Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

Use of C-reactive protein to predict outcome in dogs with systemic inflammatory response syndrome or sepsis

Background – There is a high mortality rate in patients with systemic inflammatory response syndrome (SIRS) or sepsis. Therefore, an early diagnosis and prognostic assessment is important for optimal therapeutic intervention. The objective of the study was to evaluate if baseline values and changes in serum C-reactive protein (CRP) might predict survival in dogs with SIRS and sepsis.
Design – Prospective study; July 2004 to July 2005.
Setting – Small Animal Clinic, Berlin, Clinic of Small Animal Medicine, Munich.
Animals – Sixty-one dogs.
Measurements and Main Results – For the CRP analysis blood was drawn on day 0, 1, and 2; CRP was measured using a commercial ELISA test kit. Thirteen dogs suffered from nonseptic SIRS and 48 dogs from sepsis. The 14-day survival rate was 61% (69% nonseptic SIRS, 58% sepsis). Serum CRP was higher in sick dogs compared with controls ( P <0.001). Over the 3-day period surviving dogs ( n =31) displayed a significantly greater decrease in CRP than nonsurvivors ( n =10) ( P =0.001). No correlation was found between the initial CRP concentrations and the survival rate. The changes in CRP corresponded to the survival rate ( P =0.01).
Conclusion – There was no significant relationship between the survival rate in dogs with nonseptic SIRS or sepsis and the initial serum CRP concentrations. There was a correlation between decreasing CRP concentrations and recovery from disease. However, the changes in CRP concentrations over a 3-day period correctly predicted survival in 94% of dogs and death in 30% of the dogs (false positive rate 22%).     

Synchronizing Cell Cycle of Goat Fibroblasts by Serum Starvation Causes Apoptosis

Cell cycle stage and synchronization of donor cells are important factors influencing the success of somatic cell nuclear transfer. This study examined whether serum starvation has any effect on specific cell death. We also studied the effects of serum starvation, culture to confluence, and full confluency (confluent + 72 h) on cell cycle characteristics and apoptosis of goat dermal fibroblast cells. The cells were obtained from the ear of a 1.5‐year‐old female goat. The following experimental groups were analysed for fibroblast cells: (i) normally growing, (ii) confluent, (iii) full confluency, (iv) cells starved for 48 h and (v) cells starved for 72 h. Analysis of cell cycle distribution by flow cytometry showed that 4.56 and 51.88% of normal cycling cells were at the G0 and G1 phases respectively. In the confluent group, 80% of the cells were arrested in the G0/G1 phase. Serum starvation for 48 and 72 h arrested 84.78% and 90.1% cells at the G0/G1 phase respectively which showed a significant difference when compared with the control group (p < 0.05). Double staining by PI and FITC distinguishes G0 phase from G1 phase. In the full confluency group, 91.53% of cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Under normal culture conditions, 6.39% of cells underwent early apoptosis. In the confluent group 8.93% of cells showed early apoptosis. Serum starvation for 48 and 72 h caused early apoptosis in 8.91 and 39.83% of the cells respectively. Full confluency treatment did not increase the number of apoptotic cells significantly (8.67%). After 72 h, serum starvation significantly increased early apoptosis (p < 0.05). In conclusion, the use of full confluency is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase and also induces less apoptosis in comparison with the serum starvation group.     

Cyclic CD8+ lymphopenia in dogs experimentally infected with Bartonella vinsonii subsp. berkhoffii

Until recently, it was presumed that Bartonella vinsonii only infected voles, a species of North American rodents. In April of 1993, however, our laboratory isolated a novel subspecies of B. vinsonii (B. vinsonii subsp. berkhoffii) from the blood of a dog diagnosed with vegetative valvular endocarditis. Subsequently, based on a seroepidemiologic survey of dogs from North Carolina and Virginia presenting for a variety of medical problems, we found evidence supporting a potentially important association between B. vinsonii and Ehrlichia canis co-infection in dogs. In the following study, eight dogs were infected with B. vinsonii: four specific pathogen free dogs and four dogs that had previously been infected with E. canis. Flow cytometric analysis of peripheral blood lymphocytes revealed a cyclic elevation of the CD4/CD8 T-cell ratio that correlated with cyclic CD8+ lymphopenia in all dogs infected with B. vinsonii, regardless of prior exposure to E. canis.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.