Modification of standard freezing media to limit capacitation and maximise motility of frozen-thawed equine spermatozoa

OBJECTIVE: To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. PROCEDURE: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. RESULTS: Following centrifugation, spermatozoa diluted with modified Kenney's Centrifugation Medium (MKCM) displayed a higher percentage of (normal) F pattern (94.3%) compared with spermatozoa in Kenney's Centrifugation Medium (KCM) (84.9%) and Glucose-EDTA Centrifugation Medium (GECM) (85.2%). Conversely, the percentage of spermatozoa displaying the (capacitated) B pattern was higher in the KCM (14.1%) and GECM (13.8%) than in the MKCM (5.0%). Following freezing-thawing, there were lower percentages of spermatozoa displaying the AR (acrosome reacted) pattern in modified Kenney's Freezing Medium (MKFM) (45.6%) compared with Kenney's Freezing Medium (KFM) (61.4%) and lactose-EDTA Freezing Medium (LEFM) (61.1%). There was a correspondingly higher percentage of spermatozoa displaying the B pattern in MKFM (52.3%) compared with KFM (37.9%) and LEFM (38.6%). There was no significant difference between the freezing media in the percentage of spermatozoa displaying the F pattern. The percentage of progressively motile spermatozoa was also influenced by the type of freezing medium (P < 0.001). Post-thaw percentages of progressively motile spermatozoa, frozen in MKFM, KFM, and LEFM, were 31.4, 25.8 and 23.3%, respectively. CONCLUSION: MKFM was the preferred medium for cryopreservation of equine spermatozoa due to its superior protection against changes in motility and membrane quality compared with the other freezing media studied.     

Prevalence of psammoma bodies in meninges and choroid plexuses of raccoons (Procyon lotor) from Parramore Island, Virginia.

Microscopic evidence of multifocal mineralizations (psammoma bodies) were seen in brains of 33/53 (62%) raccoons (Procyon lotor) necropsied on Parramore Island, Virginia. Most mineralized foci had concentric laminations and were present in small capillaries of meninges of the brain (15/33), in choroid plexus (3/33), or at both these sites (13/33). In 2 raccoons, the lesions were confined to the meninges of the proximal cervical spinal cord. In most cases, the affected vessels appeared to have been completely occluded. However, no evidence of ischemic changes in the brain parenchyma was seen, and none of the raccoons had abnormal neurologic signs prior to euthanasia. The condition appears to be a common incidental histopathologic finding in raccoons from the eastern United States. Although the exact cause of this condition is not known, a primary vascular insult with resultant dystrophic mineralization of the affected vessels is suspected.     

Bacterial diseases of potatoes: from classical phytobacteriology to molecular pathogenicity

Various bacterial pathogens attack potato plants and tubers. These pathogens includeErwinia, Corynebacterium (Clavibacter), Psuedomonas andStreptomyces. Over the past few years there have been significant advances in the molecular biological analysis of several of these pathogens and this is now helping us to understand the major aspects of virulence mechanisms. However, to date, such information is not sufficiently useful to allow us to intervene rationally in these potato diseases, except by the standard practises of good husbandry.     

Theileria parva: the immune status of calves born of dams immunised against East Coast fever

Eight Friesian cross cows three months pregnant to a single Friesian bull were immunised against East Coast fever by infection with Theileria parva (Muguga) sporozoite stabilate and treatment with pyrrolidino-methyl tetracycline. They were challenged with the homologous stock four times before calving and a fifth time after calving, and resisted all five challenges which killed all of the five groups of five susceptible controls. Calves born to these hyperimmunised dams were fully susceptible on challenge with the same stabilate, as were susceptible cows from the same farm and their calves. In both instances the calves died three to seven days earlier than the cows which were approximately 10 times heavier. These results show that one- to two-month-old taurine calves from artificially immunised dams are not protected from experimental T parva sporozoite challenge and that there is no inherent calfhood resistance to East Coast fever.     

Effects of heartworm and intestinal parasitic infections on hematology and peripheral lymph node cytology in louisiana dogs

Hookworm infected dogs had higher blood basophil counts than hookworm negative dogs. Dogs with occult heartworm infection (i.e., without microfilaremia) had higher blood neutrophil counts than heartworm negative dogs. The percentage of eosinophils in peripheral lymph nodes was higher in heartworm infected, microfilaremic dogs than in both occult infected dogs and heartworm negative dogs. Heartworm or intestinal parasitic infection had no other significant effects on hematologic parameters or peripheral lymph node cytology.     

Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.