Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA

To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa – including sex sorting – as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry‐based assays. After sorting, oxidative stress increased from 26% to 33% in pre‐ and post‐incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post‐sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.     

Effects of local anaesthesia of 4 to 8 hours duration on the acute cortisol response to scoop dehorning in calves

Objective To determine the effect of a long-acting local anaesthetic on the plasma cortisol response in calves dehorned using a scoop.
Design A physiological study with controls.
Procedure Seventy female calves, 3 to 4 months old, were divided into seven groups and dehorned by scoop with or without a long acting local anaesthetic (bupivacaine) which provides local anaesthesia for 3 to 4 h. In some treatment groups the local anaesthetic was given a second time, after 4 h, to give 8 h of anaesthesia. Blood samples were taken before and for 9 h after treatment and plasma cortisol concentrations were measured.
Results Scoop dehorning caused a marked rise in plasma cortisol concentration, which returned to pre-dehorning values after 7 h. The cortisol concentrations of calves given bupiva-caine were similar to those of control animals for 4 h after dehorning, but then a significant and protracted rise occurred, with concentrations returning to pretreatment values at about 9 h after dehorning. In calves given a second treatment of bupivacaine 4 h after dehorning, the plasma cortisol concentration remained similar to control values for 8 h after dehorning but then increased sharply.
Conclusions During its period of activity long-acting local anaesthetic prevented the rise usually seen after scoop dehorning in plasma cortisol concentrations; by inference it also alleviated the pain-induced distress caused by this procedure. However, when its effects had worn off the plasma cortisol values rose sharply indicating that animals still experienced pain-induced distress despite local anaesthetic being administered.     

Genetic diversity of infectious hematopoietic necrosis virus from Feather River and Lake Oroville, California, and virulence of selected isolates for Chinook salmon and rainbow trout

Infectious hematopoietic necrosis virus (IHNV) is a significant pathogen of young salmonid fishes worldwide but particularly within the historical range of the Pacific Northwest and California. In the Sacramento and San Joaquin River drainages of California, IHNV outbreaks in juvenile Chinook salmon Oncorhynchus tshawytscha have been observed regularly at large production hatcheries, including Coleman National Fish Hatchery (established in 1941) and Feather River State Fish Hatchery (FRH; established in 1967), since facility operations began. Recent severe epidemics at the FRH in 1998 and 2000-2002 prompted investigations into the characteristics and potential sources of virus at this facility. Both phylogenetic analyses of a variable portion of the glycoprotein gene and serologic comparisons based on neutralization with three polyclonal rabbit sera were used to characterize 82 IHNV isolates from the Feather River watershed between 1969 and 2004. All isolates examined were in the L genogroup and belonged to one of three serologic groups typical of IHNV from California. The IHNV isolates from the Feather River area demonstrated a maximum nucleotide sequence divergence of 4.0%, and new isolates appeared to emerge from previous isolates rather than by the introduction of more diverse subgroups from exogenous sources. The earliest isolates examined from the watershed formed the subgroup LI, which disappeared coincidently with a temporal shift to new genetic and serologic types of the larger subgroup LII. Experimental challenges demonstrated no significant differences in the virulence for juvenile Chinook salmon and rainbow trout O. mykiss from selected isolates representing the principal types of IHNV found historically and from recent epidemics at FRH. While most isolates were equally virulent for both host species, one isolate was found to be more virulent for Chinook salmon than for rainbow trout.     

Factors affecting the relationship between arterial and end-tidal carbon dioxide pressures in the anaesthetised horse

Objective To assess the effects of the duration of anaesthesia, position of recumbency, mode of ventilation, anaesthetic drug protocol, patient age and type of surgical procedure on the usefulness of capnometry as a measure of the partial pressure of arterial carbon dioxide (P_aco ₂) during general anaesthesia in horses. Design A prospective study compared the P_aco ₂ values with those of partial pressure of end-tidal carbon dioxide (ETco ₂) in horses anaesthetised for elective or emergency surgical procedures. The difference between P_aco ₂ and ETco ₂ (P_aco ₂− ETco ₂) and the physiological dead space to tidal volume ratio (V_D/V_T) were calculated. The effects of the study parameters on these variables was determined. Results The agreement between P_aco ₂ and ETco ₂ was poor. P_aco ₂− ETco ₂ and V_D/V_T during the first 60 min of anaesthesia was significantly less than after 60 min of anaesthesia. Mode of ventilation, position of recumbency, anaesthetic drug protocol, patient age and type of procedure did not have a significant affect on either value. Conclusions P_aco ₂− ETco ₂ in anaesthetised horses can be large, making ETco ₂ unreliable as a predictor of P_aco ₂ and for assessment of pulmonary ventilation. For anaesthesia lasting less than 60 min at least one blood gas analysis of an arterial blood sample is required to assess P_aco ₂− ETco ₂. Arterial blood gas analysis should be repeated after 60 min of general anaesthesia.     

Glycerol,Methyl‐Formamide and Dimethyl‐Formamide in Canine Semen Cryopreservation

The aim of the present work was to compare the efficiency of methyl‐formamide (MF), dimethyl‐formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk–TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo‐osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 ± 5.4%, 61.0 ± 7.4%, 2.9 ± 0.5 and 57.1 ± 5.0% for GL; 59.0 ± 8.9%, 50.0 ± 10.0%, 2.5 ± 0.7 and 66.9 ± 7.7% for MF; and 44.0 ± 21.0%, 37.0 ± 19.8%, 2.1 ± 0.6 and 61.1 ± 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 ± 12.4%, 35.8 ± 18.4% and 34.4 ± 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.     

Proliferation and Apoptosis in Aged and Photoregressed Mammalian Seminiferous Epithelium,with Particular Attention to Rodents and Humans

Imbalances in the proliferation and apoptosis processes are involved in numerous epithelial alterations. In the seminiferous epithelium, normal spermatogenesis is regulated by spermatogonia proliferation and germ cell apoptosis, and both processes are involved in diverse pathological alterations of the seminiferous epithelium. Other physiological phenomena including aging and short photoperiod, in which apoptosis and proliferation seem to play important roles, cause testicular changes. Aging is accompanied by diminished proliferation and increased apoptosis, the latter occurring in specific states of the seminiferous cycle and considered the cause of epithelium involution. However, there is no clear evidence concerning whether proliferation decreases in the spermatogonia themselves or is due to an alteration in the cell microenvironment that surrounds them. As regards the factors that regulate the process, the data are scant, but it is considered that the diminution of c‐kit expression in the spermatagonia, together with the diminution in antiapoptotic factors (Bcl‐x_L)) of the intrinsic molecular pathway of apoptosis play a part in epithelial regression. A short photoperiod, especially in rodents, produces a gradual involution of the seminiferous epithelium, which is related with increased apoptosis during the regression phase and a diminution of apoptosis during recrudescence. Proliferative activity varies, especially during the total regression phase, when it usually increases in the undifferentiated spermatogonia. In other species showing seasonal reproduction, however, decreased proliferation is considered the main factor in the regression of the seminiferous epithelium. Little is known about how both phenomena are regulated, although data in rodents suggest that both the intrinsic and extrinsic pathways of apoptosis contribute to the increase in this process. In conclusion, regression of the seminiferous epithelium in physiological situations, as in many pathological situations, is a result of alterations in equilibrium between the proliferation and apoptosis of germinal cell types. However, both physiological phenomena showed important differences as regard proliferation/apoptosis and their regulation pathways, probably as a result of their irreversible or reversible character.