Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa

<p>Abstract</p> <p>Background</p> <p>Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters.</p> <p>Results</p> <p>Ejaculates were collected using the artificial vagina from four Chal rams and three replicates of the ejaculates were diluted with a Tris-based extender and packed in 0.25 ml straws. Then, sample processed according to the two methods. Method 1: straws cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and equilibrated at 5°C for 80 min, then the straws were frozen at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C and plunged into liquid nitrogen for storage. Method 2: straws were transferred to refrigerator and maintained at 5°C for 3 h, then the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen for 15 min and plunged into liquid nitrogen. Computer-assisted sperm motility analysis was used to analyze sperm motion characteristics.</p> <p>Conclusions</p> <p>Controlled rate of freezing (Method 1) significantly improve the pre-freezing and post-thaw total and progressive motility compared to uncontrolled rate (Method 2). In specific kinetic parameters, Method 1 gives significantly higher value for VSL and VCL in comparison with Method 2. There are no significant differences between the two methods for VAP and LIN. In conclusion, controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared to uncontrolled rate of cooling prior to programmable freezing.</p

Effects of the Insemination of Hydrogen Peroxide-Treated Epididymal Mouse Spermatozoa on γH2AX Repair and Embryo Development

BACKGROUND: Cryopreservation of human semen for assisted reproduction is complicated by cryodamage to spermatozoa caused by excessive reactive oxygen species (ROS) generation. METHODS AND FINDINGS: We used exogenous ROS (H(2)O(2)) to simulate cryopreservation and examined DNA damage repair in embryos fertilized with sperm with H(2)O(2)-induced DNA damage. Sperm samples were collected from epididymis of adult male KM mice and treated with capacitation medium (containing 0, 0.1, 0.5 and 1 mM H(2)O(2)) or cryopreservation. The model of DNA-damaged sperm was based on sperm motility, viability and the expression of γH2AX, the DNA damage-repair marker. We examined fertility rate, development, cell cleavage, and γH2AX level in embryos fertilized with DNA-damaged sperm. Cryopreservation and 1-mM H(2)O(2) treatment produced similar DNA damage. Most of the one- and two-cell embryos fertilized with DNA-damaged sperm showed a delay in cleavage before the blastocyst stage. Immunocytochemistry revealed γH2AX in the one- and four-cell embryos. CONCLUSIONS: γH2AX may be involved in repair of preimplantation embryos fertilized with oxygen-stressed spermatozoa

Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5°C.

The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5°C. Thirty-two Merino rams (12-14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 μg/day of total AF. The EG group received commercial feed plus 2g/day of EG. The AF + EG group was given commercial feed plus 250 μg/day of total AF and 2g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5°C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5°C at 0, 24, 48, 72 and 96 h of liquid storage. During the first two weeks of the trial, the groups did not statistically differ from each other for sperm motility or rates of abnormal and nonviable spermatozoa at 0, 24, 48 and 96 h of storage. As from the third week, the short-term storage of semen produced statistically significant differences between the AF group and the other treatment groups for sperm parameters (p<0.05). The administration of aflatoxin was observed to have reduced sperm motility and to have increased the rates of abnormal and nonviable spermatozoa in comparison to the control group (p<0.05), while EG co-administered with AF was determined to have ameliorated the adverse effects of AF on sperm parameters, and this ameliorative effect continued throughout the short-term storage of semen. On the other hand, aflatoxin administration resulted in the deterioration of the sperm parameters in the following weeks, and the combined administration of EG + AF reversed this adverse effect, thus, bringing the sperm parameters closer to the values of the control group. This study demonstrated that, in rams, AF adversely affected sperm, biochemical and testis parameters, and that the combined administration of EG and AF reversibly improved these adverse effects

Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities

Ram Semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the Semen. The aim of the present work was to Study the effect of two anti-oxidants, namely, glutamine and all amino acid Solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rains were evaluated and pooled at 37 degrees C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 degrees C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0+/-4.4%) and hypo-osmotic swelling test (HOST) (64.1+/-5.5%). when compared to glutamine (2.5 mM) and BME (13 and 26%) (P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the Supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4+/-0.9 kU/g protein), compared to the other treatments (P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control (P < 0.05). Findings showed that glutamine (5 mM) supplementation ill Semen extenders, was of greater benefit to frozen-thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen-thawed ram sperm is used. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved

storage at 5 degrees C

The aim of this study was to investigate the effects of methionine and lipoic acid on ram sperm parameters during liquid storage (5 degrees C). Ejaculates collected from five Merino rams were pooled at 37 degrees C. Each pooled ejaculate was divided into five equal aliquots and diluted (37 degrees C) with five extenders, one of which was without additives, two of which contained methionine at two different doses, and the other two of which contained lipoic acid at two different doses. Sperm parameters were determined at 0, 24, 48, 72 and 96 h of liquid storage at 5 degrees C.The extenders containing 2 and 4 mM of methionine resulted in higher motility percentages, in comparison to the control, up to 96 h of storage. Methionine at doses of 2 and 4 mM led to higher viability and sperm mitochondrial activity percentages, when compared to the controls during 48, 72 and 96 h of liquid storage (P < 0.05). The findings of this study showed that methionine was of greater benefit to ram sperm parameters during liquid storage. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved

The influence of cysteine and taurine on microscopic-oxidative stress parameters and fertilizing ability of bull semen following cryopreservation

Oxidative stress significantly damages sperm functions such as motility, functional integrity, endogenous antioxidant enzyme activities and fertility due to lipid peroxidation induced by reactive oxygen species (ROS). The aim of this study was to determine the effects of antioxidants such as taurine and cysteine in Bioxcell (R) extender on standard semen parameters, fertilizing ability, lipid peroxidation (LPO) and antioxidant activities comprising reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) after the cryopreservation/thawing of bull semen. Nine ejaculates for each bull were included in the study. Three groups, namely taurine (2 mM), cysteine (2 mM), and control, were designed to analyze the antioxidants in Bioxcell (R). Insemination doses were processed so that each 0.25-ml straw contained 15 x 10(6) sperm

Protective effect of esterified glucomannan on aflatoxin-induced changes in testicular function, sperm quality, and seminal plasma biochemistry in rams.

The aim of this study was to determine the effect of aflatoxin (AF) on spermatologic, biochemical, and testis parameters in rams, and the protective efficiency of esterified glucomannan (EG) co-administered with AF. Thirty-two Merino rams (12-14 months old) were used. The experimental design consisted of four dietary treatments. The control group was fed commercial feed. The AF group was fed with commercial feed plus 250 μg/d of total AF. The EG group received commercial feed plus 2 g/d of EG. The AF + EG group was given commercial feed plus 250 μg/d of total AF and 2 g/d of EG. There were treatment, time, and treatment-by-time interaction effects on sperm motility, abnormal spermatozoa, damaged acrosome, and dead spermatozoa (P < 0.01). The percentage of motile sperm was lower and the percentages of abnormal sperm, sperm with damaged acrosomes, and dead sperm were greater in the AF group than in the control, AF+EG, and EG groups, as from week 3 until the end of week 12 (P < 0.05). As from week 3, hyaluronidase activity in the seminal plasma increased significantly in the AF group, compared with the control. The co-administration of AF+EG was found to be effective in preventing the increase in hyaluronidase activity. As week 4, malondialdehyde (MDA) levels were significantly higher in the AF group compared with the control. The combined administration of AF+EG was found to be effective in lowering the MDA levels, increased by AF, to the levels measured in the control (P < 0.05). Although glutathione (GSH) levels were determined to have significantly decreased in the AF group in comparison to the control, it was observed that, in the group co-administered with AF and EG, particularly after week 7, the GSH levels, which had decreased owing to AF, were largely ameliorated (P < 0.05). In conclusion, AF adversely affected spermatologic, biochemical, and testis parameters, and the combined administration of EG with AF reversibly eliminated these adverse effects in rams