Studies on the cryopreservation of common wombat (Vombatus ursinus) spermatozoa

In an attempt to develop a gamete-recovery protocol for the northern hairy nosed wombat (Lasiorhinus krefftii), spermatozoa were removed from the cauda epididymides of four common wombats (Vombatus ursinus) and cryopreserved following a variety of prefreeze storage conditions. Spermatozoa stored for 72 h at 4 degrees C within the testicle before cryopreservation tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability (% motile P< 0.01; rate of movement P< 0.01; % live P< 0.01) than sperm recovered on the day of post-mortem, stored in a test tube for 72 h at 4 degrees C and then frozen. The effect of post-thaw dilution with Tris citrate fructose (TCF) diluent on the survival of epididymal common wombat spermatozoa was also investigated. Motility (P< 0.05), rate of sperm movement (P< 0.01) and the percentage of live spermatozoa (P< 0.05) were all significantly greater when spermatozoa were thawed and diluted immediately in TCF than when thawed without dilution. The present study also reports, for the first time, a successful pellet method of freezing wombat spermatozoa on dry ice; volumes of 0.25 and 0.5mL resulted in higher post- thaw survival compared with 0.1-mL pellets.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei : Istiophoredae)

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa. were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation; mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

Scabies mite inactivated serine protease paralogues are present both internally in the mite gut and externally in feces

The scabies mite, Sarcoptes scabiei, is the causative agent of scabies, a disease that is common among disadvantaged populations and facilitates streptococcal infections with serious sequelae. Previously, we encountered large families of genes encoding paralogues of house dust mite protease allergens with their catalytic sites inactivated by mutation (scabies mite inactivated protease paralogues [SMIPPs]). We postulated that SMIPPs have evolved as an adaptation to the parasitic lifestyle of the scabies mite, functioning as competitive inhibitors of proteases involved in the host–parasite interaction. To propose testable hypotheses for their functions, it is essential to know their locations in the mite. Here we show by immunohistochemistry that SMIPPs exist in two compartments: 1) internal to the mite in the gut and 2) external to the mite after excretion from the gut in scybala (fecal pellets). SMIPPs may well function in both of these compartments to evade host proteases.

Total cholesterol concentrations of the muscles in castrated Boer goats

Goat meat is an important source of animal protein and Boer goats are the most widespread breed of meat goats. Cholesterol in meat is a potential cause of human disease and little is known about the cholesterol concentration in different goat muscles. As animals gain bodyweight the amount of cholesterol in their tissues generally increases and can vary for different tissues. Therefore, the aim of this experiment was to determine the effect of slaughter weight and muscle type on total cholesterol concentrations in three muscles (longissimus thoracic, infraspinatus and biceps femoris) taken from castrated male Boer goats in Australia. The goats were grazed on a mixture of native pasture and Rhodes grass (Chloris gayana) and had access to lucerne and commercial goat pellets (CP 18%; ME 12.3 MJ/kg DM). The muscles were dissected from 24 goats slaughtered in groups of 4 or 5 animals at 30, 45, 60, 75 and 90 kg liveweight. Total cholesterol concentrations were then determined using high performance liquid chromatography (HPLC). Total cholesterol concentrations decreased (P < 0.01) with an increase in slaughter weight for the longissimus thoracic, infraspinatus and biceps femoris muscles. Significant differences (P < 0.01) were recorded in the total cholesterol concentrations for the three muscles studied. The average total cholesterol concentrations ranged from 55 to 60 mg/100 g for the longissimus thoracic, 69 to 88 mg/100g for infraspinatus and 65 to 82 mg/100 g for the biceps femoris muscles. These results are important for consumers of goat meat as total cholesterol concentrations vary between different muscles taken from animals slaughtered at different weights and the concentrations are higher in muscles taken from lighter and younger animals. Therefore, health conscious consumers of meat, concerned about the cholesterol content of their diet, should consume meat from the longissimus thoracic muscles of heavier or older goats. (c) 2005 Elsevier B.V. All rights reserved.