The effects of magnetic separation on cryopreserved bovine spermatozoa motility, viability and cryo-capacitation status.

Cryopreservation is a technique used to preserve cells for long-time storage. It is widely used in agriculture to store male gametes in liquid nitrogen. The aim of this study was to determine the optimum thawing temperature and time for samples subjected to annexin V magnetic-activated cell sorting (AnMACS) as the sperm preparation technique. Pooled semen samples from three ejaculates were divided into two groups. The treatment group was subjected both to AnMACS and to being cryopreserved, whilst the control group was cryopreserved directly without MACS. Post-thaw analysis was carried out for samples thawed at either 20°C for 13 s, 37°C for 30 s, 40°C for 7 s, 60°C for 6 s or 80°C for 5 s. Sperm kinematics, viability and capacitation status were determined for samples subjected to all thawing temperatures described. Results showed that thawing at 37°C for 13 s for MACS-processed samples was a superior option compared with other thawing procedures; there was a significant difference in P < 0.05 values for curvilinear velocity (VCL µm/s) and sperm straightness (STR %) when samples were thawed at 40°C for 7 s, with fewer capacitated spermatozoa (P < 0.05) when samples were thawed at 37°C for 30 s, 40°C for 7 s or 60°C for 6 s. Hence, we can speculate that the use of AnMACS as the sperm preparation technique can somehow enhance sperm cryosurvival rate after cryopreservation, however the fertilization potential of these cells has yet to be determined.

Post mortem changes in relation to different types of clothing.

Post mortem changes are important in estimating post mortem interval (PMI). This project's aim was to study the effect of burial and type of clothing on rate of decomposition, which can contribute to estimating PMI for victims. 12 rabbits (Oryctolagus cuniculus) carcasses were separated into 3 groups: no clothing, light clothing and heavy clothing. Control subjects were placed on the ground surface while test subjects were buried at 30 cm depth graves. Soil samples prior and after decomposition were collected for soil pH and moisture analysis. Post mortem change was assessed using a Total Body Score system. The head, neck and limb regions were found to decay faster than the body trunk region. Mummifi cation occurred on body parts that were exposed directly to the atmosphere while adipocere formed on some buried subjects. Burial delayed decomposition due to lower insect activity and lower soil temperature. The soil layer also blocked the accessibility of majority of the arthropods, causing further delay in decomposition. Clothing enhanced decay for bodies on ground surface because it provided protection for maggots and retained moisture on tissues. However, clothing delayed decomposition in buried bodies because it physically separated the bodies from soil and arthropods. Higher sun exposure and repetitive exhumation showed acceleration of decomposition. The decomposition process increased soil pH and moisture percentage values. Soil pH initially increased until pH 8.0-8.4 followed by a slight decrease while soil moisture percentage changed inconsistently. Burial was significant in affecting post mortem change, F(1,11)=12.991, p<0.05 while type of clothing was not significant, F(2,9)=0.022, p=0.978 and combination of both type of clothing and burial factors were also not significant, F(2,3)=0.429, p=0.686. For validation, an accuracy of 83.33% was achieved based on soil pH and soil moisture percentage analysis.

Plasma vitamin C and tocotrienols changes in response to dietary supplementation among young male adults.

BACKGROUND AND AIMS: Intake of the antioxidant vitamins C and E lowers the oxidative stress. The study aimed to determine plasma concentrations of vitamin C and tocotrienols after supplementation of both vitamins in young male adults. MATERIALS AND METHODS: A total of 64 police recruits were randomly assigned to one of these groups: (a) 500 mg vitamin C (Vitamin C), (b) 200 mg Tocovid (Tocotrienol), (c) combination of 500 mg vitamin C and 200 mg Tocovid (Combination) or (d) placebo (Placebo) for eight-weeks of supplementation followed by six-week washout period. RESULTS: In Combination group, mean plasma vitamin C concentration significantly increased from baseline 2.86 +/- 1.19 mg/L to 10.37 +/- 1.29 mg/L and 15.63 +/- 1.27 mg/L after four- and eight-week supplementation, respectively. The corresponding figures for alpha-, delta- and gamma-tocotrienols were 9.9 +/- 2.5 ng/ml to 104.1 +/- 19.8 ng/ml and 112.8 +/- 38.0 ng/ml; 2.5 +/- 0.9 ng/ml to 29.9 +/- 7.0 ng/ml and 17.9 +/- 4.7 ng/ml; 19.2 +/- 3.1 ng/ml to 75.2 +/- 24.1 ng/ml and 161.7 +/- 49.9 ng/ml, respectively. In Vitamin C group, plasma vitamin C concentrations were significantly increased. Conversely, concentration of plasma vitamin C in Tocotrienol group increased from baseline of 2.72 +/- 0.20 mg/L to 6.80 +/- 0.63 mg/L and 8.9 +/- 0.77 mg/L respectively. Plasma concentrations of alpha-, delta- and gamma-tocotrienols in this group were significantly elevated. After 6-week washout period, all the elevated concentrations returned to basal levels. CONCLUSION: The study showed a good bioavailability of these vitamins and increment due to supplementation.

A preliminary study of reactive oxygen species activity within motile Boer buck sperm using confocal laser scanning microscope (CLSM).

