mm fructose and 20 mm galactose,) and the same disaccharides as in D (50 mm each). Ram semen samples diluted in the mentioned media were refrigerated at 5°C for 1 h, and rewarmed upto 37°C in order to mimic the temperature in the female reproductive tract. Addition of monosaccharides to the extender did not produce a better preservation of motility or viability after cooling. The supplementation with other disaccharides apart from sucrose did not enhance the viability either. Thus, after cooling and rewarming, there were no significant differences in sperm viability (membrane integrity evaluated by CFDA/PI staining) or the percentage of progressive motile and rapid sperm (evaluated by CASA) between the three media. However, the percentage of viable non‐capacitated sperm evaluated by the chlortetracycline (CTC) assay was higher and sperm oxygen consumption was lower in SM than in D and in D+M. Although the apoptosis‐like markers [phosphatidylserine exposure assessed by Annexin V/CFDA staining and DNA‐damage evaluated by TUNEL assay] showed a continuous increment throughout the process with all diluents, the percentage of sperm with damaged DNA at the end of the process was significantly lower in SM than in the other two media (p < 0.01). On the basis of these results, we would make two recommendations: the use of an extender supplemented only with sucrose and glucose for ram sperm refrigeration; the inclusion of non‐conventional methods such as oxygen consumption measure, evaluation of capacitation state and apoptosis‐like markers for revealing differences between sperm samples.     

Epidemic of blindness in kangaroos - evidence of a viral aetiology

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.     

Electrocardiographic values in Spanish-bred horses of different ages

SUMMARY The duration of electrocardiograph wave forms and intervals were determined in 179 Spanish-bred (Andalusian) horses aged from 1 month to 17 years. The values were compared with those of other breeds, and the relationship between electrocardiographic data and age was examined. High correlation coefficients were found between PR, ST and QT intervals and the age of the horses, and an inverted relation between heart rate and age was found. A multiple range analysis was made and the results suggest that significant changes in duration values and heart rate ocurred at the age of 6 months and in the second year of life.     

Serum Starvation and Full Confluency for Cell Cycle Synchronization of Domestic Cat (Felis catus) Foetal Fibroblasts

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p ≤ 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p ≤ 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.     

Caspase Activation,Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.     

Primary anconeal fracture in a boxer

A 14-month-old speyed female Boxer was presented with acute non-weight-bearing lameness in the right forelimb. Radiography revealed separation of the anconeal process, which was thence surgically removed. Histological examination of the anconeal process confirmed a primary fracture. Isolated unilateral anconeal process fracture in the dog is rare and this report includes histopathological findings of a primary anconeal fracture.