OBJECTIVE: There is an association between reactive oxygen species (ROS) and DNA damage to sperm. Researchers believe that ROS is always present at the sperm's head. The variation of ROS concentration within the area has an impact on the integrity of the DNA. MATERIALS AND METHODS: A study was conducted to confirm the location of ROS within a group of motile Boer buck sperm. A dual photon confocal laser scanning microscope (CLSM) with a 507 nm excitation and 529 nm emission was used on thawed cryopreserved sperms mixed with 10% polyvinyl pyrrolidone (PVP) and stained with 10 mmol/l dihydrorhodamine 123. ROS production was observed and captured under the CLSM at 400x magnification and a frame rate of 35 fps. All fluorescent images were then automatically overlaid with images obtained through Brightfield (BF) to the location of the sperm's membrane. A 3-D reconstruction of the sperm was also conducted to confirm the location of ROS activity. RESULTS: ROS activity was present at the acrosome, midpiece one third of the sperm's tail and whole area of the sperm head. ROS in immotile sperm was also present at the acrosome, one third of sperm tail and the whole area of the head compartment. Relatively fluorescence intensity of ROS was found lower in motile Boer buck sperm as compared to the immotile Boer buck sperm. CONCLUSION: It is concluded that sperm viability largely depends on the ability of the sperm to confine ROS within the acrosome, midpiece and one third of the sperm's tail.

An observation on the decomposition process of gasoline-ingested monkey carcasses in a secondary forest in Malaysia.

This study was designed to mimic homicide or suicide cases using gasoline. Six adult long-tailed macaque (Macaca fascicularis), weighing between 2.5 to 4.0 kg, were equally divided into control and test groups. The control group was sacrificed by a lethal dose of phenobarbital intracardiac while test group was force fed with two doses of gasoline LD50 (37.7 ml/kg) after sedation with phenobarbital. All carcasses were then placed in a decomposition site to observe the decomposition and invasion process of cadaveric fauna on the carcasses. A total of five decomposition stages were recognized during this study. This study was performed during July 2007. Fresh stage of control and test carcasses occurred between 0 to 15 and 0 to 39 hours of exposure, respectively. The subsequent decomposition stages also exhibited the similar pattern whereby the decomposition process of control carcasses were faster than tested one. The first larvae were found on control carcasses after 9 hours of death while the test group carcasses had only their first blowfly eggs after 15 hours of exposure. Blow flies, Achoetandrus rufifacies and Chrysomya megacephala were the most dominant invader of both carcasses throughout the decaying process. Diptera collected from control carcasses comprised of scuttle fly, Megaselia scalaris and flesh fly, sarcophagid. We concluded that the presence of gasoline and its odor on the carcass had delayed the arrival of insect to the carcasses, thereby slowing down the decomposition process in the carcass by 6 hours.

Alpha lipoic acid possess dual antioxidant and lipid lowering properties in atherosclerotic-induced New Zealand White rabbit.

There is accumulating data demonstrated hypercholesterolemia and oxidative stress play an important role in the development of atherosclerosis. In the present study, a protective activity of alpha-lipoic acid; a metabolic antioxidant in hypercholesterolemic-induced animals was investigated. Eighteen adult male New Zealand White (NZW) rabbit were segregated into three groups labelled as group K, AT and ALA (n=6). While group K was fed with normal chow and acted as a control, the rest fed with 100 g/head/day with 1% high cholesterol diet to induce hypercholesterolemia. 4.2 mg/body weight of alpha lipoic acid was supplemented daily to the ALA group. Drinking water was given ad-libitum. The study was designed for 10 weeks. Blood sampling was taken from the ear lobe vein at the beginning of the study, week 5 and week 10 and plasma was prepared for lipid profile estimation and microsomal lipid peroxidation index indicated with malondialdehyde (MDA) formation. Animals were sacrificed at the end of the study and the aortas were excised for intimal lesion analysis. The results showed a significant reduction of lipid peroxidation index indicated with low MDA level (p<0.05) in ALA group compared to that of the AT group. The blood total cholesterol (TCHOL) and low density lipoprotein (LDL) levels were found to be significantly low in ALA group compared to that of the AT group (p<0.05). Histomorphometric intimal lesion analysis of the aorta showing less of atheromatous plaque formation in alpha lipoic acid supplemented group (p<0.05) compared to that of AT group. These findings suggested that apart from its antioxidant activity, alpha lipoic acid may also posses a lipid lowering effect indicated with low plasma TCHOL and LDL levels and reduced the athero-lesion formation in rabbits fed a high cholesterol diet.

The effect of ∞-lipoic Acid in blood lipid levels and malondialdehyde in atherosclerotic-induced new zealand white rabbit.

∞-Lipoic acid (ALA) is a naturally occuring cofactor that serves as an acyl carrier in oxidative decarboxylation of α-keto acids in carbohydrate metabolism. Current findings suggest that ∞-lipoic acid and its reduced form, dihydrolipoic acid (DHLA) may act as antioxidants and are able to quench free radicals in vitro and in vivo. However, the mechanism underlying the process is still unknown. In this study, atherosclerotic lesions were induced in six groups of adult male NZW rabbits labelled as group K, A, B, C, D, E (n=6) by giving 100g/head/day of 2% cholesterol-rich diet for ten weeks. While group K acted as a control, the rest were supplemented with ALA orally (1.4, 2.8, 4.2, 8.0 and 10mg/kg, respectively). In week ten, venous blood samples drawn from ear lobes were analysed for complete lipid profile and peroxidation index. The results showed a significant reduction of total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C) levels in most of the treated groups as compared to the control whereas apo-A levels showed a significant increase in group C and D. However, microsomal lipid peroxidation index, malondialdehyde (MDA) was found to be not significantly different. These findings suggest that ∞-lipoic acid may act as a lipid lowering agent in dose dependent manner in premature stage of atherosclerosis but was unable to inhibit lipid peroxidation processes in matured stage of atherosclerosis in rabbits fed a high cholesterol diet.